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The following set of peer-reviewed publications demonstrates the breadth and depth of our capabilities and the vast experience of our experts.
YearAuthorsTitleJournalPageVolumeIssuePubMed IDAbstractImpact FactorSnippetLinkKeyword
2020Daniel J. Butler, Christopher Mozsary, Cem Meydan, David Danko, Jonathan Foox, Joel Rosiene, Alon Shaiber, Ebrahim Afshinnekoo, Matthew MacKay, Fritz J. Sedlazeck, Nikolay A.Ivanov, Maria Sierra, Diana Pohle, Michael Zietz, Vijendra Ramlall, Undina Gisladottir, Craig D. Westover, Krista Ryon, Benjamin Young, Chandrima Bhattacharya, Phyllis Ruggiero, Bradley W. Langhorst, Nathan Tanner, Justyna Gawrys, Dmitry Meleshko, Dong Xu, Jenny Xiang, Angelika Iftner, Daniela Bezdan, John Sipley, Lin Cong, Arryn Craney, Priya Velu, Ari M. Melnick, Iman Hajirasouliha, Thomas Iftner, Mirella Salvatore, Massimo Loda, Lars F. Westblade, Shawn Levy, Melissa Cushing, Nicholas Tatonetti, Marcin Imielinski, Hanna Rennert, Christopher E. MasonHost, Viral, and Environmental Transcriptome Profiles of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)bioRxivThe pandemic from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to hundreds of thousands of deaths, including >15,000 in New York City (NYC). This pandemic highlighted a pressing clinical and public health need for rapid, scalable diagnostics that can detect SARS-CoV-2 infection, interrogate strain evolution, and map host response in patients. To address these challenges, we designed a fast (30 minute) colorimetric test to identify SARS-CoV-2 infection and simultaneously developed a large-scale shotgun metatranscriptomic profiling platform for nasopharyngeal swabs. Both technologies were used to profile 338 clinical specimens tested for SARS-CoV-2 and 86 NYC subway samples, creating a broad molecular picture of the COVID-19 epidemic in NYC. Our results nominate a novel, NYC-enriched SARS-CoV-2 subclade, reveal specific host responses in ACE pathways, and find mediation risks associated with SARS-CoV-2 infection and ACE inhibitors. Our findings have immediate applications to SARS-CoV-2 diagnostics, public health monitoring, and therapeutic development.The pandemic from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to hundreds of thousands of deaths, including >15,000 in New York City (NYC). This pandemic highlighted a pressing clinical and public health need for rapid, scalable diagnostics that can detect SARS-CoV-2 infection, interrogate strain evolution, and map host response in patients. To address these challenges, we designed a fast (30 minute) colorimetric test to identify SARS-CoV-2 infection and simultaneously developed a large-scale shotgun metatranscriptomic profiling platform for nasopharyngeal swabs. Both technologies were used to profile 338 clinical specimens tested for SARS-CoV-2 and 86 NYC subway samples, creating a broad molecular picture of the COVID-19 epidemic in NYC. Our results nominate a novel, NYC-enriched SARS-CoV-2 subclade, reveal specific host responses in ACE pathways, and find mediation risks associated with SARS-CoV-2 infection and ACE inhibitors. Our findings have immediate applications to SARS-CoV-2 diagnostics, public health monitoring, and therapeutic development.https://www.biorxiv.org/content/10.1101/2020.04.20.048066v3Discovery Life Sciences
2020M.C.Liu, G.R.Oxnard, E.A.Klein, C.Swanton, M.V.SeidenSensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNAAnnals of Oncology745-759316142,0Background: Early cancer detection could identify tumors at a time when outcomes are superior and treatment is less morbid. This prospective case-control sub-study (from NCT02889978 and NCT03085888) assessed the performance of targeted methylation analysis of circulating cell-free DNA (cfDNA) to detect and localize multiple cancer types across all stages at high specificity. Participants and methods: The 6689 participants [2482 cancer (>50 cancer types), 4207 non-cancer] were divided into training and validation sets. Plasma cfDNA underwent bisulfite sequencing targeting a panel of >100 000 informative methylation regions. A classifier was developed and validated for cancer detection and tissue of origin (TOO) localization. Results: Performance was consistent in training and validation sets. In validation, specificity was 99.3% [95% confidence interval (CI): 98.3% to 99.8%; 0.7% false-positive rate (FPR)]. Stage I–III sensitivity was 67.3% (CI: 60.7% to 73.3%) in a pre-specified set of 12 cancer types (anus, bladder, colon/rectum, esophagus, head and neck, liver/bile-duct, lung, lymphoma, ovary, pancreas, plasma cell neoplasm, stomach), which account for ∼63% of US cancer deaths annually, and was 43.9% (CI: 39.4% to 48.5%) in all cancer types. Detection increased with increasing stage: in the pre-specified cancer types sensitivity was 39% (CI: 27% to 52%) in stage I, 69% (CI: 56% to 80%) in stage II, 83% (CI: 75% to 90%) in stage III, and 92% (CI: 86% to 96%) in stage IV. In all cancer types sensitivity was 18% (CI: 13% to 25%) in stage I, 43% (CI: 35% to 51%) in stage II, 81% (CI: 73% to 87%) in stage III, and 93% (CI: 87% to 96%) in stage IV. TOO was predicted in 96% of samples with cancer-like signal; of those, the TOO localization was accurate in 93%. Conclusions: cfDNA sequencing leveraging informative methylation patterns detected more than 50 cancer types across stages. Considering the potential value of early detection in deadly malignancies, further evaluation of this test is justified in prospective population-level studies.https://www.sciencedirect.com/science/article/pii/S0923753420360580Discovery Life Sciences
2018Cristescu, R;Mogg, R;Ayers, M;Albright, A;Murphy, E;Yearley, J;Sher, X;Liu, XQ;Lu, H;Nebozhyn, M;Zhang, C;Lunceford, JK;Joe, A;Cheng, J;Webber, AL;Ibrahim, N;Plimack, ER;Ott, PA;Seiwert, TY;Ribas, A;McClanahan, TK;Tomassini, JE;Loboda, A;Kaufman, D;Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapyScience362641130309915Programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) checkpoint blockade immunotherapy elicits durable antitumor effects in multiple cancers, yet not all patients respond. We report the evaluation of >300 patient samples across 22 tumor types from four KEYNOTE clinical trials. Tumor mutational burden (TMB) and a T cell-inflamed gene expression profile (GEP) exhibited joint predictive utility in identifying responders and nonresponders to the PD-1 antibody pembrolizumab. TMB and GEP were independently predictive of response and demonstrated low correlation, suggesting that they capture distinct features of neoantigenicity and T cell activation. Analysis of The Cancer Genome Atlas database showed TMB and GEP to have a low correlation, and analysis by joint stratification revealed biomarker-defined patterns of targetable-resistance biology. These biomarkers may have utility in clinical trial design by guiding rational selection of anti-PD-1 monotherapy and combination immunotherapy regimens. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.410,0Associations of TMB and the T cell–inflamed GEP with BOR and PFS were evaluated by using tumor samples from subgroups of patients treated with pembrolizumab in clinical trials who had WES data available. These included a discovery cohort of patients with HNSCC (KEYNOTE-012 B1), a pan-tumor validation cohort (KEYNOTE-012/028), and single-indication cohorts of patients with HNSCC (KEYNOTE-012 B1+B2) and melanoma (KN001 and 006). The discovery cohort included 34 of 297 total enrolled patients with PD-L1–selected (≥1%, modified proportion score or interface pattern, QualTek IHC) (39) HNSCC (B1 cohort). The pan-tumor cohort comprised patients with PD-L1-positive (≥1%, modified proportion score or interface pattern, QualTek IHC) (39) advanced solid tumors pooled from two multi-cohort trials, including 39 of 297 total enrolled patients in KEYNOTE-012 (cohorts A, C, and D: triple-negative breast cancer, urothelial cancer, and gastric cancer, respectively) and 80 of 450 total enrolled patients in KEYNOTE-028 (17 of 20 cohorts with anal, biliary, carcinoid, cervical, colorectal, endometrial, esophageal, estrogen receptor–positive human epidermal growth factor receptor-2–negative breast, pancreatic, salivary gland, prostate, small cell lung, thyroid, and vulvar cancers and neuroendocrine tumors, mesothelioma, and leiomyosarcomahttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718162QualTek
2020Geoerger, B;Kang, HJ;Yalon-Oren, M;Marshall, LV;Vezina, C;Pappo, A;Laetsch, TW;Petrilli, AS;Ebinger, M;Toporski, J;Glade-Bender, J;Nicholls, W;Fox, E;DuBois, SG;Macy, ME;Cohn, SL;Pathiraja, K;Diede, SJ;Ebbinghaus, S;Pinto, N;Pembrolizumab in paediatric patients with advanced melanoma or a PD-L1-positive, advanced, relapsed, or refractory solid tumour or lymphoma (KEYNOTE-051): interim analysis of an open-label, single-arm, phase 1–2 trialLancet Oncol.121-13321131812554Pembrolizumab is approved for the treatment of advanced cancer in adults; however, no information is available on safety and efficacy in paediatric patients. We aimed to establish the recommended phase 2 dose of pembrolizumab and its safety and antitumour activity in advanced paediatric cancer. KEYNOTE-051 is an ongoing phase 1-2 open-label trial. In this interim analysis, children aged 6 months to 17 years were recruited at 30 hospitals located in Australia, Brazil, Canada, France, Germany, Israel, Italy, South Korea, Sweden, the UK, and the USA. Patients with melanoma or a centrally confirmed, PD-L1-positive, relapsed or refractory solid tumour or lymphoma, and a Lansky Play/Karnofsky Performance status score of 50 or higher, received intravenous pembrolizumab at an initial dose of 2 mg/kg every 3 weeks. Pharmacokinetics and dose-limiting toxicities were used to establish the recommended phase 2 dose, and the safety and antitumour activity of this dose were assessed. Primary endpoints were determination of dose-limiting toxicities at the maximum administered dose, safety and tolerability, and the proportion of patients with objective response to pembrolizumab for each tumour type according to the Response Evaluation Criteria in Solid Tumours version 1.1 or the International Neuroblastoma Response Criteria. Safety and efficacy were assessed in all treated patients who received at least one dose of pembrolizumab. Separate reporting of the cohort of patients with relapsed or refractory classical Hodgkin lymphoma was a post-hoc decision. The data cutoff for this interim analysis was Sept 3, 2018. This trial is still enrolling patients and is registered with ClinicalTrials.gov, number NCT02332668. Of 863 patients screened between March 23, 2015, and Sept 3, 2018, 796 had tumours that were evaluable for PD-L1 expression (278 [35%] were PD-L1-positive); 155 eligible patients were enrolled and 154 had at least one dose of pembrolizumab. The median age of the enrolled patients was 13 years (IQR 8-15). Median follow-up was 8·6 months (IQR 2·5-16·4). No dose-limiting toxicities were reported in phase 1, and pembrolizumab plasma concentrations were consistent with those previously reported in adults; the recommended phase 2 dose was therefore established as 2 mg/kg every 3 weeks. Of the 154 patients treated, 69 (45%) experienced grade 3-5 adverse events, most commonly anaemia in 14 (9%) patients and decreased lymphocyte count in nine (6%) patients. 13 (8%) of the 154 patients had grade 3-5 treatment-related adverse events, most commonly decreased lymphocyte count in three (2%) patients and anaemia in two (1%) patients. 14 (9%) patients had serious treatment-related adverse events, most commonly pyrexia (four [3%]), and hypertension and pleural effusion (two [1%] each). Four patients (3%) discontinued treatment because of treatment-related adverse events, and two (1%) died (one due to pulmonary oedema and one due to pleural effusion and pneumonitis). Of 15 patients with relapsed or refractory Hodgkin lymphoma, two had complete and seven had partial responses; thus, nine patients achieved an objective response (60·0%; 95% CI 32·3-83·7). Of 136 patients with solid tumours and other lymphomas, eight had partial responses (two patients each with adrenocortical carcinoma and mesothelioma, and one patient each with malignant ganglioglioma, epithelioid sarcoma, lymphoepithelial carcinoma, and malignant rhabdoid tumour); the proportion of patients with an objective response was 5·9% (95% CI 2·6-11·3). Pembrolizumab was well tolerated and showed encouraging antitumour activity in paediatric patients with relapsed or refractory Hodgkin lymphoma, consistent with experience in adult patients. Pembrolizumab had low antitumour activity in the majority of paediatric tumour types, and responses were observed in only a few rare PD-L1-positive tumour types, suggesting that PD-L1 expression alone is not sufficient as a biomarker for the selection of paediatric patients who are likely to respond to PD-1 checkpoint inhibitors. Final results of KEYNOTE-051, expected by September, 2022, with the possibility for extension, will report further on the activity of pembrolizumab in Hodgkin lymphoma, microsatellite instability-high tumours, and melanoma. Merck Sharp & Dohme, a subsidiary of Merck & Co. Copyright © 2020 Elsevier Ltd. All rights reserved.354,0In all patients, baseline PD-L1 expression in tumour samples (positivity defined as any staining of the stroma or PD-L1 expression on at least 1% of tumour cells) was assessed centrally at QualTek Molecular Laboratories (Goleta, CA, USA) using a prototypehttps://www.sciencedirect.com/science/article/pii/S1470204519306710QualTek
2017Alley, EW;Lopez, J;Santoro, A;Morosky, A;Saraf, S;Piperdi, B;van Brummelen, E;Clinical safety and activity of pembrolizumab in patients with malignant pleural mesothelioma (KEYNOTE-028): preliminary results from a non-randomised, open-label, phase 1b trialLancet Oncol.623-63018528291584Malignant pleural mesothelioma is a highly aggressive cancer with poor prognosis and few treatment options following progression on platinum-containing chemotherapy. We assessed the safety and efficacy of pembrolizumab (an anti-programmed cell death receptor 1 [PD-1] antibody) in advanced solid tumours expressing programmed cell death ligand 1 (PD-L1) and report here on the interim analysis of the malignant pleural mesothelioma cohort. Previously treated patients with PD-L1-positive malignant pleural mesothelioma were enrolled from 13 centres in six countries. Patients received pembrolizumab (10 mg/kg every 2 weeks) for up to 2 years or until confirmed progression or unacceptable toxicity. Key eligibility criteria included measurable disease, failure of standard therapy, and Eastern Cooperative Oncology Group performance status of 0 or 1. PD-L1 positivity was defined as expression in 1% or more of tumour cells by immunohistochemistry. Response was assessed based on investigator review using the Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1). Primary endpoints were safety and tolerability, analysed in the all-patients-as-treated population, and objective response, analysed for the full-analysis set. This trial is registered with ClinicalTrials.gov, number NCT02054806, and is ongoing but not recruiting participants. As of June 20, 2016, 25 patients received pembrolizumab. 16 (64%) patients reported a treatment-related adverse event; the most common adverse event were fatigue (six [24%]), nausea (six [24%]), and arthralgia (five [20%]). Five (20%) patients reported grade 3 treatment-related adverse events. Three (12%) patients required dose interruption because of immune-related adverse events: one (4%) of 25 each had grade 3 rhabdomyolysis and grade 2 hypothyroidism; grade 3 iridocyclitis, grade 1 erythema multiforme, and grade 3 erythema; and grade 2 infusion-related reaction. No treatment-related deaths or discontinuations occurred. Five (20%) patients had a partial response, for an objective response of 20% (95% CI 6·8-40·7), and 13 (52%) of 25 had stable disease. Responses were durable (median response duration 12·0 months [95% CI 3·7 to not reached]); two patients remained on treatment at data cutoff. Pembrolizumab appears to be well tolerated and might confer anti-tumour activity in patients with PD-L1-positive malignant pleural mesothelioma. Response durability and efficacy in this patient population warrants further investigation. Merck. Copyright © 2017 Elsevier Ltd. All rights reserved.354,0PD-L1 expression was assessed in archival or new tumour samples by a central laboratory (QualTek Molecular Laboratories, Goleta, CA, USA) with a prototype assay and the 22C3 antibody (Merck, Kenilworth, NJ, USA).22, 23 PD-L1 positivity was defined by membranous PDhttps://www.sciencedirect.com/science/article/pii/S1470204517301699QualTek
2017Plimack, ER;Bellmunt, J;Gupta, S;Berger, R;Chow, LQ;Juco, J;Lunceford, J;Saraf, S;Perini, RF;O'Donnell, PH;Safety and activity of pembrolizumab in patients with locally advanced or metastatic urothelial cancer (KEYNOTE-012): a non-randomised, open-label, phase 1b studyLancet Oncol.212-22018228081914PD-1 and its ligands are expressed in urothelial cancer, and findings have shown that inhibition of the PD-1 pathway has clinical benefit. We aimed to assess the safety and activity of an anti-PD-1 antibody pembrolizumab in patients with locally advanced or metastatic urothelial cancer. This study was part of the non-randomised, multi-cohort, open-label, phase 1b KEYNOTE-012 basket trial. We enrolled patients aged 18 years and older with a histologically or cytologically confirmed diagnosis of locally advanced or metastatic urothelial cancer, including cancers of the renal pelvis, ureter, bladder, or urethra, from eight hospitals in the USA and Israel. Patients were required to have at least 1% PD-L1 expression detected on the tumour cells or in tumour stroma, as determined by immunohistochemistry. Patients were given 10 mg/kg intravenous pembrolizumab every 2 weeks until disease progression, unacceptable toxic effects, or the end of the study (ie, 24 months of treatment). Primary endpoints were safety and overall response (defined by Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1), as assessed by a masked, independent central review. Safety was assessed in patients who received one or more doses of pembrolizumab (all-patients-as-treated population); activity was assessed in patients who received pembrolizumab, had measurable disease at baseline, and had one or more post-baseline scans, or discontinued because of progressive disease or treatment-related adverse events (full analysis set). This study is registered with ClinicalTrials.gov, number NCT01848834, and is no longer enrolling patients; follow-up is ongoing. Between May 14, 2013, and Dec 10, 2013, 115 patients were tissue pre-screened as part of a two-part consent process. 61 (53%) patients were PD-L1 positive, of whom 33 were enrolled in this study. All enrolled patients received at least one dose of pembrolizumab and were included in the safety analyses. 27 patients comprised the full analysis set and were deemed assessable for activity. Six patients were not assessable: three discontinued study drug because of a non-treatment-related adverse event before the first post-baseline scan, two withdrew before the first post-baseline scan, and one had no measurable disease at baseline. The most common treatment-related adverse events were fatigue (six [18%] of 33 patients) and peripheral oedema (4 [12%]). Five (15%) patients had 11 grade 3 treatment-related adverse events; no single event occurred in more than one patient. Three (9%) patients experienced five serious treatment-related adverse events. After median follow-up of 13 months (range 1-26, IQR 5-23), an overall response was achieved in seven (26% [95% CI 11-46]) of 27 assessable patients, with three (11% [2-29]) complete and four (15% [4-34]) partial responses. Of the four deaths that occurred during the study (cardiac arrest, pneumonia, sepsis, and subarachnoid haemorrhage), none were deemed treatment related. Pembrolizumab showed anti-tumour activity and acceptable safety in patients with advanced urothelial cancer, supporting ongoing phase 2 and 3 studies of pembrolizumab in this population. Merck & Co., Inc. Copyright © 2017 Elsevier Ltd. All rights reserved.354,0Initially, we used a prototype PD-L1 immunohistochemical assay to establish the eligibility of patients in KEYNOTE-012, which was done at a laboratory site at QualTek (Goleta, CA, USA) accredited by the College of American Pathologists and Clinical Laboratory Improvementhttps://www.sciencedirect.com/science/article/pii/S1470204517300074QualTek
2016Goldberg, SB;Gettinger, SN;Mahajan, A;Chiang, AC;Herbst, RS;Sznol, M;Tsiouris, AJ;Cohen, J;Vortmeyer, A;Jilaveanu, L;Yu, J;Hegde, U;Speaker, S;Madura, M;Ralabate, A;Rivera, A;Rowen, E;Gerrish, H;Yao, X;Chiang, V;Kluger, HM;Pembrolizumab for patients with melanoma or non-small-cell lung cancer and untreated brain metastases: early analysis of a non-randomised, open-label, phase 2 trialLancet Oncol.976-98317727267608Immunotherapy targeting the PD-1 axis has activity in several tumour types. We aimed to establish the activity and safety of the PD-1 inhibitor pembrolizumab in patients with untreated brain metastases from melanoma or non-small-cell lung cancer (NSCLC). In this non-randomised, open-label, phase 2 trial, we enrolled patients aged 18 years or older with melanoma or NSCLC with untreated brain metastases from the Yale Cancer Center. Patients had at least one untreated or progressive brain metastasis between 5 and 20 mm in diameter without associated neurological symptoms or the need for corticosteroids. Patients with NSCLC had tumour tissue positive for PD-L1 expression; this was not required for patients with melanoma. Patients were given 10 mg/kg pembrolizumab every 2 weeks until progression. The primary endpoint was brain metastasis response assessed in all treated patients. The trial is ongoing and here we present an early analysis. The study is registered with ClinicalTrials.gov, number NCT02085070. Between March 31, 2014, and May 31, 2015, we screened 52 patients with untreated or progressive brain metastases (18 with melanoma, 34 with NSCLC), and enrolled 36 (18 with melanoma, 18 with NSCLC). A brain metastasis response was achieved in four (22%; 95% CI 7-48) of 18 patients with melanoma and six (33%; 14-59) of 18 patients with NSCLC. Responses were durable, with all but one patient with NSCLC who responded showing an ongoing response at the time of data analysis on June 30, 2015. Treatment-related serious and grade 3-4 adverse events were grade 3 elevated aminotransferases (n=1 [6%]) in the melanoma cohort, and grade 3 colitis (n=1 [6%]), grade 3 pneumonitis (n=1 [6%]), grade 3 fatigue (n=1 [6%]), grade 4 hyperkalemia (n=1 [6%]), and grade 2 acute kidney injury (n=1 [6%]) in the NSCLC cohort. Clinically significant neurological adverse events included transient grade 3 cognitive dysfunction and grade 1-2 seizures (n=3 [17%]) in the melanoma cohort. Pembrolizumab shows activity in brain metastases in patients with melanoma or NSCLC with an acceptable safety profile, which suggests that there might be a role for systemic immunotherapy in patients with untreated or progressive brain metastases. Merck and the Yale Cancer Center. Copyright © 2016 Elsevier Ltd. All rights reserved.354,0In patients with NSCLC, PD-L1 staining was done by Qualtek (Goleta, CA, USA) on tissue from any disease site after the most recent systemic therapy employing previously described methods 6 using 1% staining as a cutpoint for positivityhttps://www.sciencedirect.com/science/article/pii/S1470204516300535QualTek
2016Seiwert, TY;Burtness, B;Mehra, R;Weiss, J;Berger, R;Eder, JP;Heath, K;McClanahan, T;Lunceford, J;Gause, C;Cheng, JD;Chow, LQ;Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trialLancet Oncol.956-96517727247226Patients with recurrent or metastatic squamous cell carcinoma of the head and neck have few treatment options. We aimed to assess the safety, tolerability, and antitumour activity of pembrolizumab, a humanised anti-programmed death receptor 1 (PD-1) antibody, in patients with PD-L1-positive recurrent or metastatic squamous cell carcinoma of the head and neck. This study was an open-label, multicentre, phase 1b trial of patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Patients were eligible for enrolment if they were aged 18 years or older, had a confirmed diagnosis of recurrent or metastatic squamous cell carcinoma of the head and neck, and had any level of PD-L1 expression (ie, at least 1% of tumour cells or stroma that were PD-L1-positive by immunohistochemistry). Patients received pembrolizumab 10 mg/kg intravenously every 2 weeks. Primary outcomes were safety in the per-protocol population and the proportion of patients with centrally reviewed overall response per Response Evaluation Criteria In Solid Tumors (RECIST, version 1.1). Overall response was analysed in the full analysis set, which was defined as all patients who had received at least one dose of pembrolizumab, had measurable disease at baseline, and one post-baseline scan or patients without a post-baseline scan who discontinued therapy because of disease progression or a drug-related adverse event. The study is registered with ClinicalTrials.gov, number NCT01848834 and is ongoing, but no longer enrolling patients. Of the 104 patients screened between June 7, 2013, and Oct 3, 2013, 81 (78%) were PD-L1-positive. Of these, 60 patients with PD-L1-positive squamous cell carcinoma of the head and neck were enrolled and treated: 23 (38%) were HPV-positive and 37 (62%) were HPV-negative. Pembrolizumab was well tolerated, with 10 (17%) of 60 patients having grade 3-4 drug-related adverse events, the most common of which were increases in alanine aminotransferase and in aspartate aminotransferase, and hyponatraemia, each occurring in two of 60 patients; one patient developed a grade 3 drug-related rash. 27 (45%) of 60 patients experienced a serious adverse event. There were no drug-related deaths. The proportion of patients with an overall response by central imaging review was 18% (eight of 45 patients; 95% CI 8-32) in all patients and was 25% (four of 16 patients; 7-52) in HPV-positive patients and 14% (four of 29 patients; 4-32) in HPV-negative patients. Pembrolizumab was well tolerated and demonstrated clinically meaningful antitumour activity in recurrent or metastatic squamous cell carcinoma of the head and neck, supporting further study of pembrolizumab as anticancer therapy for advanced head and neck cancers. Merck & Co. Copyright © 2016 Elsevier Ltd. All rights reserved.354,0Funding for this research was provided by Merck & Co, Inc, Kenilworth, NJ, USA. The authors thank the patients and their families and caregivers for participating in the study. The authors also thank Mark Ayers, Michael Nebozhyn, Erin Murphy, and Jennifer Yearley for their contributions to the NanoString analysis, and QualTek Molecular Laboratories (Newtown, PA, USA) for development of the PD-L1 immunohistochemistry assay. Medical writing assistance was provided by Matthew Grzywacz, PhD, of ApotheCom, Yardley, PA, USA. This assistance was funded by Merck & Co, Inc, Kenilworth, NJ, USAhttps://www.sciencedirect.com/science/article/pii/S1470204516300663QualTek
2016Muro, K;Chung, HC;Shankaran, V;Geva, R;Catenacci, D;Gupta, S;Eder, JP;Golan, T;Le, DT;Burtness, B;McRee, AJ;Lin, CC;Pathiraja, K;Lunceford, J;Emancipator, K;Juco, J;Koshiji, M;Bang, YJ;Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trialLancet Oncol.717-72617627157491Expression of PD-L1 has been shown to be upregulated in some patients with gastric cancer. As part of the phase 1b KEYNOTE-012 study, we aimed to assess the safety and activity of the anti-PD-1 antibody pembrolizumab in patients with PD-L1-positive recurrent or metastatic adenocarcinoma of the stomach or gastro-oesophageal junction. This study was a multicentre, open-label, phase 1b trial done at 13 cancer research centres in the USA, Israel, Japan, South Korea, and Taiwan. We enrolled patients with PD-L1-positive recurrent or metastatic adenocarcinoma of the stomach or gastro-oesophageal junction. Patients received intravenous pembrolizumab at 10 mg/kg once every 2 weeks for 24 months or until progression or unacceptable toxic effects occurred. Response was assessed every 8 weeks in accordance with Response Evaluation Criteria in Solid Tumors version 1.1. The primary objectives were safety in patients who received at least one dose of pembrolizumab and the proportion of patients achieving overall responses in patients who received at least one pembrolizumab dose and who either had a post-baseline scan or who discontinued therapy because of clinical disease progression or a treatment-related adverse event before the first post-baseline scan. The study is registered with ClinicalTrials.gov, number NCT01848834, and is ongoing but no longer enrolling patients. From Oct 23, 2013, to May 5, 2014, 39 patients were enrolled. 36 were evaluable for response by central assessment. Eight (22%, 95% CI 10-39) patients were judged to have had an overall response at central review; all responses were partial. All 39 patients were included in the safety analyses. Five (13%) patients had a total of six grade 3 or 4 treatment-related adverse events, consisting of two cases of grade 3 fatigue, one case each of grade 3 pemphigoid, grade 3 hypothyroidism, and grade 3 peripheral sensory neuropathy, and one case of grade 4 pneumonitis. No treatment-related deaths occurred. In this population of patients with recurrent or metastatic PD-L1-positive gastric cancer, pembrolizumab had a manageable toxicity profile and promising antitumour activity, warranting further study in phase 2 and 3 trials. Merck & Co. Copyright © 2016 Elsevier Ltd. All rights reserved.354,0To screen for PD-L1 expression at enrolment, archival tumour samples were assessed with a laboratory-developed prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, CA, USA), which included the 22C3 antibody (Merck & Co, Kenilworth, NJ, USAhttps://www.sciencedirect.com/science/article/pii/S1470204516001753QualTek
2019Loi, S;Giobbie-Hurder, A;Gombos, A;Bachelot, T;Hui, R;Curigliano, G;Campone, M;Biganzoli, L;Bonnefoi, H;Jerusalem, G;Bartsch, R;Rabaglio-Poretti, M;Kammler, R;Maibach, R;Smyth, M;Di Leo, A;Colleoni, M;Viale, G;Regan, M;André, F;Fumagalli, D;Gelber, R;Goulioti, T;Hiltbrunner, A;Hui, R;Roschitzki, H;Ruepp, B;Boyle, F;Stahel, R;Aebi, S;Coates, A;Goldhirsch, A;Karlsson, P;Kössler, I;Fournarakou, S;Gasca, A;Pfister, R;Ribeli-Hofmann, S;Weber, M;Celotto, D;Comune, C;Frapolli, M;Sánchez-Hohl, M;Huang, H;Mahoney, C;Price, K;Scott, K;Shaw, H;Fischer, S;Greco, M;King, C;Andrighetto, S;Piccart-Gebhart, M;Findlay, H;Jenkins, M;Karantza, V;Mejia, J;Schneier, P;Pembrolizumab plus trastuzumab in trastuzumab-resistant, advanced, HER2-positive breast cancer (PANACEA): a single-arm, multicentre, phase 1b–2 trialThe Lancet Oncology371-382203Background HER2-positive breast cancers usually contain large amounts of T-cell infiltrate. We hypothesised that trastuzumab resistance in HER2-positive breast cancer could be mediated by immune mechanisms. We assessed the safety and anti-tumour activity of pembrolizumab, a programmed cell death protein 1 (PD-1) inhibitor, added to trastuzumab in trastuzumab-resistant, advanced HER2-positive breast cancer. Methods We did this single-arm, multicentre, phase 1b–2 trial in 11 centres based in five countries. Eligible participants were women aged 18 years or older, who had advanced, histologically confirmed, HER2-positive breast cancer; documented progression during previous trastuzumab-based therapy; an Eastern Cooperative Oncology Group performance status of 0 or 1; and a formalin-fixed, paraffin-embedded metastatic tumour biopsy for central assessment of programmed cell death 1 ligand 1 (PD-L1) status. In phase 1b, we enrolled patients with PD-L1-positive tumours in a 3 + 3 dose-escalation of intravenous pembrolizumab (2 mg/kg and 10 mg/kg, every 3 weeks) plus 6 mg/kg of intravenous trastuzumab. The primary endpoint of the phase 1b study was the incidence of dose-limiting toxicity and recommended phase 2 dose; however, a protocol amendment on Aug 28, 2015, stipulated a flat dose of pembrolizumab of 200 mg every 3 weeks in all Merck-sponsored trials. In phase 2, patients with PD-L1-positive and PD-L1-negative tumours were enrolled in parallel cohorts and received the flat dose of pembrolizumab plus standard trastuzumab. The primary endpoint of the phase 2 study was the proportion of PD-L1-positive patients achieving an objective response. This trial is registered in ClinicalTrials.gov, number NCT02129556, and with EudraCT, number 2013-004770-10, and is closed. Findings Between Feb 2, 2015, and April 5, 2017, six patients were enrolled in phase 1b (n=3 received 2 mg/kg pembrolizumab, n=3 received 10 mg/kg pembrolizumab) and 52 patients in phase 2 (n=40 had PD-L1-positive tumours, n=12 had PD-L1-negative tumours). The data cutoff for this analysis was Aug 7, 2017. During phase 1b, there were no dose-limiting toxicities in the dose cohorts tested. Median follow-up for the phase 2 cohort was 13·6 months (IQR 11·6–18·4) for patients with PD-L1-positive tumours, and 12·2 months (7·9–12·2) for patients with PD-L1-negative tumours. Six (15%, 90% CI 7–29) of 40 PD-L1-positive patients achieved an objective response. There were no objective responders among the PD-L1-negative patients. The most common treatment-related adverse event of any grade was fatigue (12 [21%] of 58 patients). Grade 3–5 adverse events occurred in 29 (50%) of patients, treatment-related grade 3–5 adverse events occurred in 17 (29%), and serious adverse events occurred in 29 (50%) patients. The most commonly occurring serious adverse events were dyspnoea (n=3 [5%]), pneumonitis (n=3 [5%]), pericardial effusion (n=2 [3%]), and upper respiratory infection (n=2 [3%]). There was one treatment-related death due to Lambert-Eaton syndrome in a PD-L1-negative patient during phase 2. Interpretation Pembrolizumab plus trastuzumab was safe and showed activity and durable clinical benefit in patients with PD-L1-positive, trastuzumab-resistant, advanced, HER2-positive breast cancer. Further studies in this breast cancer subtype should focus on a PD-L1-positive population and be done in less heavily pretreated patients. Funding Merck, International Breast Cancer Study Group.354,0PD-L1 status for the first 79 patients was assessed with the QualTek immunohistochemistry assay (QualtTek Molecular Laboratories, Santa Barbara, CA, USA) in which PD-L1 staining in at least 1% of tumour cells or any staining in stroma were defined as positivehttps://www.sciencedirect.com/science/article/pii/S147020451830812XQualTek
2020Dudek, AZ;Liu, LC;Gupta, S;Logan, TF;Singer, EA;Joshi, M;Zakharia, YN;Lang, JM;Schwarz, JK;Al-Janadi, A;Alva, AS;Phase Ib/II Clinical Trial of Pembrolizumab With Bevacizumab for Metastatic Renal Cell Carcinoma: BTCRC-GU14-003J. Clin. Oncol.1138-1145381132097091We hypothesized that bevacizumab will potentiate activity of pembrolizumab. We conducted a phase Ib/II, single-arm, multisite clinical trial of the combination in metastatic renal cell carcinoma (RCC). Patients with metastatic clear cell RCC who experienced progression after at least one systemic therapy (phase Ib) or were treatment naïve (phase II) were enrolled. In phase Ib, pembrolizumab (200 mg) and bevacizumab (10 or 15 mg/kg) were given intravenously every 3 weeks. The primary end point for phase II was overall response rate (ORR). With an 80% statistical power and a type I error probability of 0.1, 48 patients were to be accrued to detect an ORR of 42%. Thirteen patients (ages 33-68 years; median, 55 years) were enrolled in the phase Ib study. No dose-limiting toxicities were reported. Pembrolizumab 200 mg and bevacizumab 15 mg/kg were chosen for phase II. Forty-eight patients (ages 42-84 years; median age, 61 years; 33 males) were accrued for the phase II study. The primary end point was met, with the ORR reaching 60.9% (95% CI, 45.4% to 74.9%), consisting of 1 complete response (CR), 2 CRs in target lesions, 25 partial responses, 18 responses of stable disease, 2 unevaluable responses. Median progression-free survival was 20.7 months (95% CI, 11.3 to 27.4 months). Median overall survival was not reached at the median follow-up of 28.3 months. The most common treatment-related grade 3 toxicities were hypertension and proteinuria. There were two grade 4 toxicities: duodenal ulcer and hyponatremia. Presence of tumor-infiltrating T cells, but not programmed death-ligand 1 expression, in tumor tissue correlated with response. The combination of 200 mg of pembrolizumab and a 15 mg/kg dose of bevacizumab given every 3 weeks is safe and active in metastatic RCC.282,0Events (CTCAE; version 4.0). Exploratory Studies. Expression of PD-L1 in archived diagnostic tumor tissue was determined by immunohistochemistry (IHC) using a 22C3 antibody (Qualtek Electronics, Newton, PA). Both a modified ;logy Criteria for Adverse Events (CTCAE; version 4.0). Exploratory Studies Expression of PD-L1 in archived diagnostic tumor tissue was determined by immunohistochemistry (IHC) using a 22C3 antibody (Qualtek Electronics, Newton, PA). Both a modified percent score (percentage of tumor and any tumor-infiltrating mononuclear inflammatory cells that had membrane staining at 1+ intensities or greater)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145584/QualTek
2019Nghiem, P;Bhatia, S;Lipson, EJ;Sharfman, WH;Kudchadkar, RR;Brohl, AS;Friedlander, PA;Daud, A;Kluger, HM;Reddy, SA;Boulmay, BC;Riker, AI;Burgess, MA;Hanks, BA;Olencki, T;Margolin, K;Lundgren, LM;Soni, A;Ramchurren, N;Church, C;Park, SY;Shinohara, MM;Salim, B;Taube, JM;Bird, SR;Ibrahim, N;Fling, SP;Homet Moreno, B;Sharon, E;Cheever, MA;Topalian, SL;Durable Tumor Regression and Overall Survival in Patients With Advanced Merkel Cell Carcinoma Receiving Pembrolizumab as First-Line TherapyJ. Clin. Oncol.693-70237930726175Merkel cell carcinoma (MCC) is an aggressive skin cancer often caused by the Merkel cell polyomavirus. Clinical trials of programmed cell death-1 pathway inhibitors for advanced MCC (aMCC) demonstrate increased progression-free survival (PFS) compared with historical chemotherapy data. However, response durability and overall survival (OS) data are limited. In this multicenter phase II trial (Cancer Immunotherapy Trials Network-09/Keynote-017), 50 adults naïve to systemic therapy for aMCC received pembrolizumab (2 mg/kg every 3 weeks) for up to 2 years. Radiographic responses were assessed centrally per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Among 50 patients, the median age was 70.5 years, and 64% had Merkel cell polyomavirus-positive tumors. The objective response rate (ORR) to pembrolizumab was 56% (complete response [24%] plus partial response [32%]; 95% CI, 41.3% to 70.0%), with ORRs of 59% in virus-positive and 53% in virus-negative tumors. Median follow-up time was 14.9 months (range, 0.4 to 36.4+ months). Among 28 responders, median response duration was not reached (range, 5.9 to 34.5+ months). The 24-month PFS rate was 48.3%, and median PFS time was 16.8 months (95% CI, 4.6 months to not estimable). The 24-month OS rate was 68.7%, and median OS time was not reached. Although tumor viral status did not correlate with ORR, PFS, or OS, there was a trend toward improved PFS and OS in patients with programmed death ligand-1-positive tumors. Grade 3 or greater treatment-related adverse events occurred in 14 (28%) of 50 patients and led to treatment discontinuation in seven (14%) of 50 patients, including one treatment-related death. Here, we present the longest observation to date of patients with aMCC receiving first-line anti-programmed cell death-1 therapy. Pembrolizumab demonstrated durable tumor control, a generally manageable safety profile, and favorable OS compared with historical data from patients treated with first-line chemotherapy.282,021,22. PD-L1 IHC. PD-L1 staining (anti-PD-L1 clone 22C3; Merck Research Laboratories, Kenilworth, NJ) was performed at QualTek Molecular Laboratories (Newtown, PA) on formalin-fixed paraffin-embedded sections from pretreatment MCC biopsies, as previouslyhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424137QualTek
2018Doi, T;Piha-Paul, SA;Jalal, SI;Saraf, S;Lunceford, J;Koshiji, M;Bennouna, J;Safety and Antitumor Activity of the Anti-Programmed Death-1 Antibody Pembrolizumab in Patients With Advanced Esophageal CarcinomaJ. Clin. Oncol.61-6736129116900Purpose The anti-programmed death-1 antibody pembrolizumab was evaluated in KEYNOTE-028, a multicohort, phase IB study of patients with programmed death ligand-1 (PD-L1)-positive advanced solid tumors. Results from the esophageal carcinoma cohort are reported herein. Patients and Methods Eligible patients with squamous cell carcinoma or adenocarcinoma of the esophagus or gastroesophageal junction in whom standard therapy failed and who had PD-L1-positive tumors received pembrolizumab 10 mg/kg every 2 weeks for up to 2 years or until confirmed disease progression or intolerable toxicity. Response was assessed every 8 weeks up to 6 months and every 12 weeks thereafter. Primary end points were safety and overall response rate, determined by investigator review per Response Evaluation Criteria in Solid Tumors (version 1.1). Results Among 83 patients with esophageal carcinoma and samples evaluable for PD-L1 expression, 37 (45%) had PD-L1-positive tumors, and 23 were enrolled. Median age was 65 years; 78% had squamous histology; and 87% received ≥ two prior therapies for advanced/metastatic disease. As of the data cutoff (February 20, 2017), median follow-up was 7 months (range, 1 to 33 months). Nine patients (39%) experienced treatment-related adverse events, most commonly decreased appetite, decreased lymphocyte count, generalized rash, and rash (two patients [9%] each). No grade 4 adverse events or deaths were attributed to pembrolizumab. Overall response rate was 30% (95% CI, 13% to 53%); median duration of response was 15 months (range, 6 to 26 months). A six-gene interferon-γ gene expression signature analysis suggested that delayed progression and increased response occur among pembrolizumab-treated patients with higher interferon-γ composite scores. Conclusion Pembrolizumab demonstrated manageable toxicity and durable antitumor activity in patients with heavily pretreated, PD-L1-positive advanced esophageal carcinoma.282,0PD-L1 Expression Analysis PD-L1 expression was assessed at screening by a central laboratory on the basis of a laboratory-developed prototype immunohistochemical assay (QualTek Molecular Laboratories, Goleta, CA) using either an archived (formalin-fixed, paraffinhttps://scholarworks.iupui.edu/handle/1805/17316QualTek
2017Frenel, JS;Le Tourneau, C;O'Neil, B;Ott, PA;Piha-Paul, SA;Gomez-Roca, C;van Brummelen, EMJ;Rugo, HS;Thomas, S;Saraf, S;Rangwala, R;Varga, A;Safety and Efficacy of Pembrolizumab in Advanced, Programmed Death Ligand 1-Positive Cervical Cancer: Results From the Phase Ib KEYNOTE-028 TrialJ. Clin. Oncol.4035-4041353629095678Purpose The KEYNOTE-028 trial ( ClinicalTrials.gov identifier: NCT02054806) was designed to assess the safety and efficacy of pembrolizumab in 20 programmed death ligand 1-positive, advanced solid tumor cohorts. Here, we present the results from the cohort of patients with advanced cervical cancer. Methods Patients were treated with pembrolizumab 10 mg/kg every 2 weeks for up to 24 months. Response was assessed every 8 weeks for the first 6 months and every 12 weeks thereafter. The primary end point was overall response rate per Response Evaluation Criteria in Solid Tumors, version 1.1, by investigator review. Safety was a secondary end point. Results Twenty-four patients were enrolled in the cervical cancer cohort. The median age was 42 years (range, 26 to 62 years), 22 patients (92%) had received prior radiation therapy, and 15 patients (63%) had received two or more lines of therapy, including bevacizumab (10 of 24 patients), for advanced disease. At the data cutoff, median follow-up duration was 11.0 months (range, 1.3 to 32.2 months). Overall response rate was 17% (95% CI, 5% to 37%); four patients (17%) achieved a confirmed partial response, and three patients (13%) had stable disease. Median duration of response for the four patients who achieved a partial response was 5.4 months (4.1 to 7.5 months). Treatment related adverse events (AEs) were experienced by 18 patients (75%); only rash (n = 5; 21%) and pyrexia (n = 4; 17%) and occurred in ≥ 10% of patients. Five patients experienced grade 3 treatment-related AEs. No grade 4 treatment-related AEs or deaths were observed. Conclusion In patients with programmed death ligand 1-positive advanced cervical cancer, pembrolizumab demonstrated antitumor activity and exhibited a safety profile consistent with that seen in other tumor types.282,0tumor sample or newly obtained core or excisional biopsy sample, was defined as membranous staining on $ 1% modified proportion score or interface pattern as assessed using a laboratory-developed prototype immunohistochemistry assay (QualTek Molecular Laboratorieshttps://pdfs.semanticscholar.org/c58f/ea9db94d55be500c8f684518e18a643edcba.pdfQualTek
2017Hsu, C;Lee, SH;Ejadi, S;Even, C;Cohen, RB;Le Tourneau, C;Mehnert, JM;Algazi, A;van Brummelen, EMJ;Saraf, S;Thanigaimani, P;Cheng, JD;Hansen, AR;Safety and Antitumor Activity of Pembrolizumab in Patients With Programmed Death-Ligand 1-Positive Nasopharyngeal Carcinoma: Results of the KEYNOTE-028 StudyJ. Clin. Oncol.4050-4056353628837405Purpose To establish the safety profile and antitumor activity of the anti-programmed death 1 receptor monoclonal antibody, pembrolizumab, in patients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) that expressed programmed death-ligand 1 (PD-L1). Patients and Methods KEYNOTE-028 (NCT02054806) is a nonrandomized, multicohort, phase Ib trial of pembrolizumab in patients with PD-L1-positive advanced solid tumors. Key eligibility criteria for the NPC cohort included unresectable or metastatic disease, failure on prior standard therapy, and PD-L1 expression in 1% or more of tumor cells or tumor-infiltrating lymphocytes. Patients received pembrolizumab 10 mg/kg every 2 weeks up to 2 years or until disease progression or unacceptable toxicity. Primary end point was objective response rate (ORR) per investigator review. Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) every 8 weeks for the first 6 months and every 12 weeks thereafter. Results Twenty-seven patients received pembrolizumab. Median age was 52.0 years (range, 18 to 68 years); 92.6% received prior therapies for RM-NPC; 70.4% had received three or more therapies. Partial response and stable disease were observed in seven and 14 patients, respectively, for an ORR of 25.9% (95% CI, 11.1 to 46.3) over a median follow-up of 20 months. ORR by central review was similar (26.3%). Drug-related adverse events that occurred in 15% or more of patients included rash (25.9%), pruritus (25.9%), pain (22.2%), hypothyroidism (18.5%), and fatigue (18.5%). Grade ≥ 3 drug-related adverse events occurred in eight patients (29.6%), and there was one drug-related death (sepsis). As of the data cutoff (June 20, 2016), two patients remained on pembrolizumab treatment. Conclusion Pembrolizumab demonstrated antitumor activity and a manageable safety profile in patients with RM-NPC.282,0Tumor PD-L1 positivity was established at a central laboratory at baseline on an archived formalin-fixed, paraffin-embedded tumor sample or a newly obtained biopsy sample using a laboratory-developed prototype immunohistochemical assay (QualTek Molecular Laboratorieshttp://dx.doi.org/10.1200/JCO.2017.73.3675QualTek
2017Ott, PA;Elez, E;Hiret, S;Kim, DW;Morosky, A;Saraf, S;Piperdi, B;Mehnert, JM;Pembrolizumab in Patients With Extensive-Stage Small-Cell Lung Cancer: Results From the Phase Ib KEYNOTE-028 StudyJ. Clin. Oncol.3823-3829353428813164Purpose The safety and efficacy of pembrolizumab, a humanized monoclonal antibody against programmed death 1 (PD-1), were assessed in patients with programmed death ligand 1 (PD-L1)-expressing extensive-stage small-cell lung cancer (SCLC) in the multicohort, phase Ib open-label KEYNOTE-028 study ( ClinicalTrials.gov identifier: NCT02054806). Methods Patients with SCLC received pembrolizumab 10 mg/kg every 2 weeks for 24 months or until disease progression or intolerable toxicity occurred. PD-L1 expression was assessed by immunohistochemistry. PD-L1-positive patients had membranous PD-L1 expression in ≥ 1% of tumor and associated inflammatory cells or positive staining in stroma. Response was assessed by investigator per Response Evaluation Criteria in Solid Tumors version 1.1 every 8 weeks for the first 6 months and every 12 weeks thereafter. Adverse events (AEs) were reported per the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. Primary end points were safety, tolerability, and objective response rate (ORR). Secondary end points included progression-free survival, overall survival, and duration of response. Results Twenty-four patients with PD-L1-expressing SCLC were enrolled and received at least one pembrolizumab dose. At the data cutoff date (June 20, 2016), the median follow-up duration was 9.8 months (range, 0.5 to 24 months). All 24 patients experienced AEs; the most common were asthenia (n = 7), fatigue (n = 7), and cough (n = 6). Two patients experienced grade 3 to 5 treatment-related AEs: one patient had elevated bilirubin, and one patient had asthenia, grade 5 colitis, and intestinal ischemia. One patient had a complete response, and seven patients had partial responses, resulting in an ORR of 33% (95% CI, 16% to 55%). Conclusion The safety of pembrolizumab was consistent with the known safety profile in other tumor types. Pembrolizumab demonstrated promising antitumor activity in patients with pretreated, PD-L1-expressing SCLC.282,0Tumor PD-L1 expression was required to be eligible for treatment in the study; it was evaluated in archival or fresh tumor samples by a central laboratory (QualTek Molecular Laboratories, Goleta, CA) by using a prototype assay and the 22C3 antibody (Merck & Co, Kenilworthhttps://pdfs.semanticscholar.org/cd2e/31a68e3041ba0d4285a211b67f575d073915.pdfQualTek
2017Chen, R;Zinzani, PL;Fanale, MA;Armand, P;Johnson, NA;Brice, P;Radford, J;Ribrag, V;Molin, D;Vassilakopoulos, TP;Tomita, A;von Tresckow, B;Shipp, MA;Zhang, Y;Ricart, AD;Balakumaran, A;Moskowitz, CH;, ;Phase II Study of the Efficacy and Safety of Pembrolizumab for Relapsed/Refractory Classic Hodgkin LymphomaJ. Clin. Oncol.2125-2132351928441111Purpose Hodgkin Reed-Sternberg cells harbor alterations in chromosome 9p24.1, leading to overexpression of programmed death-ligand 1 (PD-L1) and PD-L2. Pembrolizumab, a programmed death 1-blocking antibody, demonstrated a high overall response rate (ORR) in patients with relapsed or refractory classic Hodgkin lymphoma (rrHL) in phase I testing. Methods KEYNOTE-087 ( ClinicalTrials.gov identifier, NCT02453594) was a single-arm phase II study of pembrolizumab in three cohorts of patients with rrHL, defined on the basis of lymphoma progression after (1) autologous stem cell transplantation (ASCT) and subsequent brentuximab vedotin (BV); (2) salvage chemotherapy and BV, and thus, ineligible for ASCT because of chemoresistant disease; and (3) ASCT, but without BV after transplantation. Patients received pembrolizumab 200 mg once every 3 weeks. Response was assessed every 12 weeks. The primary end points were ORR by central review and safety. Results A total of 210 patients were enrolled and treated (69 in cohort 1, 81 in cohort 2, and 60 in cohort 3). At the time of analysis, patients received a median of 13 treatment cycles. Per central review, the ORR was 69.0% (95% CI, 62.3% to 75.2%), and the complete response rate was 22.4% (95% CI, 16.9% to 28.6%). By cohort, ORRs were 73.9% for cohort 1, 64.2% for cohort 2, and 70.0% for cohort 3. Thirty-one patients had a response ≥ 6 months. The safety profile was largely consistent with previous pembrolizumab studies. Conclusion Pembrolizumab was associated with high response rates and an acceptable safety profile in patients with rrHL, offering a new treatment paradigm for this disease.282,0PD-L1 expression was determined as previously described, 16 using fresh or archival formalin-fixed, paraffin-embedded pretreatment tissue sectioned at 4 to 5 microns, with a proprietary immunohistochemical assay developed at QualTek Molecular Laboratories (Newtownhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791843/QualTek
2016Armand, P;Shipp, MA;Ribrag, V;Michot, JM;Zinzani, PL;Kuruvilla, J;Snyder, ES;Ricart, AD;Balakumaran, A;Rose, S;Moskowitz, CH;Programmed Death-1 Blockade With Pembrolizumab in Patients With Classical Hodgkin Lymphoma After Brentuximab Vedotin FailureJ. Clin. Oncol.3733-3739343127354476Purpose Classical Hodgkin lymphoma (HL) frequently exhibits genetic alterations leading to overexpression of the programmed death-1 (PD-1) ligands, suggesting a possible vulnerability to PD-1 blockade. The phase Ib study KEYNOTE-013 (NCT01953692) tested the safety and efficacy of the anti-PD-1 antibody pembrolizumab in patients with hematologic malignancies. Based on its genetics, HL was included as an independent cohort. Methods We enrolled patients with relapsed or refractory HL whose disease progressed on or after treatment with brentuximab vedotin. Patients received pembrolizumab, 10 mg/kg every 2 weeks, until disease progression occurred. Response to treatment was assessed at week 12 and every 8 weeks thereafter. Principal end points were safety and complete remission (CR) rate. Results Thirty-one patients were enrolled; 55% had more than four lines of prior therapy, and 71% had relapsed after autologous stem cell transplantation. Five patients (16%) experienced grade 3 drug-related adverse events (AEs); there were no grade 4 AEs or deaths related to treatment. The CR rate was 16% (90% CI, 7% to 31%). In addition, 48% of patients achieved a partial remission, for an overall response rate of 65% (90% CI, 48% to 79%). Most of the responses (70%) lasted longer than 24 weeks (range, 0.14+ to 74+ weeks), with a median follow-up of 17 months. The progression-free survival rate was 69% at 24 weeks and 46% at 52 weeks. Biomarker analyses demonstrated a high prevalence of PD-L1 and PD-L2 expression, treatment-induced expansion of T cells and natural killer cells, and activation of interferon-γ, T-cell receptor, and expanded immune-related signaling pathways. Conclusions Pembrolizumab was associated with a favorable safety profile. Pembrolizumab treatment induced favorable responses in a heavily pretreated patient cohort, justifying further studies.282,0PD-L1 expression was determined using either fresh or archival formalin-fixed, paraffin-embedded tissue sectioned at 4 to 5 microns, with a proprietary assay developed at QualTek Molecular Laboratories (Newtown, PA) in collaboration with Merck (Kenilworth, NJ)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791838/QualTek
2016Nanda, R;Chow, LQ;Dees, EC;Berger, R;Gupta, S;Geva, R;Pusztai, L;Pathiraja, K;Aktan, G;Cheng, JD;Karantza, V;Buisseret, L;Pembrolizumab in Patients With Advanced Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 StudyJ. Clin. Oncol.2460-2467342127138582Immune checkpoint inhibition has been demonstrated to be an effective anticancer strategy. Several lines of evidence support the study of immunotherapy in triple-negative breast cancer (TNBC). We assessed the safety and antitumor activity of the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab in patients with advanced TNBC. KEYNOTE-012 (ClinicalTrials.gov identifier: NCT01848834) was a multicenter, nonrandomized phase Ib trial of single-agent pembrolizumab given intravenously at 10 mg/kg every 2 weeks to patients with advanced PD-L1-positive (expression in stroma or ≥ 1% of tumor cells by immunohistochemistry) TNBC, gastric cancer, urothelial cancer, and head and neck cancer. This report focuses on the TNBC cohort. Among 111 patients with TNBC whose tumor samples were screened for PD-L1 expression, 58.6% had PD-L1-positive tumors. Thirty-two women (median age, 50.5 years; range, 29 to 72 years) were enrolled and assessed for safety and antitumor activity. The median number of doses administered was five (range, 1 to 36 doses). Common toxicities were mild and similar to those observed in other tumor cohorts (eg, arthralgia, fatigue, myalgia, and nausea), and included five (15.6%) patients with grade ≥ 3 toxicity and one treatment-related death. Among the 27 patients who were evaluable for antitumor activity, the overall response rate was 18.5%, the median time to response was 17.9 weeks (range, 7.3 to 32.4 weeks), and the median duration of response was not yet reached (range, 15.0 to ≥ 47.3 weeks). This phase Ib study describes preliminary evidence of clinical activity and a potentially acceptable safety profile of pembrolizumab given every 2 weeks to patients with heavily pretreated, advanced TNBC. A single-agent phase II study examining a 200-mg dose given once every 3 weeks (ClinicalTrials.gov identifier: NCT02447003) is ongoing. © 2016 by American Society of Clinical Oncology.282,0Filhart and Kenneth Emancipator (Merck & Co., Kenilworth, NJ) for immunohistochemistry expertise and study support, Roger Dansey (Merck & Co.) for critical manuscript review and study support, Karl Heath (Merck & Co.) for study support, and QualTek Molecular Laboratorieshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816000/QualTek
2018Kanjanapan, Y;Lien, S;Yang, C;Clouthier, D;Elston, S;Jiang, H;Butler, M;Hogg, D;Ohashi, P;Pugh, T;Siu, L;Spreafico, A;Genomic and immune landscape of metastatic melanoma (MM) treated with pembrolizumab (PEM).JCO184-184365_suppl184 Background: Predictive biomarkers for PEM in MM is an active area of research. Methods: INSPIRE (NCT02644369) is a biomarker-driven Princess Margaret Cancer Centre initiative to evaluate genomic and immunologic changes in tumor and blood of patient (pts) treated with PEM at 200 mg IV Q3W. Baseline (BL) and on treatment (post 2-3 doses of PEM) fresh tumor biopsies were collected for DNA/RNA sequencing, immune-profiling, and tumor PD-L1 expression by immunohistochemistry (QualTek), with longitudinal blood collection for immunophenotyping. Results: As of 9/2017, 11 MM pts (9 cutaneous, 1 choroidal, 1 mucosal) were enrolled. Median treatment duration was 6 months (0.75 – 14.25); 7 pts remain on study. 7/11 (64%) pts had partial response (PR), 3/11 (27%) progressive disease (PD) and 1 was not evaluable (NE) per RECIST 1.1. Based on data for 8 pts (6 PR, 2 PD), higher percentage (%) PD-1 (mean 70 vs 12%), TIGIT (82 vs 43%) and 4-1BB (23 vs 4%) positivity on CD8 T cells in tumor vs blood was seen (all p < 0.05). In BL blood, lower % TIGIT positive CD8 T cells was detected in pts with PR vs PD (38 vs 58%, p < 0.05). T cell profile of 4 pts with paired BL and post PEM samples is shown in Table 1. Mean tumor PD-L1 score was 24% (0 - 90%), with no correlation to PEM response. Genomic analysis on 8 pts (5 PR, 2 PD, 1 NE) found mean tumor mutation burden (TMB) of 538. B2M deletion was seen in 2/2 PD and 0/6 PR pts. Among pts with PRs, 4/6 (67%) had TAP1 and TAP2 amplification post PEM therapy. HLA-A and HLA-B amplifications were each found in 3/6 (50%) PR pts, with only 1 present at BL. TMB was not associated with response to PEM. Conclusions: In this exploratory analysis of MM pts treated with PEM, there was significantly discordant T cell phenotype in tumor vs blood at BL. Our findings suggest B2M deletion to be associated with PEM resistance. The observed development of HLA-A/B and TAP1/2 amplification in 50-67% pts with PR post PEM suggests potential predictive significance, although further validation is warranted. Clinical trial information: NCT02644369. [Table: see text]282,0Baseline (BL) and on treatment (post 2-3 doses of PEM) fresh tumor biopsies were collected for DNA/RNA sequencing, immune-profiling, and tumor PD-L1 expression by immunohistochemistry (QualTek), with longitudinal blood collection for immunophenotypinghttps://ascopubs.org/doi/abs/10.1200/JCO.2018.36.5_suppl.184QualTek
2018Graff, J;Alumkal, J;Thompson, R;Moran, A;Thomas, G;Wood, M;Drake, C;Slottke, R;Beer, T;Pembrolizumab (Pembro) plus enzalutamide (Enz) in metastatic castration resistant prostate cancer (mCRPC): Extended follow up.JCO5047-50473615_supplBackground: Anti-PD1 treatment with Pembro is a promising treatment strategy in many solid tumors. Here, we report clinical outcomes of adding Pembro to men with mCRPC progressing on Enz. Methods: We enrolled 28 patients with mCRPC who had not previously had chemotherapy for mCRPC or checkpoint inhibitors. Pembro was given 200 mg IV every 3 weeks for 4 doses, which could be repeated for the patients who had stable or responsive disease. The primary endpoint was prostate specific antigen (PSA) response (PSA decline of ≥ 50%). Secondary endpoints were radiographic objective response (RECIST 1.1), PSA progression free survival, time to subsequent treatment, and time to death from any cause. Baseline tumor biopsies were done if there was a metastatic deposit amenable to biopsy. We are presenting updated clinical outcomes and evaluation of samples for presence of Program Death-Ligand 1 (PD-L1), microsatellite instability (MSI) and DNA repair defects. Results: Five of 28 patients (18%) had a PSA decline of ≥ 50%. Three of 12 patients (25%) with measurable disease at baseline achieved an objective response on radiographs. Of the responders (R), one passed away from an unrelated cause without PSA recurrence after 14.2 mos, and one relapsed after 10 mos and did not respond to a second course of Pembro. The other 3 continue to be in response (range 21.9-33.8 mos). For the entire cohort, the median follow up was 22.7 mos, and the median PSA-PFS time was 3.8 mos (95% CI: 2.8 – 9.9 mos). Time to subsequent treatment was 8.2 mos (95% CI: 5.1 – 12.8 mos). Median overall survival was 22.2 mos (95% CI: 14.7 – 28 .4 mos). Median radiographic PFS was 10.8 mos (5-22 mos). There were 8 immune related adverse events in 7 unique patients (hypothyroid 3, hyperthyroid 1, myositis 2, colitis 2). Seventeen patients had baseline biopsies of a metastatic deposit: 3 R, 14 non-responders (NR). Of the 3 R who had baseline biopsies, one had MSI and DNA repair defects. The other 2 had neither. Of the NR who had sequencing, 4 had a DNA repair defects, and none had MSI. None of the biopsies showed tumoral PD-L1 expression (Qualtek, 22C3). Conclusions: Pembro has activity in mCRPC when added to Enz. Responses were deep and durable in a subset of patients282,0The other 2 had neither. Of the NR who had sequencing, 4 had a DNA repair defects, and none had MSI. None of the biopsies showed tumoral PD-L1 expression (Qualtek, 22C3). Conclusions: Pembro has activity in mCRPC when added to Enzhttps://ascopubs.org/doi/abs/10.1200/JCO.2018.36.15_suppl.5047QualTek
2018Kelly, C;Bowler, T;Munhoz, R;Chi, P;Dickson, M;Gounder, M;Keohan, M;Dholakia, R;Condy, M;Singer, S;Crago, A;Yoon, S;Ariyan, C;Hwang, S;Erinjeri, J;Antonescu, C;Qin, L;Tap, W;D'Angelo, S;A phase II study of talimogene laherparepvec (T-VEC) and pembrolizumab in patients with metastatic sarcoma.JCO11516-115163615_supplBackground: T-VEC is an oncolytic immunotherapy derived from HSV type-1 that is modified to selectively replicate within tumors and produce GM-CSF. Pembrolizumab (P) demonstrated activity in selective sarcoma (SAR) subtypes. The combination of T-VEC and P has shown favorable safety and efficacy in melanoma. We performed an open-label, single-center, phase II study evaluating T-VEC and P in patients (pts) with advanced SAR who failed at least one standard systemic therapy where available. Methods: Pts received P (200mg/dose) and ≤4ml of T-VEC injected into palpable tumor site(s). Both drugs were administered on day 1 of a 21-day cycle. The primary endpoint was best objective response rate (RR) (complete response and partial response [PR]) at 24 weeks by RECIST 1.1, with 30% as promising and 5% as not promising. If ≥ 3 responses were observed in 20 pts the combination would be claimed to be positive. Secondary endpoints included adverse events (AEs), RR by irRECIST, progression free and overall survival. Correlative studies included PD-L1 expression by IHC (Qualtek), multiplex IHC, characterization of tumor infiltrating lymphocytes, mutational burden/neoantigen analysis, and T cell receptor clonality. Results: 20 pts were enrolled [median age 63.5 yrs (range, 24-90), 60% female]. Represented histological subtypes included: leiomyosarcoma (25%), cutaneous angiosarcoma (15%), SAR not otherwise specified (15%), undifferentiated pleomorphic SAR (10%) and “other” SAR subtype (35%). Pts were refractory to 0 (10%), 1 (20%), and ≥ 2 (70%) prior regimens. Grade (G) 3 treatment related AEs (TRAEs) occurred in 2 pts (10%); fever from TVEC & pneumonitis from P. No G4/5 TRAEs were seen. 1pt stopped P due to G3 TRAE. Among 19 evaluable pts 4 confirmed PRs (21%), 9 stable diseases (SD) (47%) and 6 progressions (32%) were observed by RECIST 1.1. Two of the SDs had decrease in tumor burden of 17% and 28.6% at their first (week8) interval scan. PRs were seen in 3 histologies. The time to PR ranged from 8 to 32 weeks (wks) and all PRs are ongoing (range 1 -36 wks). Conclusions: TVEC and P demonstrated acceptable safety and promising anti-tumor activity across a range of SAR histologies. 4 pts maintain a PR. Correlative analyses are ongoing282,0overall survival. Correlative studies included PD-L1 expression by IHC (Qualtek), multiplex IHC, characterization of tumor infiltrating lymphocytes, mutational burden/neoantigen analysis, and T cell receptor clonality. Resultshttps://ascopubs.org/doi/abs/10.1200/JCO.2018.36.15_suppl.11516QualTek
2017Colombo, I;Lien, S;Yang, C;Clouthier, D;Bonilla, L;Cyriac, S;Ethier, J;Lee, Y;Kanjanapan, Y;Mandilaras, V;Dhani, N;Butler, M;Oza, A;Quintos, J;Chow, H;Pugh, T;Ohashi, P;Siu, L;Lheureux, S;Immunologic and genomic characterization of high grade serous ovarian cancer (HGSOC) in patients (pts) treated with pembrolizumab (Pembro) in the phase II INSPIRE trial.JCO5581-55813515_suppl5581 Background: Checkpoint inhibitors have shown to be effective in different tumors and are under investigation in HGSOC. Methods: INSPIRE (NCT02644369) is a prospective multi-cohort study investigating tumor genomic and immune landscapes in pts treated with Pembro at 200 mg IV Q3W. Patients underwent tumor biopsy pre, on-treatment and at progression for DNA/RNA sequence, immune-profile, and PD-L1 expression by immunohistochemistry (IHC). Serial blood samples for immunophenotyping were collected. Correlative data are available for 6 pts: 3 with shrinkage in target lesion and 3 with progressive disease (PD). Results: At interim analysis as of January 2017, 18 pts with HGSOC have been enrolled and 16 have platinum-resistant disease, with median 3 prior lines of treatment (range 1-7). Of 14 evaluable pts, best response by RECIST 1.1 was stable disease (SD) in 5 (36%) and PD in 9 (64%). Mean Tumor Proportion Score of PD-L1 by IHC (Qualtek) was 6.4% (range 0-30%). Grade 3/4 adverse events possibly related to Pembro were observed in 4/18 (22%) pts; none was fatal and the most common were fatigue and hyponatremia. Preliminary correlative data showed no significant change in CD4, CD8 and myeloid-derived suppressor cells in peripheral blood after Pembro treatment. Mean PD-1 expression on CD4 and CD8 T cells on baseline tumor tissue (measured as product of PD-1+ cells and the per cell expression of PD-1 [% of mean fluorescence intensity]) was significantly higher in pts with tumor shrinkage compared to pts with PD (CD4: 2658 vs 678, p = .02; CD8: 1999 vs 451, p = .048). Genomic analysis of baseline tumor tissue was available for 3 pts with tumor shrinkage and 2 with PD. Mean mutation burden was higher for pts with tumor shrinkage (2.38 vs 1.0 mutations/Mb covered). The pt with the longest SD in our cohort (6 months) had the highest mutation burden (2.72), including somatic POLE (c.6331-6C > G) and germline BRCA2mutations. Conclusions: In HGSOC, pts with higher PD-1 level on tumor CD4 and CD8 T cells and higher mutation burden at baseline may have a better outcome following treatment with Pembro. POLE mutation is rare in HGSOC but may correlate with checkpoint inhibitor activity. Clinical trial information: NCT02644369.282,0Mean Tumor Proportion Score of PD-L1 by IHC (Qualtek) was 6.4% (range 0-30%). Grade 3/4 adverse events possibly related to Pembro were observed in 4/18 (22%) pts; none was fatal and the most common were fatigue and hyponatremiahttps://ascopubs.org/doi/abs/10.1200/JCO.2017.35.15_suppl.5581QualTek
2018D'Angelo, SP;Melchiori, L;Merchant, MS;Bernstein, D;Glod, J;Kaplan, R;Grupp, S;Tap, WD;Chagin, K;Binder, GK;Basu, S;Lowther, DE;Wang, R;Bath, N;Tipping, A;Betts, G;Ramachandran, I;Navenot, JM;Zhang, H;Wells, DK;Van Winkle, E;Kari, G;Trivedi, T;Holdich, T;Pandite, L;Amado, R;Mackall, CL;Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 c259T Cells in Synovial SarcomaCancer Discov944-9578829891538We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR.See related commentary by Keung and Tawbi, p. 914This article is highlighted in the In This Issue feature, p. 899. ©2018 American Association for Cancer Research.264,0Serial 5-μm sections of formalin-fixed, paraffin-embedded tissues were analyzed for the following single markers by IHC: NY-ESO-1, Pan-CK (Clone AE1/AE3/PCK26, Ventana), CD45 (Clone 2B11 + PD7/26, Ventana), CD3 (Clone 2GV6, Ventana), CD8 (Clone C8/144B, Dako), CD4 (Clone 1F6, Novocastra), PD-L1 (Clone SP142, Ventana), and PD-1 (Clone SP269, Spring Bioscience). Staining was performed either at a CLIA-certified clinical laboratory (QualTek Labs) or a CLIA-certified and Belgian Accreditation Organization and College of American Pathologists–accredited laboratory (HistoGeneX). For some samples, NY-ESO-1 IHC testing was performed at the NCI (Bethesda, MD)https://cancerdiscovery.aacrjournals.org/content/8/8/944.shortQualTek
2019Leighl, NB;Hellmann, MD;Hui, R;Carcereny, E;Felip, E;Ahn, MJ;Eder, JP;Balmanoukian, AS;Aggarwal, C;Horn, L;Patnaik, A;Gubens, M;Ramalingam, SS;Lubiniecki, GM;Zhang, J;Piperdi, B;Garon, EB;Pembrolizumab in patients with advanced non-small-cell lung cancer (KEYNOTE-001): 3-year results from an open-label, phase 1 studyLancet Respir Med347-3577430876831The anti-programmed death 1 monoclonal antibody pembrolizumab has shown antitumour activity and is a first-line and second-line treatment option for patients with programmed death ligand 1 (PD-L1)-expressing advanced non-small-cell lung cancer. We report updated 3-year safety and efficacy outcomes from the phase 1 study, KEYNOTE-001. KEYNOTE-001 is a multicohort, open-label, phase 1 study of pembrolizumab (2 mg/kg every 3 weeks or 10 mg/kg every 2 or 3 weeks) in treatment naive or previously treated patients with locally advanced or metastatic non-small-cell lung cancer with measurable disease at baseline. Two cohorts were randomly assigned to a pembrolizumab dose by use of a computer-generated randomisation schedule at cohort-dependent ratios, and a further four cohorts were assigned to a pembrolizumab dose without randomisation. We present 3-year outcomes for the full analysis set of patients who received at least one dose of study treatment, pooled for all pembrolizumab doses. The primary efficacy endpoint was proportion of patients with objective response, analysed here as investigator-assessed response according to immune-related response criteria. Secondary efficacy endpoints included overall survival, duration of response, and progression-free survival. Safety endpoints included incidence of adverse events. This study is registered at ClinicalTrials.gov, number NCT01295827, and is ongoing. Between May 8, 2012 and July 13, 2014, 550 patients (101 treatment naive and 449 previously treated) were enrolled. Median follow-up was 34·5 months at data cutoff (Sept 1, 2016). At 36 months, investigator-assessed objective response according to immune-related response criteria was achieved for 41 of 101 treatment naive patients (41% [95% CI 30·9-50·8]; median duration of response was 16·7 months [95% CI 12·6-not reached]) and 102 of 449 previously treated patients (23% [18·9-26·9]; 33·3 ([22·5-not reached]). The Kaplan-Meier estimate of overall survival at 36 months was 26·4% (95% CI 14·3-40·1) for treatment naive patients and 19·0% (15·0-23·4) for previously treated patients, with median overall survival of 22·3 months (95% CI 17·1-31·5) and 10·5 months (8·6-13·2). PD-L1 tumour proportion score ≥50% was associated with longer median overall survival (95% CI) versus tumour proportion score 1-49% (treatment naive: 34·9 [20·3-not reached] vs 19·5 [10·7-26·3] months; previously treated: 15·4 [10·5-18·5] vs 8·5 [6·0-12·7] months). Grade 3-5 treatment-related adverse events occurred in 66 patients (12%), and 30 (6%) discontinued owing to a treatment-related adverse event. The most frequent grade 3-4 treatment-related adverse events were pneumonitis (10 [2%] of 550) and fatigue (5 [1%] of 550). Overall, 227 patients (41%) of 550 had serious adverse events, of which 50 (9%) were treatment related. Pembrolizumab provides durable response and long-term effects on overall survival, with tolerable safety, for treatment naive and previously treated patients with advanced non-small-cell lung cancer expressing PD-L1. Merck Sharp & Dohme Corp. Copyright © 2019 Elsevier Ltd. All rights reserved.230,0NR=not reached. PD-L1=programmed death ligand 1. TPS=tumour proportion score. *PD-L1 TPS 1% on the basis of the QualTek assay (Molecular Laboratories, Goletahttps://www.sciencedirect.com/science/article/pii/S2213260018305009QualTek
2020Jabbour, SK;Berman, AT;Decker, RH;Lin, Y;Feigenberg, SJ;Gettinger, SN;Aggarwal, C;Langer, CJ;Simone, CB;Bradley, JD;Aisner, J;Malhotra, J;Phase 1 Trial of Pembrolizumab Administered Concurrently With Chemoradiotherapy for Locally Advanced Non-Small Cell Lung Cancer: A Nonrandomized Controlled TrialJAMA Oncol32077891Consolidative programmed death ligand-1 (PD-L) inhibition after chemoradiotherapy improves overall survival and progression-free survival (PFS) for stage III non-small cell lung cancer (NSCLC) and requires safety evaluation for incorporation of programmed cell death 1 (PD-1) inhibition at the onset of chemoradiotherapy. To determine the safety and tolerability of PD-1 inhibition concurrently with definitive chemoradiotherapy for NSCLC. This phase 1 prospective multicenter nonrandomized controlled trial using a 3 plus 3 design was performed from August 30, 2016, to October 24, 2018, with a median follow-up of 16.0 (95% CI, 12.0-22.6) months and data locked on July 25, 2019. Twenty-one participants had locally advanced, unresectable, stage III NSCLC as determined by multidisciplinary review, Eastern Cooperative Oncology Group performance status 0 or 1, and adequate hematologic, renal, and hepatic function. Data were analyzed from October 17, 2016, to July 19, 2019. Pembrolizumab was combined with concurrent chemoradiotherapy (weekly carboplatin and paclitaxel with 60 Gy of radiation in 2 Gy per d). Dose cohorts evaluated included full-dose pembrolizumab (200 mg intravenously every 3 weeks) 2 to 6 weeks after chemoradiotherapy (cohort 1); reduced-dose pembrolizumab (100 mg intravenously every 3 weeks) starting day 29 of chemoradiotherapy (cohort 2); full-dose pembrolizumab starting day 29 of chemoradiotherapy (cohort 3); reduced-dose pembrolizumab starting day 1 of chemoradiotherapy (cohort 4); and full-dose pembrolizumab starting day 1 of chemoradiotherapy (cohort 5). A safety expansion cohort of 6 patients was planned based on the maximum tolerated dose of pembrolizumab. Dose-limiting toxic effects were defined as pneumonitis of at least grade 4 within cycle 1 of pembrolizumab treatment. Safety and tolerability of PD-1 inhibition with chemoradiotherapy for NSCLC. Secondary outcomes included PFS and pneumonitis rates. Among the 21 patients included in the analysis (11 female [52%]; median age, 69.5 [range, 53.0-85.0] years), no dose-limiting toxic effects in any cohort were observed. One case of grade 5 pneumonitis occurred in the safety expansion cohort with the cohort 5 regimen. Immune-related adverse events of at least grade 3 occurred in 4 patients (18%). Median PFS for patients who received at least 1 dose of pembrolizumab (n = 21) was 18.7 (95% CI, 11.8-29.4) months, and 6- and 12-month PFS were 81.0% (95% CI, 64.1%-97.7%) and 69.7% (95% CI, 49.3%-90.2%), respectively. Median PFS for patients who received at least 2 doses of pembrolizumab (n = 19) was 21.0 (95% CI, 15.3 to infinity) months. These findings suggest that combined treatment with PD-1 inhibitors and chemoradiotherapy for stage III NSCLC is tolerable, with promising PFS of 69.7% at 12 months, and requires further study. ClinicalTrials.gov Identifier: NCT02621398.224,0Exploratory analyses included PD-L1 status, by mean proportion score. Immunohistochemical staining for PD-L1 used a mouse monoclonal anti-PD-L1 antibody (clone 22C3).21 Furthermore, TILs were scored on a continuum from 0 to 3 (0 indicates absent TILs; 3, high profusion of TILs) (performed by QualTek Molecular Laboratory)https://jamanetwork.com/journals/jamaoncology/article-abstract/2761665QualTek
2020Kelly, CM;Antonescu, CR;Bowler, T;Munhoz, R;Chi, P;Dickson, MA;Gounder, MM;Keohan, ML;Movva, S;Dholakia, R;Ahmad, H;Biniakewitz, M;Condy, M;Phelan, H;Callahan, M;Wong, P;Singer, S;Ariyan, C;Bartlett, EK;Crago, A;Yoon, S;Hwang, S;Erinjeri, JP;Qin, LX;Tap, WD;D'Angelo, SP;Objective Response Rate Among Patients With Locally Advanced or Metastatic Sarcoma Treated With Talimogene Laherparepvec in Combination With Pembrolizumab: A Phase 2 Clinical TrialJAMA Oncol31971541Patients with advanced sarcoma have limited treatment options. Talimogene laherparepvec (T-VEC) has been shown to increase tumor-specific immune activation via augmenting antigen presentation and T-cell priming. To examine whether T-VEC in combination with pembrolizumab is associated with increased tumor-infiltrating lymphocyte infiltration and programmed death-ligand 1 expression and thus with increased antitumor activity in patients with locally advanced or metastatic sarcoma. This open-label, single-institution phase 2 interventional trial of T-VEC plus pembrolizumab enrolled 20 patients with locally advanced or metastatic sarcoma between March 16 and December 4, 2017, for whom at least 1 standard systemic therapy had failed. The median duration of therapy was 16 weeks (range, 7-67 weeks). Reported analyses include data through December 14, 2018. Patients received pembrolizumab (200-mg flat dose) intravenously and T-VEC (first dose, ≤4 mL × 106 plaque-forming units [PFU]/mL; second and subsequent doses, ≤4 mL × 108 PFU/mL) injected into palpable tumor site(s) on day 1 of each 21-day cycle. The primary end point was objective response rate (ORR; complete response and partial response) at 24 weeks determined by Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, criteria. Secondary end points included best ORR by immune-related RECIST criteria, progression-free survival rate at 24 weeks, overall survival, and safety. All 20 patients (12 women [60%]; median age, 63.5 years [range, 24-90 years]) were evaluable for response. The study met its primary end point of evaluating the best ORR at 24 weeks determined by RECIST, version 1.1, criteria; the best ORR was 30% (95% CI, 12%-54%; n = 6). The ORR overall was 35% (95% CI, 15%-59%; n = 7). The incidence of grade 3 treatment-related adverse events was low (4 patients [20%]). There were no grade 4 treatment-related adverse events or treatment-related deaths. In this phase 2 clinical trial, treatment with T-VEC plus pembrolizumab was associated with antitumor activity in advanced sarcoma across a range of sarcoma histologic subtypes, with a manageable safety profile. This combination therapy met its predefined primary study end point; further evaluation of T-VEC in combination with pembrolizumab for patients with select sarcoma subtypes is planned. ClinicalTrials.gov identifier: NCT03069378.224,0Paraffin-embedded tumor material taken before and after treatment was examined for TIL immune biomarker expression, including PD-L1, PD-1, CD3, CD4, FOXP3, CD8, CD68, CD163, and MHC1 by Qualtek Laboratory. Programmed death-ligand 1 tumor membranous expression was determined using the Merck 22C3 antibody. The threshold for positive PD-L1 expression was greater than 1%. The immunohistochemistry for all other immune biomarkers was performed on automated strainers. Slides were imaged using a Mirax slide scanner (Zeiss Inc). Images were generated using Caseviewer software (3DHistech)https://jamanetwork.com/journals/jamaoncology/article-abstract/2758838QualTek
2014Locke, D;Lynch, F;Bernstein, S;Siami-Namini, K;Yarranton, G;An IHC Screen for EphA3 Positive Myelofibrosis (MF) and Other Myeloproliferative Neoplasms (MPNs)Blood5588-558812421Abstract MF is a neoplastic stem cell disorder in which a multipotent hematopoietic stem cell acquires a clonal proliferative advantage, and its progeny inappropriately releases fibrogenic factors into the bone marrow microenvironment, leading to secondary bone marrow fibrosis. The cytokines implicated in the pathogenesis of MF include transforming growth factor β (TGF-β), basic fibroblastic growth factor (b-FGF), and platelet-derived growth factor (PDGF). Some of these cytokines, such as b-FGF and PDGF can act as angiogenic factors. Patients with MF have higher concentrations of circulating vascular endothelial growth factor (VEGF) and b-FGF than do control subjects. In addition, a higher degree of bone marrow angiogenesis has been reported in patients with MF. Eph receptors form the largest subgroup of receptor tyrosine kinases (RTK). The Ephrins are the ligands of the Ephs and stimulate bi-directional signaling allowing cell movement and shape change. EphA3 is a member of the Eph family and is expressed mainly during fetal development. Aberrant expression of EphA3 is detected in some solid and hematologic tumors. Recently, the expression of EphA3 has been detected in bone samples from subjects with MF. Antibody targeting of EphA3 may therefore constitute a novel approach to treating MF and other hematologic malignancies. Using well-characterized normal and diseased FFPE bone marrow biopsies, an IHC assay for EphA3 expression, which was developed and validated previously for use in AML and MDS patient screening, was evaluated further for use on MPNs, such as MF, polycythemia vera (PV) and essential thrombocythemia (ET). This IHC assay for EphA3 expression was validated (intra-assay variability/precision and inter-assay variability/reproducibility established by variance of SHS for serial tissue sections) using MPNs with negative, low, medium and high intensity and/or percent positive expression of EphA3. A numerical scoring scheme (semi-quantitative simplified H-Score [SHS]) was used by a-board certified anatomic & clinical pathologist to capture tumor & non-tumor (specifically, fibroblast) reactivity of EphA3-specific monoclonal antibody. A survey of ten (10) normal bone marrow (NLBM), thirty five (35) cases of primary MF, five (5) post PV-MF cases, six (6) PV cases and four (4) cases with ET was made with the validated IHC assay. For NLBM, there was low level reactivity in a subset of immature-blast cells that represent a small fraction (1-5% or less) of the total population of nucleated cells. There was no reactivity in fibroblasts, if present. Low EphA3 immunoreactivity of NLBM samples per se is consistent with published RT-PCR data. For MPNs, significant EphA3 expression in both tumor and tumor-associated stromal fibroblasts was noted (not solely in areas of fibrosis, although not all MPN samples studied were in the fibrotic phase). Interpretation of EphA3 status in these NLBM and MPNs was used to refine the semi-quantitative SHS scheme in anticipation of setting patient sample cut-off. Between 60-70% of MF samples were deemed EphA3+ using this IHC assay for EphA3 expression. Targeting EphA3 tumor cells and/or tumor stromal fibroblasts may therefore constitute a novel approach to treating MF and other MPNs. The Phase 2 component of a clinical study is ongoing in which the activity of KB004, a non-fucosylated anti-EphA3 Humaneered® antibody, will be characterized in disease specific cohorts including AML, MDS, MF and other MPNs. Disclosures Locke: QualTek Molecular Labs: Employment. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership.166,0Neoplasms (MPNs). Darren Locke, PhD Darren Locke, PhD. 1. QualTek Molecular Laboratories, Newtown,. 1. Search for other works by this author on: This Site. PubMed MPNs. Disclosures. Locke:QualTek Molecular Labs: Employment. Lynchhttps://ashpublications.org/blood/article-abstract/124/21/5588/101175QualTek
2013Locke, D;Bernstein, S;Lynch, F;Siami-Namini, K;Walling, J;Yonker, T;Yarranton, G;An IHC Screen For EphA3 Positive FFPE TumorsBlood4965-496512221Abstract Eph receptors are the largest subgroup of the receptor tyrosine kinases (RTK). Unlike other RTK, these function principally during development. Quiescent in postembryonic tissues, Eph expression by adult tissues is abnormal and implicated in tumor initiation, invasion and metastasis. Aberrant EphA3 expression is seen in solid and hematologic tumors, particularly advanced stage lymphoproliferative and myeloproliferative diseases. In this regard, targeting EphA3 may constitute a novel treatment for hematological malignancy. With clinical utility in mind, i.e., patient selection for anti-EphA3 therapy, a panel of commercial and proprietary (KaloBios) antibodies was screened by Western Blot for reactivity to recombinant Eph receptors (EphA3, EphA4, EphA5, EphA7, EphB2, EphB3). Those with EphA3 binding selectivity were further screened (QualTek) by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded (FFPE) normal and diseased human bone marrow (NLBM, AML) as well the LK63 pre-B-ALL cell line from which EphA3 was originally isolated. Different tissue pretreatments were used for antigen/epitope retrieval of EphA3, including steam-heating in citrate-based or Tris & chelator-based buffers, subsequent/or protease digestion, or neither. Following each antigen/epitope retrieval procedure, antibody reactivity for EphA3 was assessed by light microscopy using enzymatic biotin, tyramide and polymer-based detection techniques. In each instance, the location of the EphA3/antibody complex was visualized with 3,3-diaminobenzidine that precipitates a discrete insoluble reaction product in presence of enzyme (HRP). Nuclei were counterstained with hematoxylin to assess cell/tissue morphology. From this screen, one antibody (with an epitope in the cytoplasmic CT-domain of EphA3) was chosen for further assay optimization based on its selective reactivity towards LK63 and AML and low selective reactivity towards NLBM. Assay optimization included, amongst other aspects, evaluation of EphA3 expression in a panel of NLBM and hematopoietic tumors (200+ specimens inclusive of acute & chronic leukemia, peripheral T-cell & B-cell neoplasm, Hodgkin & non-Hodgkin lymphoma, multiple myeloma, myelodysplasic syndrome, myeloproliferative neoplasm) by a board-certified hematopathologist. In NLBM, the frequency of EphA3+ immature-blast cells (CD34+ or CD117+) was insignificant. Less than 10% CD34+/CD117+ cells were EphA3+. Elevated EphA3 expression (percentage EphA3+ nucleated cells) was observed in most hematopoietic tumors. For example, in multiple myeloma, tumor cells were typically EphA3+/CD138+ plasma cells. For AML, leukemic CD34+ or CD117+ blasts/initiating cells were typically EphA3+. Some CD138- plasma cells or leukemic CD34-/CD117- cells were also EphA3+. Correlation was made between EphA3 expression and specific tumor maturation stages, differentiation status and/or tumor aggressiveness. Tumors in blast crisis presented elevated EphA3 expression, e.g., CML in accelerated or blastic crisis but not chronic phase. Elevated EphA3 expression was noted in pre-B-ALL & pro-B-ALL (early pre-B-ALL) rather than mature B-cell ALL. EphA3 expression for some peripheral B-cell neoplasms correlated well with tumor grade: high grade, poorly differentiated (typically aggressive) B-cell lymphomas or follicular lymphomas were EphA3+. A similar relationship was noted for non-Hodgkin lymphoma (Hodgkin lymphoma was EphA3-). Preclinical screening also provided evidence for EphA3 expression by stroma/fibroblast (mostly lymphoma) and vasculature/endothelium, further rationale for development of reliable tools for profiling EphA3 in hematologic tumors and other malignancies. Using well-characterized normal and diseased FFPE bone marrow biopsies, this IHC assay for EphA3 has subsequently been validated (QualTek) to provide data that is not directly available from routine histopathology review and that supports use of the assay for profiling EphA3 in specific hematologic tumors and for patient selection in early Phase clinical trial/s of an anti-EphA3 monoclonal antibody (KB004, KaloBios). Beyond this, EphA3 targeted therapy with KB004 is anticipated for treatment of solid tumors. Recent genome-wide surveys identify EphA3 amongst the most frequently overexpressed genes in pancreatic, colon and lung carcinoma, melanoma and glioblastoma. Disclosures: Locke: QualTek Molecular Labs: Employment. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Walling:KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; Amgen: Equity Ownership; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity’s Board of Directors or advisory committees. Yonker:KaloBios: Employment, Equity Ownership. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.166,0November 15, 2013. An IHC Screen For EphA3 Positive FFPE Tumors. Darren Locke, PhD Darren Locke, PhD. 1. QualTek Molecular Laboratories, Newtown, USA,. 1. Search for other works by this author on: This Site. PubMed. Googlehttps://ashpublications.org/blood/article-abstract/122/21/4965/14583QualTek
2018Smith, S;Lynch, R;Till, B;Cowan, A;Wu, Q;Voutsinas, J;Shadman, M;Blue, K;Shustov, A;Kanan, S;Low, L;Cassaday, R;Fromm, J;Gopal, A;Pembrolizumab in Combination with Standard RCHOP Therapy for Previously Untreated Diffuse Large B-Cell LymphomaBlood1686-1686132Supplement 1Abstract Background: Rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) remains standard therapy for previously untreated diffuse large B-cell lymphoma (DLBCL). However, a substantial proportion of patients (pts) will relapse. The anti-PD-1 monoclonal antibody pembrolizumab has shown modest efficacy in previously treated DLBCL. We postulated that the first-line setting, with relatively intact host immunity and coexistence of malignant cells with T-cells in the microenvironment, may represent an opportunity for effective immune checkpoint inhibition. We carried out the first known prospective trial of anti-PD1 therapy with anthracycline chemotherapy in lymphoma, to evaluate the safety of this combination. Methods: Pts age 18 years or older with previously untreated DLBCL, transformed lymphoma and grade 3B follicular lymphoma with a plan for 6-cycle, curative-intent RCHOP were eligible. Steroids within 7 days of treatment, active autoimmune disease, or history of pneumonitis were excluded. This phase I study combined pembrolizumab (200 mg IV q 3 weeks x 6) to RCHOP x 6, with supportive care per standard practice. A 30-pt sample size permitted estimation of the rate of clinically relevant grade 3-5 toxicity, assumed to occur in 40% of pts with RCHOP alone. A stopping rule for more than 3 instances of non-relapse death or inability to complete 6 cycles of RCHOP for any reason was applied. Secondary endpoints included response rates, overall (OS) and progression-free survival (PFS). Baseline PDL1 expression was analyzed centrally (Merck/Qualtek). Peripheral blood flow cytometry for changes in PD1, PDL-1, and PDL-2 subsets were performed. After PET-based response assessment, pts were followed for relapse and survival. Results: Since March 2016, 29 pts were treated on study; 28 have finished all therapy. One is currently on therapy, and 1 remains to be enrolled. Pt characteristics are in Table 1. In 29 pts to date, 18 grade 3-5 clinically significant AE's occurred in 13 unique pts (13/29, 45%). 16 SAE's occurred in 11 pts (11/29, 38%); none qualified as unanticipated events. Two deaths occurred: One in the first accured patient, who had extensive gastric involvement by DLBCL, and died during cycle 1 of bleeding from the responding tumor bed despite maximal inpatient intervention. The other death occurred due to steroid-refractory GVHD after haploidenticial allogeneic transplant for relapsed disease. Four immune-related adverse events (IRAE) occurred: grade 3 rash, resolving with oral steroids and not recurring with further pembrolizumab, grade 1 hyperthyroidism, grade 2 colitis, and 1 episode of grade 3 pneumonitis. This case of pneumonitis was the only immune-related SAE, occurring in a 78 yo with a history of tobacco use and COPD, and resulted in stopping therapy after cycle 3. Among 27 completing treatment (excluding the pt who died during cycle 1), average anthracycline dose was 95% of expected; anthracycline relative dose intensity (RDI: delivered dose intensity/standard dose intensity) was 94%. PET response assessment among 26 evaluable pts showed 18 CR (69%), 7 PR, and 1 primary refractory disease. Among 7 PR, 1 relapsed, 3 had negative biopsies, and 3 remain in remission. Median follow-up of survivors is 13 months and 2 relapses (1 primary progressive disease, 1 after PET PR) occurred. One-year PFS is 87% (Figure 1). Baseline PDL-1 staining available for 19 pts showed a median % tumor cell staining of 30% (moderate 2+ plus strong 3+ staining; range, 0-100%). Tumor PDL1 was ≤5% in 4, 10-29% in 5, 30-49% in 4, and ≥50% in 6 pts. Both EBV+ DLBCL had 60% tumor PDL1 expression. 1 relapsing pt had no detectable tumor PDL1, and the THRLBL failed for technical reasons. Conclusions: RCHOP with pembrolizumab 200 mg q 3 weeks x 6 cycles is safe, with toxicity similar to RCHOP alone and only 4 IRAE's. Anthracycline dose intensity, CR rate, and PFS are favorable. PDL1 tumor cell expression of 10% or more was observed in 15/19 pts. These data support further study of pembrolizumab with chemotherapy in untreated lymphoid malignancies. Final response/progression, correlative studies, and survival data will be presented in December after final accrual. Disclosures Smith: Merck Sharp and Dohme Corp.: Consultancy, Research Funding; Pharmacyclics: Research Funding; Portola Pharmaceuticals: Research Funding; Acerta Pharma BV: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Incyte Corporation: Research Funding. Lynch:Johnson Graffe Keay Moniz & Wick LLP: Consultancy; Incyte Corporation: Research Funding; Takeda Pharmaceuticals: Research Funding; T.G. Therapeutics: Research Funding; Rhizen Pharmaceuticals S.A.: Research Funding. Till:Mustang Bio: Patents & Royalties, Research Funding. Cowan:Sanofi: Research Funding; Juno Therapeutics: Research Funding; Abbvie: Research Funding; Janssen: Research Funding. Shadman:Acerta Pharma: Research Funding; Genentech: Research Funding; Verastem: Consultancy; Gilead Sciences: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy; Celgene: Research Funding; Genentech: Consultancy; Beigene: Research Funding; Pharmacyclics: Research Funding; AbbVie: Consultancy; Mustang Biopharma: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Consultancy. Shustov:Seattle Genetics: Research Funding. Cassaday:Amgen: Consultancy, Research Funding; Incyte: Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Merck: Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptive Biotechnologies: Consultancy; Pfizer: Consultancy, Research Funding; Kite Pharma: Research Funding. Gopal:Pfizer: Research Funding; Janssen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Research Funding; Incyte: Consultancy; Spectrum: Research Funding; Gilead: Consultancy, Research Funding; Merck: Research Funding; Aptevo: Consultancy; Asana: Consultancy; Seattle Genetics: Consultancy, Research Funding; Brim: Consultancy; Teva: Research Funding.166,0Secondary endpoints included response rates, overall (OS) and progression-free survival (PFS). Baseline PDL1 expression was analyzed centrally (Merck/Qualtek). Peripheral blood flow cytometry for changes in PD1, PDL-1, and PDL-2 subsets were performedhttps://ashpublications.org/blood/article/132/Supplement%201/1686/272992QualTek
2019Petermann, F;Pękowska, A;Johnson, CA;Jankovic, D;Shih, HY;Jiang, K;Hudson, WH;Brooks, SR;Sun, HW;Villarino, AV;Yao, C;Singleton, K;Akondy, RS;Kanno, Y;Sher, A;Casellas, R;Ahmed, R;O'Shea, JJ;The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1Mol. Cell1229-1242.e5756Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection. Published by Elsevier Inc.145,0For LCMV experiments RNA was isolated from sorted cells of LCMV-infected mice with the QIAGEN AllPrep Micro Kit. Libraries were prepared and sequenced by the HudsonAlpha Genomic Services Lab (paired-end, 150 bp reads). For all other experiments total RNA was prepared using the RNeasy kit (QIAGEN). 500 ng of total RNA was subsequently used to prepare RNA-seq libraries by a combination of NEBNext RNA library prep kit (New England BioLabs) and Ovation SP Ultralow DR Multiplex system (Nugen) following the manufacturer’s protocol. The libraries were sequenced for 50 cycles (single read) using the HiSeq 2500 (Illumina)https://www.sciencedirect.com/science/article/pii/S1097276519304812"HudsonAlpha Genomic Services"
2020Taylor, WC;Comment on \"Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA\" by M. C. Liu et alAnn. Oncol.32371122142,0cfDNA [n = 2628 (1493 cancer; 1135 non-cancer)], formalin fixed paraffin embedded (FFPE) tumor biopsies (n = 242), and WBCs (n = 70) from the first CCGA sub-study; commercial tissue or cells [n = 227; Discovery Life Sciences (formerly Conversant Biologics, Inc.); Huntsvillehttps://www.sciencedirect.com/science/article/pii/S0923753420360580"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Ott, PA;Piha-Paul, SA;Munster, P;Pishvaian, MJ;van Brummelen, EMJ;Cohen, RB;Gomez-Roca, C;Ejadi, S;Stein, M;Chan, E;Simonelli, M;Morosky, A;Saraf, S;Emancipator, K;Koshiji, M;Bennouna, J;Safety and antitumor activity of the anti-PD-1 antibody pembrolizumab in patients with recurrent carcinoma of the anal canalAnn. Oncol.1036-104128528453692Safety and efficacy of pembrolizumab, a humanized programmed death 1 monoclonal antibody, was assessed in KEYNOTE-028, a multicohort, phase Ib trial for patients with programmed death ligand 1 (PD-L1)-positive advanced solid tumors. We report results for the cohort of patients with advanced anal carcinoma. Patients with PD-L1-positive tumors (≥1%) received intravenous pembrolizumab 10 mg/kg once every 2 weeks for up to 2 years or until confirmed progression or unacceptable toxicity. Response was assessed every 8 weeks for the first 6 months and every 12 weeks thereafter per Response Evaluation Criteria In Solid Tumors, version 1.1. Primary endpoints were safety and overall response rate per investigator review. Secondary endpoints included progression-free survival, overall survival, and response duration. Data cutoff date was 1 July 2015. Of the 43 patients with advanced anal carcinoma evaluable for PD-L1 expression, 32 (74%) had PD-L1-positive tumors as assessed with the 22C3 prototype assay, of whom 25 were enrolled between April and September 2014. Sixteen patients (64%) experienced treatment-related adverse events; the most common ones were diarrhea and fatigue in four patients (16%) each and nausea in three patients (12%). There were no treatment-related deaths or discontinuations as of the data cutoff date. Among the 24 patients with squamous cell carcinoma histology, four had confirmed partial response, for an overall response rate of 17% [95% confidence interval (CI), 5%-37%) and 10 (42%) had confirmed stable disease, for a disease control rate of 58%. One additional patient with non-squamous histology had confirmed stable disease. In this population of patients with PD-L1-positive advanced squamous cell anal carcinoma, pembrolizumab demonstrated a manageable safety profile and encouraging antitumor activity. These data support further study of pembrolizumab for this patient population. NCT02054806. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.142,0mbedded tumor sample or a newly obtained biopsy specimen was assessed at a central laboratory for PD-L1 expression at screening with a laboratory-developed prototype immunohistochemistry (IHC) assay (QualTek Molecular Laboratories, Goleta, CA) [15] using the 22C3 antibody (Merck & Co., Inc., Kenilworth, NJ). PD-L1 positivity was defined as membrane staining of ≥1% of scorable cells, including bo;mbedded tumor sample or a newly obtained biopsy specimen was assessed at a central laboratory for PD-L1 expression at screening with a laboratory-developed prototype immunohistochemistry (IHC) assay (QualTek Molecular Laboratories, Goleta, CA) [15] using the 22C3 antibody (Merck & Co., Inc., Kenilworth, NJ). PD-L1 positivity was defined as membrane staining of ≥1% of scorable cells, including bohttps://academic.oup.com/annonc/article-abstract/28/5/1036/2972137QualTek
2017Hui, R;Garon, EB;Goldman, JW;Leighl, NB;Hellmann, MD;Patnaik, A;Gandhi, L;Eder, JP;Ahn, MJ;Horn, L;Felip, E;Carcereny, E;Rangwala, R;Lubiniecki, GM;Zhang, J;Emancipator, K;Roach, C;Rizvi, NA;Pembrolizumab as first-line therapy for patients with PD-L1-positive advanced non-small cell lung cancer: a phase 1 trialAnn. Oncol.874-88128428168303Pembrolizumab improved survival as first- and second-line therapy compared with chemotherapy in patients with highly programmed death ligand 1 (PD-L1) expressing advanced non-small cell lung cancer (NSCLC). We report the long-term safety and clinical activity of pembrolizumab as first-line therapy for patients with advanced NSCLC and the correlation between PD-L1 expression and efficacy. In the open-label phase 1b KEYNOTE-001 trial, treatment-naive patients with advanced NSCLC whose tumors expressed PD-L1 (≥1% staining, assessed using a prototype assay) were randomly assigned to intravenous pembrolizumab 2 or 10 mg/kg every 3 (Q3W) or 2 (Q2W) weeks. Response was assessed per central RECIST v1.1 every 9 weeks in all patients who received ≥1 pembrolizumab dose. Using pre-treatment tumor tissue, a clinical assay quantified the percentage of tumor cells expressing PD-L1 as tumor proportion score (TPS). Between 1 March 2013 and 18 September 2015, 101 patients received pembrolizumab 2 mg/kg Q3W (n = 6), 10 mg/kg Q3W (n = 49), or 10 mg/kg Q2W (n = 46). Of these, 27 (26.7%) had TPS ≥50%, 52 (51.5%) had TPS 1%-49%, and 12 (11.9%) had TPS <1%. The objective response rate (ORR) was 27% (27/101, 95% CI 18-37) and median overall survival was 22.1 months (95% CI 17.1-27.2). In patients with PD-L1 TPS ≥50%, ORR, 12-month PFS, and 12-month OS were higher [14/27 (51.9%; 95% CI 32%-71%), 54%, and 85%, respectively] than the overall population [27/101 (26.7%; 95% CI 18.4%-36.5%), 35%, 71%]. Pembrolizumab was well tolerated, with only 12 (11.9%) patients experiencing grade 3/4 treatment-related adverse events and no treatment-related deaths. Pembrolizumab provides promising long-term OS benefit with a manageable safety profile for PD-L1-expressing treatment-naive advanced NSCLC, with greatest efficacy observed in patients with TPS ≥50%. KEYNOTE-001 (ClinicalTrials.gov, NCT01295827). © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.142,0As previously described [5], PD-L1 expression for enrollment was assessed with a prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, CA) and the relationship between PD-L1 expression and efficacy was investigated using a clinical trial immunohistochemistry assay (early version of the PD-L1 22C3 IHC pharmDx assay, Dako North America, Carpinteria, CA). Both assays used the murine 22C3 anti-human PD-L1 antibody (Merck & Co., Inc., Kenilworth, NJ). PD-L1 positivity was defined as membrane staining on ≥1% of cells within tumor nests, including both neoplastic cells and intercalated mononuclear inflammatory cells, or a distinctive pattern of staining caused by mononuclear inflammatory cells infiltrating the stroma, forming a banding pattern adjacent to tumor nests [6]. Tumors were categorized based on TPS, the percentage of tumor cells demonstrating membranous PD-L1 staining [7]https://academic.oup.com/annonc/article-abstract/28/4/874/2972135QualTek
2020Chung, HC;Piha-Paul, SA;Lopez-Martin, J;Schellens, JHM;Kao, S;Miller, WH;Delord, JP;Gao, B;Planchard, D;Gottfried, M;Zer, A;Jalal, SI;Penel, N;Mehnert, JM;Matos, I;Bennouna, J;Kim, DW;Xu, L;Krishnan, S;Norwood, K;Ott, PA;Pembrolizumab After Two or More Lines of Previous Therapy in Patients With Recurrent or Metastatic SCLC: Results From the KEYNOTE-028 and KEYNOTE-158 StudiesJ Thorac Oncol618-62715431870883Pembrolizumab has shown clinical benefit in patients with previously treated recurrent or metastatic SCLC in the phase 1b multicohort study KEYNOTE-028 (NCT02054806) and the phase 2 multicohort study KEYNOTE-158 (NCT02628067). We present a pooled analysis of patients from KEYNOTE-028 and KEYNOTE-158 who had received two or more lines of previous therapy for SCLC. Eligible patients were aged 18 years and above, had histologically or cytologically confirmed incurable recurrent or metastatic SCLC, had an Eastern Cooperative Oncology Group performance status of 1 and below, and had received two or more lines of previous therapy. Patients in KEYNOTE-028 were required to have a programmed death ligand 1 (PD-L1)-positive tumor. Patients received pembrolizumab (10 mg/kg every 2 weeks in KEYNOTE-028 or 200 mg every 3 weeks in KEYNOTE-158) for up to 2 years. The primary end point was objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1, which is presented here per independent review. Eighty-three patients who had received two or more lines of previous therapy (KEYNOTE-028, n = 19; KEYNOTE-158, n = 64) were included. Median follow-up duration was 7.7 (range, 0.5-48.7) months. Objective response rate was 19.3% (95% confidence interval: 11.4-29.4); two patients had complete response (one with a PD-L1-positive tumor), and 14 patients had partial response (13 with PD-L1-positive tumors). The median duration of response was not reached (range, 4.1‒35.8+ mo; plus sign indicates ongoing response); 61% of responders had responses lasting 18 months or longer. Fifty-one patients (61.4%) experienced any-grade treatment-related adverse events; eight patients (9.6%) had grade 3 or higher events. Pembrolizumab exhibited durable antitumor activity in a subset of patients with recurrent or metastatic SCLC who had undergone two or more previous lines of therapy, regardless of PD-L1 expression. Pembrolizumab was well tolerated. Copyright © 2019. Published by Elsevier Inc.125,0In KEYNOTE-028, tumor samples were assessed using a laboratory-developed prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, CA) with the 22C3 antibody clone (Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ)https://www.sciencedirect.com/science/article/pii/S155608641933850XQualTek
2018Gadgeel, SM;Pennell, NA;Fidler, MJ;Halmos, B;Bonomi, P;Stevenson, J;Schneider, B;Sukari, A;Ventimiglia, J;Chen, W;Galasso, C;Wozniak, A;Boerner, J;Kalemkerian, GP;Phase II Study of Maintenance Pembrolizumab in Patients with Extensive-Stage Small Cell Lung Cancer (SCLC)J Thorac Oncol1393-139913929775808The aim of this study was to assess the efficacy of maintenance pembrolizumab in patients with extensive-stage SCLC after treatment with platinum and etoposide. Patients with extensive-stage SCLC with a response or stable disease after induction chemotherapy were eligible. Pembrolizumab at a dose of 200 mg administered intravenously every 3 weeks was initiated within 8 weeks of the last cycle of chemotherapy. The primary end point of the study was progression-free survival (PFS) from study registration, with overall survival (OS) as a key secondary end point. Available tumor tissue was assessed for expression of programmed death ligand 1 (PD-L1) both in the tumor cells and in the surrounding stroma. Blood for circulating tumor cells was collected before the first, second, and third cycles of pembrolizumab. Of the 45 patients enrolled, 56% were male and 22% had treated brain metastases. The median PFS was 1.4 months (95% confidence interval [CI]: 1.3-2.8), with a 1-year PFS of 13%. The median OS was 9.6 months (95% CI: 7.0-12), with a 1-year OS of 37%. Of the 30 tumors that could be assessed, three had PD-L1 expression (≥1%) in the tumor cells. A total of 20 tumors could be assessed for PD-L1 expression in the stroma. The median PFS in the eight patients with tumors positive for expression of PD-L1 at the stromal interface was 6.5 months (95% CI: 1.1-12.8) compared with 1.3 months (95% CI: 0.6-2.5) in 12 patients with tumors negative for this marker. No unexpected toxicities were observed. Maintenance pembrolizumab did not appear to improve median PFS compared with the historical data. However, the 1-year PFS rate of 13% and OS rate of 37% suggest that a subset of patients did benefit from pembrolizumab. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.125,0than every four cycles. Retrieval of pretreatment biopsy specimens for assessment of PD-L1 expression in tumor cells and stromal tissue was conducted by Qualtek Clinical Laboratories (Newton, PA). Assessment of tumor PDhttps://www.sciencedirect.com/science/article/pii/S1556086418306002QualTek
2016Sun, JM;Zhou, W;Choi, YL;Choi, SJ;Kim, SE;Wang, Z;Dolled-Filhart, M;Emancipator, K;Wu, D;Weiner, R;Frisman, D;Kim, HK;Choi, YS;Shim, YM;Kim, J;Prognostic Significance of PD-L1 in Patients with Non-Small Cell Lung Cancer: A Large Cohort Study of Surgically Resected CasesJ Thorac Oncol1003-101111727103510The aim of our analysis was to evaluate the prognostic effect of programmed cell death ligand-1 (PD-L1) expression in patients with non-small cell lung cancer (NSCLC). PD-L1 expression among 1070 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Data were analyzed using Cox proportional hazard models adjusting for age, sex, smoking status, histologic type, stage, and performance status. Sixty-eight patients (6%) were strongly PD-L1 positive and 410 (38%) were weakly PD-L1 positive. A significantly higher prevalence of PD-L1 positivity was observed among patients with squamous cell carcinoma and among stage IIIB and IV patients. PD-L1 expression may be associated with poorer overall survival, with an adjusted hazard ratio of 1.56 (95% confidence interval [CI]: 1.08-2.26, p = 0.02) for strong PD-L1 positivity, 1.18 (95% CI: 0.96-1.46; p = 0.12) for weak PD-L1 positivity, and 1.23 (95% CI: 1.00-1.51; p = 0.05) for the combined strongly and weakly positive groups compared with PD-L1 negativity. Negative prognostic effect of PD-L1 expression was not statistically significant after adjustment for postoperative chemotherapy or radiotherapy. Similar results were observed for progression-free survival. Among stage I patients, the disease recurrence rate was higher in the PD-L1-positive versus in the PD-L1-negative group (48% versus 27%, p < 0.001), with an adjusted hazard ratio for disease-free survival of 2.01 (95% CI, 1.08-3.73; p = 0.03) for strong PD-L1 positivity and 1.57 (95% CI, 1.17-2.11; p = 0.003) for weak PD-L1 positivity compared with PD-L1 negativity. Tumor PD-L1 expression may be associated with poor prognosis in patients with NSCLC, although its significance weakens when postoperative therapy is considered. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.125,0Automated staining (QualTek TekMate 500, QualTek Molecular Laboratories, Newtown, PA) included rinses with 1× wash buffer (#K8012 Envision FLEX+ kit, Dako), 10-minute proteinase K incubation (#S3020, Dako), 1× wash buffer rinse, and peroxidase blocking (#K8012https://www.sciencedirect.com/science/article/pii/S1556086416300958QualTek
2008Ramalingam, S;Forster, J;Naret, C;Evans, T;Sulecki, M;Lu, H;Teegarden, P;Weber, MR;Belani, CP;Dual inhibition of the epidermal growth factor receptor with cetuximab, an IgG1 monoclonal antibody, and gefitinib, a tyrosine kinase inhibitor, in patients with refractory non-small cell lung cancer (NSCLC): a phase I studyJ Thorac Oncol258-2643318317068To determine the optimal doses of the antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody cetuximab and the EGFR tyrosine kinase inhibitor gefitinib when administered as a combination for patients with advanced/metastatic non-small cell lung cancer (NSCLC) previously treated with platinum-based chemotherapy. Patients with advanced/metastatic NSCLC treated with prior platinum-based chemotherapy received escalating doses of weekly cetuximab (100, 200, and 250 mg/m(2), IV) and fixed doses of gefitinib (250 mg/d, PO) until disease progression or unacceptable toxicity. Available tumor samples were analyzed for EGFR expression, EGFR gene copy number and mutations, and K-RAS mutations. Thirteen patients were enrolled in three cohorts. Treatment was generally well-tolerated at all doses. One grade 3 headache, observed on the first treatment cycle was initially considered dose-limiting toxicity (DLT); this event was eventually determined to be caused by a brain metastasis, not toxicity. Three cases of grade 3/4 hypomagnesemia and 1 case of grade 3 skin rash occurred in the highest-dose cohort. Grade 1/2 infusion reactions occurred in three patients without requiring treatment discontinuation. Four patients (31%) achieved stable disease, no responses were observed. None of the patients had EGFR mutations or gene amplification in their tumor samples. Dual EGFR inhibition with cetuximab and gefitinib is feasible; the combination can be safely administered and may have modest activity in advanced/metastatic NSCLC. Cetuximab 250 mg/m(2) weekly IV and gefitinib 250 mg/d PO is the recommended phase II dose, although the potential for late-onset hypomagnesemia warrants close monitoring of patients receiving this combined dosage.125,038. FFPE tumor samples were submitted to QualTek (Newtown, PA) for EGFR expression analysis by immunohistochemistry. The intensity of staining was scored as: 3 = high, 2 = moderate, 1 = low/minimal, and 0 = no staining. RESULTS. Patient Demographicshttps://www.sciencedirect.com/science/article/pii/S1556086415313691QualTek
2020Freeman, ZT;Nirschl, TR;Hovelson, DH;Johnston, RJ;Engelhardt, JJ;Selby, MJ;Kochel, CM;Lan, RY;Zhai, J;Ghasemzadeh, A;Gupta, A;Skaist, AM;Wheelan, SJ;Jiang, H;Pearson, AT;Snyder, LA;Korman, AJ;Tomlins, SA;Yegnasubramanian, S;Drake, CG;A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer targetJ. Clin. Invest.1405-1416130332015231Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types.123,0Study approval. All human tumors were collected with approval from the Johns Hopkins University IRB with the following protocol IDs: renal clear cell carcinoma (IRB00033839), prostate cancer (NA_00082175), bladder cancer (NA_00026693), and glioblastoma (IRB00049987). Human tumor samples for protein analysis were purchased from Conversant Bio. All animal experiments were reviewed and approved by local IACUCS as follows. In vivo antibody blockade experiments were reviewed and approved by the Bristol-Myers Squibb IACUC under protocol 1311-01. Generation of Foxp3 Cre+ Tnfrsf9fl/fl mice was reviewed and approved by the University of Michigan IACUC under protocol PRO00008577https://www.jci.org/articles/view/128672"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2016Resendez, SL;Jennings, JH;Ung, RL;Namboodiri, VM;Zhou, ZC;Otis, JM;Nomura, H;McHenry, JA;Kosyk, O;Stuber, GD;Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lensesNat Protoc566-59711326914316Genetically encoded calcium indicators for visualizing dynamic cellular activity have greatly expanded our understanding of the brain. However, owing to the light-scattering properties of the brain, as well as the size and rigidity of traditional imaging technology, in vivo calcium imaging has been limited to superficial brain structures during head-fixed behavioral tasks. These limitations can now be circumvented by using miniature, integrated microscopes in conjunction with an implantable microendoscopic lens to guide light into and out of the brain, thus permitting optical access to deep brain (or superficial) neural ensembles during naturalistic behaviors. Here we describe steps to conduct such imaging studies using mice. However, we anticipate that the protocol can be easily adapted for use in other small vertebrates. Successful completion of this protocol will permit cellular imaging of neuronal activity and the generation of data sets with sufficient statistical power to correlate neural activity with stimulus presentation, physiological state and other aspects of complex behavioral tasks. This protocol takes 6-11 weeks to complete.113,0Dust-off compressed gas (Falcon Safety Products, DPSXL) Gelfoam absorbable sponge (Pfizer Injectables, cat. no. 00009-0342-01) 12-well culture plates (Falcon, cat. no. 353043) Skull screws (Glass and Watch Screws, 900 piece set, JT69900) 1/16-inch diameter heat shrink tubing (Qualtek, cat. no. Q2-F3X-1/16-01-MS100FT) Carbon filter (ReFresh, cat. no. EZ-258) Parafilm (Parafilm, PM-999)https://www.nature.com/nprot/journal/v11/n3/abs/nprot.2016.021.htmlQualTek
2019Zeng, Q;Liao, C;Terhune, J;Wang, L;Impacts of florfenicol on the microbiota landscape and resistome as revealed by metagenomic analysisMicrobiome1557131818316Drug-resistant fish pathogens can cause significant economic loss to fish farmers. Since 2012, florfenicol has become an approved drug for treating both septicemia and columnaris diseases in freshwater fish. Due to the limited drug options available for aquaculture, the impact of the therapeutical florfenicol treatment on the microbiota landscape as well as the resistome present in the aquaculture farm environment needs to be evaluated. Time-series metagenomic analyses were conducted to the aquatic microbiota present in the tank-based catfish production systems, in which catfish received standard therapeutic 10-day florfenicol treatment following the federal veterinary regulations. Results showed that the florfenicol treatment shifted the structure of the microbiota and reduced the biodiversity of it by acting as a strong stressor. Planctomycetes, Chloroflexi, and 13 other phyla were susceptible to the florfenicol treatment and their abundance was inhibited by the treatment. In contrast, the abundance of several bacteria belonging to the Proteobacteria, Bacteroidetes, Actinobacteria, and Verrucomicrobia phyla increased. These bacteria with increased abundance either harbor florfenicol-resistant genes (FRGs) or had beneficial mutations. The florfenicol treatment promoted the proliferation of florfenicol-resistant genes. The copy number of phenicol-specific resistance genes as well as multiple classes of antibiotic-resistant genes (ARGs) exhibited strong correlations across different genetic exchange communities (p < 0.05), indicating the horizontal transfer of florfenicol-resistant genes among these bacterial species or genera. Florfenicol treatment also induced mutation-driven resistance. Significant changes in single-nucleotide polymorphism (SNP) allele frequencies were observed in membrane transporters, genes involved in recombination, and in genes with primary functions of a resistance phenotype. The therapeutical level of florfenicol treatment significantly altered the microbiome and resistome present in catfish tanks. Both intra-population and inter-population horizontal ARG transfer was observed, with the intra-population transfer being more common. The oxazolidinone/phenicol-resistant gene optrA was the most prevalent transferred ARG. In addition to horizontal gene transfer, bacteria could also acquire florfenicol resistance by regulating the innate efflux systems via mutations. The observations made by this study are of great importance for guiding the strategic use of florfenicol, thus preventing the formation, persistence, and spreading of florfenicol-resistant bacteria and resistance genes in aquaculture.105,0Carlsbad, CA) according to the manufacturer's instructions. The library construction and sequencing were conducted at the HudsonAlpha Genomic Services Lab (Huntsville, AL, USA). Genomic libraries were prepared with thehttps://link.springer.com/article/10.1186/s40168-019-0773-8"HudsonAlpha Genomic Services"
2020Fong, LY;Taccioli, C;Palamarchuk, A;Tagliazucchi, GM;Jing, R;Smalley, KJ;Fan, S;Altemus, J;Fiehn, O;Huebner, K;Farber, JL;Croce, CM;Abrogation of esophageal carcinoma development in miR-31 knockout ratsProc. Natl. Acad. Sci. U.S.A.6075-60851171132123074MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden following N-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P = 0.038) and miR-31 gene knockout abrogated development of ESCC (P = 1 × 10-6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB-controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31-associated EGLN3/NF-κB-controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31-/- rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development. Copyright © 2020 the Author(s). Published by PNAS.96,0lia isolated using the DNeasy Blood & Tissue Kit (Qiagen). WGS. DNA library preparation, library quality control, and nonhuman WGS to yield ∼30× coverage were performed on the Illumina HiSeq X at HudsonAlpha Genomic Services Laboratory. WGS Analysis. WGS reads were mapped against the Rnor6.0 (Rattus norvegicus) reference using BWA (50). The read postprocessing analysis was performed after thehttps://www.pnas.org/content/117/11/6075.short"HudsonAlpha Genomic Services"
1999Hubert, RS;Vivanco, I;Chen, E;Rastegar, S;Leong, K;Mitchell, SC;Madraswala, R;Zhou, Y;Kuo, J;Raitano, AB;Jakobovits, A;Saffran, DC;Afar, DE;STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumorsProc. Natl. Acad. Sci. U.S.A.14523-14528962510588738In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell-cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.96,0Human tissues for RNA and protein analyses were obtained from the Human Tissue Resource Center at the University of California, Los Angeles; from the National Disease Research Interchange (Philadelphia); and from QualTek Molecular Labs (Santa Barbara, CA). SSH. Tumor tissue and cell lines were homogenized in Trizol reagent (GIBCO/BRL) by using 10 ml/g of tissue or 10 ml/108 cells to isolate total RNA. Poly(A)-RNA was purified from total RNA by using Qiagen’s Oligotex mRNA Mini and Midi kits (Chatsworth, CA)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC24469QualTek
2019Lee, CR;Yonk, AJ;Wiskerke, J;Paradiso, KG;Tepper, JM;Margolis, DJ;Opposing Influence of Sensory and Motor Cortical Input on Striatal Circuitry and Choice BehaviorCurr. Biol.1313-1323.e529830982651The striatum is the main input nucleus of the basal ganglia and is a key site of sensorimotor integration. While the striatum receives extensive excitatory afferents from the cerebral cortex, the influence of different cortical areas on striatal circuitry and behavior is unknown. Here, we find that corticostriatal inputs from whisker-related primary somatosensory (S1) and motor (M1) cortex differentially innervate projection neurons and interneurons in the dorsal striatum and exert opposing effects on sensory-guided behavior. Optogenetic stimulation of S1-corticostriatal afferents in ex vivo recordings produced larger postsynaptic potentials in striatal parvalbumin (PV)-expressing interneurons than D1- or D2-expressing spiny projection neurons (SPNs), an effect not observed for M1-corticostriatal afferents. Critically, in vivo optogenetic stimulation of S1-corticostriatal afferents produced task-specific behavioral inhibition, which was bidirectionally modulated by striatal PV interneurons. Optogenetic stimulation of M1 afferents produced the opposite behavioral effect. Thus, our results suggest opposing roles for sensory and motor cortex in behavioral choice via distinct influences on striatal circuitry. Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.92,0Light was delivered to the cortical afferents and PV interneurons in the striatum through an optical fiber patchcord (Thorlabs; length 1 m; 0.50 nA; 200 mm diameter) connected to an optical cannula (described above) via a mating sleeve (Thorlabs). A small piece of heat shrink resistant tubing (Qualtek) was placed over the cannula during LED testing to prevent stray light from illuminating the texture during the task. A Pulse Pal [82] activated the LED current driver with 5 ms pulses at 25 Hz for two seconds beginning when the texture was presented. Corticostriatal neurons are capable of following this 25 Hz firing rate [83] Go-NoGo Tactile Discrimination Task Head-fixed mice were trained to utilize their whiskers to discrimihttps://www.sciencedirect.com/science/article/pii/S0960982219303264QualTek
2020Weiss, J;Sheth, S;Deal, AM;Grilley Olson, JE;Patel, S;Hackman, TG;Blumberg, JM;Galloway, T;Patel, S;Zanation, AM;Shen, CJ;Hayes, DN;Hilliard, C;Mehra, R;McKinnon, KP;Wang, HH;Weissler, MC;Bauman, JR;Chera, BS;Vincent, BG;Concurrent definitive immunoradiotherapy for patients with Stage III-IV Head and Neck Cancer and Cisplatin contraindicationClin. Cancer Res.32371539Although cisplatin plus radiotherapy is a standard treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC), cisplatin contraindication is common. Radiation elicits and promotes tumor-directed immune-stimulation, which may potentiate anti-PD-1 therapy. We provide the first efficacy report of combined pembrolizumab and definitive radiotherapy in LA-HNSCC. This single-arm, multi-institution, phase II study (NCT02609503) enrolled 29 cisplatin ineligible patients. Patients received radiotherapy concurrently with 3 cycles of pembrolizumab 200mg q3 weeks followed by 3 adjuvant cycles. The primary endpoint was a PFS of >16 months. Correlative studies included peripheral blood flow cytometry and Luminex cytokine profiling. Reasons for cisplatin ineligibility included otopathy (69.0%), nephropathy (20.7%), and neuropathy (6.9%). With median follow-up of 21 months, estimated twenty-four month PFS and OS rates were 71% (95% CI 49-84) and 75% (51-88). The primary PFS endpoint has exceeded the hypothesis and its median has not been reached. Toxicities were typical of radiotherapy; however high rates of grade 3/4 lymphopenia (58.6%) were observed. Flow cytometry revealed a relative decline in CD4 T cells and B cells, but not CD8 T cells. Upon treatment, frequencies of transitional B cells and tissue-like memory B cells increased while resting memory B cells decreased. Patients with progression had greater percentages of baseline naïve B cells and fewer marginal zone B cells. Pembrolizumab and radiotherapy is efficacious in LA-HNSCC and should be evaluated in a randomized trial. The observed changes in B-cell markers deserve further study both as potential biomarkers and as therapeutic targets. Copyright ©2020, American Association for Cancer Research.89,0withdrawal of permission. Correlative Analysis PDL1 staining was conducted by Qualtek Molecular Laboratories (Newtown, PA) as previously described 5 and required by Merck in supported investigator initiated trials. For bothhttps://clincancerres.aacrjournals.org/content/early/2020/05/05/1078-0432.CCR-20-0230.abstractQualTek
2020Lokeshwar, VB;Morera, DS;Hasanali, SL;Yates, TJ;Hupe, MC;Knapp, J;Lokeshwar, SD;Wang, J;Hennig, MJP;Baskar, R;Escudero, DO;Racine, RR;Dhir, N;Jordan, AR;Hoye, K;Azih, I;Manoharan, M;Klaassen, Z;Kavuri, S;Lopez, LE;Ghosh, S;Lokeshwar, BL;A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Patients with Bladder CancerClin. Cancer Res.32094233Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer. In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder. HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically. Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer. ©2020 American Association for Cancer Research.89,0Present address: Travis Yates: QualTek Molecular Laboratories, King of Prussia, PA; Diogo Escudero: George Washington University Kelly Hoye: University of North Carolina Lineberger Comprehensive Cancer Center; Ronny Racine: Astellas Institute for Regenerativehttps://clincancerres.aacrjournals.org/content/early/2020/04/27/1078-0432.CCR-19-2912.abstractQualTek
2020Rodriguez, CP;Wu, QV;Voutsinas, J;Fromm, JR;Jiang, X;Pillarisetty, VG;Lee, SM;Santana-Davila, R;Goulart, B;Baik, CS;Chow, LQM;Eaton, K;Martins, R;A Phase II Trial of Pembrolizumab and Vorinostat in Recurrent Metastatic Head and Neck Squamous Cell Carcinomas and Salivary Gland CancerClin. Cancer Res.837-84526431796519This clinical trial combined pembrolizumab and vorinostat in recurrent/metastatic squamous cell carcinomas of the head and neck (HN), and salivary gland cancer (SGC). Patients with progressing incurable HN and SGC, Eastern Cooperative Oncology Group (ECOG) ≤1, no prior immunotherapy, RECIST1.1 measurable disease, and normal organ function were eligible. Pembrolizumab 200 mg was given intravenous every 21 days, and vorinostat 400 mg given orally 5 days on and 2 days off during each 21-day cycle. Primary endpoints were safety and objective response rates. From November 2015 to August 2017, 25 patients with HN and 25 SGC were enrolled. Median age was 61 (range, 33-86) years, 39 (78%) were male, 21 (62%) were never smokers, and 27 (54%) had ECOG 0. In HN, 13 (52%) were p16+ oropharynx. Most common SGC histologies were adenoid cystic 12 (48%), acinic cell 3 (12%), and mucoepidermoid 3 (12%). Adverse events (AEs) in all patients were: 27 (54%) with grade ≥ 1 and 18 (36%) with grade ≥ 3. The most common AEs in all patients were renal insufficiency in seven, (14%), fatigue in six, (12%), and nausea in three (6%). There were three (12%) deaths on study. Responses in HN were complete response (CR) 0, partial response (PR) eight (32%), and stable disease (SD) five (20%). Efficacy in SGCs was CR 0, PR four (16%) in one lymphoepithelioma-like carcinoma, two acinic cell, one adenoid cystic, and SD 14 (56%). In the HN group, median follow-up (mFUP) was 12.6 months, median overall survival (mOS) was 12.6 months, and median progression-free survival (mPFS) was 4.5 months. In SGC, mFUP was 13.1 months, mOS was 14.0 months, and mPFS was 6.9 months. This combination demonstrated activity in HN, with fewer responses in SGC. Toxicities were higher than reported with pembrolizumab alone. ©2019 American Association for Cancer Research.89,0Submission of archived tissue was required prior to study entry. In patient with no available archived tissue, fresh tissue biopsies were recommended. Biopsies were attempted after three cycles of therapy were completed, in patients who consented. PD-L1 membrane expression was centrally assessed at Qualtek Laboratories using the Merck 22C3 antibody and reported through a modified percent score (MPS) ranging from 0 to 100. At the time of study design and patient accrual, data on the clinical relevance of CPS was not yet available and therefore CPS not utilized for correlative analysis. The MPS includes PD-L1 expression among tumor cells, as well as macrophages and lymphocytes within tumor nests. TheMPS differsfrom CPS, in that it excludes PD-L1 expression in stromal cells half a high-power field from tumor nests (17), because of this, based on the amount of stromal cells present in the tumor, MPS either approximates or underestimates the CPS score. For instance, a sample with an MPS of 20 would be predicted to have a CPS of 20 or higher. In addition, tumor slice culture was created from patients' tumors when feasible, and described in an accompanying supplement (18). Correlative blood samples were also collected from patients at baseline and after three cycles of therapy, with the intent of examining circulating immune cell phenotypes. Modified LSRII flow cytometry (BD Biosciences) was performed on these samples using Woodlist 3.1 software. Both, tissue and blood correlative analysis were exploratory and had no prespecified statistical plan for analysishttps://clincancerres.aacrjournals.org/content/26/4/837.abstractQualTek
2019Spino, M;Kurz, SC;Chiriboga, L;Serrano, J;Zeck, B;Sen, N;Patel, S;Shen, G;Vasudevaraja, V;Tsirigos, A;Suryadevara, CM;Frenster, JD;Tateishi, K;Wakimoto, H;Jain, R;Riina, HA;Nicolaides, TP;Sulman, EP;Cahill, DP;Golfinos, JG;Isse, K;Saunders, LR;Zagzag, D;Placantonakis, DG;Snuderl, M;Chi, AS;Cell Surface Notch Ligand DLL3 is a Therapeutic Target in Isocitrate Dehydrogenase-mutant GliomaClin. Cancer Res.1261-127125430397180Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%-90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody-drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma. We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay. Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 × 10-15) and protein by IHC (P = 0.0014 and P < 4.3 × 10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens. ©2018 American Association for Cancer Research.89,0For the discovery set, 20 nontumor brain tissue samples and 63 glioma tumor samples were obtained from Cooperative Human Tumor Network (CHTN), Conversant Bio, and RS Diagnostics. This set of tumors included the following diagnoses: glioblastoma, WHO grade IV (n ¼ 30 including 1 recurrent tumor), oligodendroglioma, grade II (n ¼ 11), anaplastic oligodendroglioma, grade III (n¼4, including 1 recurrent), astrocytoma, grade II (n ¼ 3, including 1 recurrent), anaplastic astrocytoma, grade III (n ¼ 4, including 1 recurrent), "oligoastrocytoma", grade III (n ¼ 5), and one each of pilocytic astrocytoma, pleomorphic xanthoastrocytoma grade II, ependymoma grade II, mixed ependymomasubependymoma, ganglioglioma, and recurrent glioma NOS. For the validation set, 62 gliomas with known IDH1/2 mutation status were obtained from the NYU pathology database to compare DLL3 expression in IDH-mutant glioma and IDH wildtype glioblastoma. This set included 26 IDH wild-type glioblastomas, including 1 recurrent tumor, and 36 IDH-mutant glioma tumors from 25 patients. Within IDH-mutant gliomas, there were 14 recurrent tumors, 11 of which had paired original tumors from the same patient. The pathologic diagnoses in this set included astrocytoma grade II (n ¼ 17), anaplastic astrocytomas (n ¼ 8), oligodendroglioma, grade II (n ¼ 7), and anaplastic oligodendrogliomas, grade III (n ¼ 4). All tumor samples, pathologic information, and molecular data were collected under NYU institutional review board–approved protocolshttps://clincancerres.aacrjournals.org/content/25/4/1261.abstract"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2018Subbiah, V;Murthy, R;Hong, DS;Prins, RM;Hosing, C;Hendricks, K;Kolli, D;Noffsinger, L;Brown, R;McGuire, M;Fu, S;Piha-Paul, S;Naing, A;Conley, AP;Benjamin, RS;Kaur, I;Bosch, ML;Cytokines Produced by Dendritic Cells Administered Intratumorally Correlate with Clinical Outcome in Patients with Diverse CancersClin. Cancer Res.3845-3856241630018119Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice.Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection.Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01).Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes. Clin Cancer Res; 24(16); 3845-56. ©2018 AACR. ©2018 American Association for Cancer Research.89,0Biopsied tumors were formalin fixed and paraffin embedded (FFPE) using standard methods. All immunohistochemistry was performed by QualTek Molecular Laboratories (Santa Barbara, CA). In situ detection of IFNγ and TNFα transcripts in FFPE specimens was performed using the RNAscope assay with probes Hs-IFNγ and Hs-TNFα (cat#310501 and 310421, respectively, Advanced Cell Diagnostics [ACD], USA), as well as positive control probe PPIB (cat#313901), and the RNAscope 2.0 HD Reagent kit (Brown, cat#310035, ACD, USA) following procedures recommended by the manufacturer. To verify IFNγ and TNFα RNAscope specificity, PBMCs from three healthy donors were tested before and after T-cell stimulation. To stimulate T cells, PBMCs were isolated using Ficoll-Paque (Sigma-Aldrich), resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum, and treated with 50 ng/mL phorbol myristate acetate and 1 μg/mL ionomycin (Sigma-Aldrich) for 5 hrs at 37°C and 5% CO2. Cells were fixed in 10% neutral buffered formalin in Histogel, processed, and embedded into FFPE blocks. Sections (5 μm) were then tested using RNAscope as indicated above. The stimulated T cells demonstrated a strong increase in both IFNγ and TNFα compared with untreated cells. Digital images of the stained slides were acquired with an Aperio ScanScope XT digital slide scannerhttps://clincancerres.aacrjournals.org/content/24/16/3845.abstractQualTek
2018Joseph, RW;Elassaiss-Schaap, J;Kefford, R;Hwu, WJ;Wolchok, JD;Joshua, AM;Ribas, A;Hodi, FS;Hamid, O;Robert, C;Daud, A;Dronca, R;Hersey, P;Weber, JS;Patnaik, A;de Alwis, DP;Perrone, A;Zhang, J;Kang, SP;Ebbinghaus, S;Anderson, KM;Gangadhar, TC;Baseline Tumor Size Is an Independent Prognostic Factor for Overall Survival in Patients with Melanoma Treated with PembrolizumabClin. Cancer Res.4960-4967242029685882Purpose: The purpose of this study was to assess the association of baseline tumor size (BTS) with other baseline clinical factors and outcomes in pembrolizumab-treated patients with advanced melanoma in KEYNOTE-001 (NCT01295827).Experimental Design: BTS was quantified by adding the sum of the longest dimensions of all measurable baseline target lesions. BTS as a dichotomous and continuous variable was evaluated with other baseline factors using logistic regression for objective response rate (ORR) and Cox regression for overall survival (OS). Nominal P values with no multiplicity adjustment describe the strength of observed associations.Results: Per central review by RECIST v1.1, 583 of 655 patients had baseline measurable disease and were included in this post hoc analysis. Median BTS was 10.2 cm (range, 1-89.5). Larger median BTS was associated with Eastern Cooperative Oncology Group performance status 1, elevated lactate dehydrogenase (LDH), stage M1c disease, and liver metastases (with or without any other sites; all P ≤ 0.001). In univariate analyses, BTS below the median was associated with higher ORR (44% vs. 23%; P < 0.001) and improved OS (HR, 0.38; P < 0.001). In multivariate analyses, BTS below the median remained an independent prognostic marker of OS (P < 0.001) but not ORR. In 459 patients with available tumor programmed death ligand 1 (PD-L1) expression, BTS below the median and PD-L1-positive tumors were independently associated with higher ORR and longer OS.Conclusions: BTS is associated with many other baseline clinical factors but is also independently prognostic of survival in pembrolizumab-treated patients with advanced melanoma. Clin Cancer Res; 24(20); 4960-7. ©2018 AACRSee related commentary by Warner and Postow, p. 4915. ©2018 American Association for Cancer Research.89,0Tumor PD-L1 expression was assessed by a prototype IHC assay (QualTek Molecular Laboratories; ref. 14) in pretreatment tumor biopsy samples using the 22C3 antibody (Merck & Co., Inc.). PD-L1 positivity was defined as membranous staining in 1% of tumor and/or immune cells in tumor nesthttps://clincancerres.aacrjournals.org/content/24/20/4960.abstractQualTek
2018Rugo, HS;Delord, JP;Im, SA;Ott, PA;Piha-Paul, SA;Bedard, PL;Sachdev, J;Le Tourneau, C;van Brummelen, EMJ;Varga, A;Salgado, R;Loi, S;Saraf, S;Pietrangelo, D;Karantza, V;Tan, AR;Safety and Antitumor Activity of Pembrolizumab in Patients with Estrogen Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast CancerClin. Cancer Res.2804-2811241229559561Purpose: We investigated the safety and antitumor activity of the anti-programmed death 1 monoclonal antibody pembrolizumab in patients with estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer with programmed death ligand 1-positive (PD-L1-positive) tumors in the phase Ib open-label, multicohort KEYNOTE-028 (NCT02054806) study.Patients and Methods: Patients with ER+/HER2- advanced breast cancer with PD-L1-positive tumors (combined positive score ≥1) received pembrolizumab (10 mg/kg every 2 weeks) up to 2 years or until confirmed progression/intolerable toxicity. Primary endpoints were safety and overall response rate (ORR), based on Response Evaluation Criteria in Solid Tumors, version 1 (RECIST v1.1) as assessed by investigator review.Results: Between April 2014 and January 2015, 25 patients were enrolled. Median number of prior therapies for breast cancer, including endocrine agents, was 9 (range, 3-15). Median follow-up was 9.7 months (range, 0.7-31.8 months). Three patients experienced partial response (PR) and none experienced complete response (CR), resulting in an ORR of 12.0% (95% CI, 2.5%-31.2%); 16% of patients had stable disease (SD) and clinical benefit rate (CR + PR + [SD for ≥24 weeks]) was 20% (95% CI, 7-41). Median duration of response was 12.0 months (range, 7.4-15.9 months). The incidence of treatment-related adverse events was 64%; nausea (20%) and fatigue (12%) were most common and were predominantly grade 1/2. No treatment-related discontinuations or deaths occurred.Conclusions: Pembrolizumab was well tolerated with modest but durable overall response in certain patients with previously treated, advanced, PD-L1-positive, ER+/HER2- breast cancer. Clin Cancer Res; 24(12); 2804-11. ©2018 AACR. ©2018 American Association for Cancer Research.89,0An archival or newly obtained core or excisional biopsy specimen from a nonirradiated tumor lesion was collected from each patient for assessment of PD-L1 expression. Tumor PD-L1 expression was assessed by immunohistochemistry at a central laboratory using a prototype assay (QualTek Molecular Laboratories; ref. 28) and the 22C3 antibody (Merck & Co. Inc., ref. 29). Imaging was performed every 8 weeks until month 6, and every 12 weeks thereafter for 2 years or until documented disease progression, start of new anticancer treatment, withdrawal of consent, death, or the end of the study. Response was based on RECIST v1.1, as assessed by the investigator. The primary efficacy endpoint was ORR, defined as the proportion of patients with a best overall response of CR or PR at any time during the study. Secondary efficacy endpoints were progression-free survival (PFS), defined as the time from enrollment to disease progression or death, whichever occurred first; overall survival (OS), defined as the time from enrollment to death from any cause; and duration of response, defined as the time from initial radiographic evidence of response based on RECIST v1.1 to disease progression in patients who experienced PR or CR. Adverse events (AE) were graded per the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 during study treatment and for up to 30 days after the last dose of trial treatment; data on serious AEs were collected up to 90 days after the last pembrolizumab dosehttps://clincancerres.aacrjournals.org/content/24/12/2804.abstractQualTek
2011Jänne, PA;Boss, DS;Camidge, DR;Britten, CD;Engelman, JA;Garon, EB;Guo, F;Wong, S;Liang, J;Letrent, S;Millham, R;Taylor, I;Eckhardt, SG;Schellens, JH;Phase I dose-escalation study of the pan-HER inhibitor, PF299804, in patients with advanced malignant solid tumorsClin. Cancer Res.1131-113917521220471PF299804 is a potent, orally available, irreversible inhibitor of tyrosine kinase human epidermal growth factor receptors (HER) 1 (EGFR), HER2, and HER4. This first-in-human study investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of PF299804 in patients with advanced solid malignancies. PF299804 was administered once daily continuously (schedule A) and intermittently (schedule B). Dose escalation proceeded until intolerable toxicities occurred. Skin biopsies were taken predose and after 14 days of treatment to establish a pharmacokinetic/pharmacodynamic relationship. Tumor response was measured once every 2 cycles. Efficacy was correlated with tumor genotypes in non-small cell lung cancer (NSCLC) patients. 121 patients were included (111 in schedule A, 10 in schedule B). The maximum tolerated dose (MTD) was 45 mg/d. Dose-limiting toxicities included stomatitis and skin toxicities. Most adverse events were mild and comprised skin toxicities, fatigue, and gastrointestinal side-effects including diarrhea, nausea, and vomiting. Pharmacokinetic analyses revealed dose-dependent increases in PF299804 exposure associated with target inhibition in skin biopsy samples. Fifty-seven patients with non-small cell lung cancer (NSCLC) were treated in this study. Four patients, all previously treated with gefitinib or erlotinib (2 with exon 19 deletions, 1 with exon 20 insertion, 1 mutational status unknown), had a partial response to PF299804. The MTD of PF299804 is 45 mg/d. Both continuous and intermittent treatment schedules were well tolerated, and encouraging signs of antitumor activity were observed in gefitinib/erlotinib treated NSCLC patients. ©2011 AACR.89,0Immunohistochemistry analysis was performed by QualTek (Newtown, PA) and was automated throughout using a TechMate 500 or TechMate 1000 (BioTek Solutions/Ventana Medical Systems). Antibodies used were: pERK 1/2 clone 20G11 (Cell Signaling Cat # 4376); p27 clone SX53G8 (Dako Cat # M7203); pSTAT3 (Cell Signaling Cat # 9131); Ki67 clone MIB-1 (Dako Cat # M7240). Data were reported as percentage of positive nuclei of the cells of the epidermis only. Wheneverhttps://clincancerres.aacrjournals.org/content/17/5/1131.shortQualTek
2009Nemunaitis, J;Clayman, G;Agarwala, SS;Hrushesky, W;Wells, JR;Moore, C;Hamm, J;Yoo, G;Baselga, J;Murphy, BA;Menander, KA;Licato, LL;Chada, S;Gibbons, RD;Olivier, M;Hainaut, P;Roth, JA;Sobol, RE;Goodwin, WJ;Biomarkers Predict p53 Gene Therapy Efficacy in Recurrent Squamous Cell Carcinoma of the Head and NeckClin. Cancer Res.7719-7725152419996201PURPOSE: Most recurrent squamous cell carcinomas of the head and neck have a dysfunctional p53 tumor suppressor pathway contributing to treatment resistance. We hypothesized that tumor p53 biomarkers may predict the efficacy of normal p53 delivered by gene therapy in these patients. EXPERIMENTAL DESIGN: Tumor p53 biomarkers were evaluated in 116 patients, including 29 treated with methotrexate in a phase III randomized controlled trial. Profiles favorable for p53 gene therapy efficacy were hypothesized to have either normal p53 gene sequences or low-level p53 protein expression, whereas unfavorable p53 inhibitor profiles were predicted to have high-level expression of mutated p53 that can inhibit normal p53 protein function. RESULTS: A statistically significant increase in tumor responses was observed for patients with favorable p53 efficacy profiles compared with those with unfavorable p53 inhibitor profiles [phase I/II trials: favorable (34 of 46, 74%) versus unfavorable (1 of 5, 20%), P = 0.0290; phase III trial: favorable (17 of 24, 71%) versus unfavorable (2 of 11, 18%), P = 0.0088]. In the phase III trial, there was statistically significant increased time to progression (TTP) and survival following p53 gene therapy in patients with favorable p53 profiles compared with unfavorable p53 inhibitor profiles (median TTP, 2.7 months versus 1.4 months, P = 0.0121; median survival, 7.2 months versus 2.7 months, P < 0.0001). In contrast, the biomarker profiles predictive of p53 gene therapy efficacy did not predict methotrexate response, TTP, or survival outcomes. CONCLUSIONS: These results indicate that tumor p53 biomarker profiles may predict p53 gene therapy efficacy in recurrent squamous cell carcinoma of the head and neck. (Clin Cancer Res 2009;15(24):7719-25).89,0iplex primers (Affymetrix, Inc.). The resultant PCR products were analyzed using the Affymetrix p53 GeneChip, which encompasses all coding exons, 2 to 11, as well as immediate flanking regions. Confirmation of the results was carried out by sequencing of the particular locations indicated by gene chip screening analysis or all exons in the cases where no mutation was detected using the gene chip. Immunohistochemical staining was done at QualTek Molecular Laboratories following Good Laboratory Practices. The DO-7 monoclonal antibody (Lab Vision Corp.) was used for p53 detection. This antibody binds at the NH2 terminus of p53 and thus can be used to detect both wild-type and mutant protein. The characteristics of the monoclonal antibodies specific for HDM2 and HDM4 have been described previously (4). Criteria for positive p53 overexpression were nuclear staining in ≥20% of tumor cells, whereas the criteria for HDM2 and HDM4 positivity were 10% as described (4). Samples from patients in trial INT-002 were analyzed as reported (9). The crihttps://clincancerres.aacrjournals.org/content/15/24/7719.shortQualTek
2019Ramachandran, I;Lowther, DE;Dryer-Minnerly, R;Wang, R;Fayngerts, S;Nunez, D;Betts, G;Bath, N;Tipping, AJ;Melchiori, L;Navenot, JM;Glod, J;Mackall, CL;D'Angelo, SP;Araujo, DM;Chow, WA;Demetri, GD;Druta, M;Van Tine, BA;Grupp, SA;Abdul Razak, AR;Wilky, B;Iyengar, M;Trivedi, T;Winkle, EV;Chagin, K;Amado, R;Binder, GK;Basu, S;Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcomaJ Immunother Cancer2767131651363Gene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells. Four cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay. Responses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients. Our studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy. ClinicalTrials.gov, NCT01343043 , Registered 27 April 2011.87,0d Cross (Philadelphia, PA). NY-ESO-1 testing was performed via IHC at a Clinical Laboratory Improvement Amendments certified pathology laboratory at the National Cancer Institute (Bethesda, MD) or at QualTek Labs (Goleta, CA). Disease response was classified according to RECIST v1.1, and radiological disease assessments were performed at weeks 4, 8, 12 and every 3 months thereafter. Patients who;d Cross (Philadelphia, PA). NY-ESO-1 testing was performed via IHC at a Clinical Laboratory Improvement Amendments certified pathology laboratory at the National Cancer Institute (Bethesda, MD) or at QualTek Labs (Goleta, CA). Disease response was classified according to RECIST v1.1, and radiological disease assessments were performed at weeks 4, 8, 12 and every 3 months thereafter. Patients whohttps://jitc.biomedcentral.com/articles/10.1186/s40425-019-0762-2QualTek
2019Habra, MA;Stephen, B;Campbell, M;Hess, K;Tapia, C;Xu, M;Rodon Ahnert, J;Jimenez, C;Lee, JE;Perrier, ND;Boraddus, RR;Pant, S;Subbiah, V;Hong, DS;Zarifa, A;Fu, S;Karp, DD;Meric-Bernstam, F;Naing, A;Phase II clinical trial of pembrolizumab efficacy and safety in advanced adrenocortical carcinomaJ Immunother Cancer2537131533818Adrenocortical carcinoma (ACC) is a rare malignancy without good treatment options. There are limited data about the use of immunotherapy in ACC. We investigated the efficacy and safety of pembrolizumab in patients with metastatic ACC. This is a pre-specified cohort of a single-center, investigator-initiated, phase II clinical trial using pembrolizumab monotherapy in patients with rare malignancies. Patients must have had prior treatment fail in the past 6 months before study enrollment. Patients were enrolled from August 2016 to October 2018. Follow-up data were updated as of March 26, 2019. Patients received 200 mg pembrolizumab intravenously every 3 weeks without concomitant oncologic therapy. The primary endpoint was non-progression rate (NPR) at 27 weeks. Other endpoints included adverse events, tumor responses measured independently by objective radiologic criteria, and select immunological markers. Sixteen patients with ACC (including eight women [50%]) were included in this cohort. Ten patients (63%) had evidence of hormonal overproduction (seven had cortisol-producing ACC). Non-progression rate at 27 weeks was evaluable in 14 patients, one patient was lost to follow-up, and one patient left the study because of an adverse event. Five of 14 patients were alive and progression-free at 27 weeks (non-progression rate at 27 weeks was 36, 95% confidence interval 13-65%). Of the 14 patients evaluable for imaging response by immune-related Response Evaluation Criteria in Solid Tumors, two had a partial response (including one with cortisol-producing ACC), seven had stable disease (including three with cortisol-producing ACC), and five had progressive disease, representing an objective response rate of 14% (95% confidence interval 2-43%). Of those who had stable disease, six had disease stabilization that lasted ≥4 months. Severe treatment-related adverse events (≥grade 3) were seen in 2 of 16 patients (13%) and resulted in one patient discontinuing study participation. All studied tumor specimens (14/14) were negative for programmed cell death ligand-1 expression. Thirteen of 14 tumor specimens (93%) were microsatellite-stable. Eight of 14 patients (57%) had a high tumor-infiltrating lymphocyte score on immunohistochemistry staining. Single-agent pembrolizumab has modest efficacy as a salvage therapy in ACC regardless of the tumor's hormonal function, microsatellite instability status, or programmed cell death ligand-1 status. Treatment was well tolerated in most study participants, with a low rate of severe adverse events. ClinicalTrials.gov identifier: NCT02721732 , Registered March 29, 2016.87,0PD-L1 staining was performed by Qualtek using Merck 22C3 antibody for PD-L1 and scored by a board-certified pathologist. Based on the percentage and intensity of membrane staining, H-score, ranging from 0 to 300, was assigned to tumor samples ;ilable) was evaluated for PD-L1 expression on tumor cells, including tumor-infiltrating mononuclear inflammatory cells, which were analyzed using immunohistochemistry. PD-L1 staining was performed by Qualtek using Merck 22C3 antibody for PD-L1 and scored by a board-certified pathologist. Based on the percentage and intensity of membrane staining, H-score, ranging from 0 to 300, was assigned to tumhttps://link.springer.com/article/10.1186/s40425-019-0722-xQualTek
2019Lv, JW;Li, JY;Luo, LN;Wang, ZX;Chen, YP;Comparative safety and efficacy of anti-PD-1 monotherapy, chemotherapy alone, and their combination therapy in advanced nasopharyngeal carcinoma: findings from recent advances in landmark trialsJ Immunother Cancer1597131238988Recent phase 1-2 trials reported manageable safety profiles and promising antitumor activities of anti-PD-1 drugs (pembrolizumab, nivolumab, camrelizumab and JS001) with/without chemotherapy in recurrent/metastatic nasopharyngeal carcinoma (RM-NPC), however head-to-head comparison among these regimens is lacking. We aimed to comprehensively compare the efficacy and safety of different anti-PD-1 drugs, standard chemotherapy, and their combination therapy in RM-NPC. Adverse event (AE) and objective response rate (ORR) were assessed. The pooled incidence rates of grade 1-5/3-5 AEs were 74.1%/29.6, 54.2%/17.4, 92.3%/24.5, 96.8%/16.1, 91.2%/42.8, and 100%/87.9% for pembrolizumab, nivolumab, JS001, camrelizumab, chemotherapy and camrelizumab+chemotherapy, respectively, which suggested that nivolumab and pembrolizumab exhibited the optimal safety regarding grade 1-5 AEs whereas camrelizumab and nivolumab regarding grade 3-5 AEs. As second- or later-line therapy, ORR was higher with camrelizumab (34.1%), followed by pembrolizumab (26.3%), JS001 (23.3%), and nivolumab (19.0%); whereas ORR with first-line nivolumab reached 40%. Additionally, first-line camrelizumab+chemotherapy achieved a dramatically higher ORR than that with chemotherapy alone (90.9% vs. 64.1%). Pooled ORR was 28.4 and 17.4% for PD-L1-positive and PD-L1-negative patients, respectively (P = 0.11). Here, we represent preliminary evidence for the comparative safety and efficacy of existing anti-PD-1 agents with/without chemotherapy in RM-NPC, which indicated that camrelizumab has the least toxicity profile and merits future investigation. Our findings might provide insights into the future design of immunotherapy trials in RM-NPC.87,0D-L1 expression was assessed baseline on an archived formalin-fixed, paraffin-embedded tumor sample or a newly obtained biopsy sample using a laboratory-developed prototype immunohistochemical assay (QualTek Molecular Laboratories, Goleta, CA). PD-L1 positivity was defined as membranous staining on 1% or more of a modified proportion score or interface pattern. NCI-9742 trial: The PD-L1 expression;D-L1 expression was assessed baseline on an archived formalin-fixed, paraffin-embedded tumor sample or a newly obtained biopsy sample using a laboratory-developed prototype immunohistochemical assay (QualTek Molecular Laboratories, Goleta, CA). PD-L1 positivity was defined as membranous staining on 1% or more of a modified proportion score or interface pattern. NCI-9742 trial: The PD-L1 expressionhttps://jitc.biomedcentral.com/articles/10.1186/s40425-019-0636-7QualTek
2019Clouthier, DL;Lien, SC;Yang, SYC;Nguyen, LT;Manem, VSK;Gray, D;Ryczko, M;Razak, ARA;Lewin, J;Lheureux, S;Colombo, I;Bedard, PL;Cescon, D;Spreafico, A;Butler, MO;Hansen, AR;Jang, RW;Ghai, S;Weinreb, I;Sotov, V;Gadalla, R;Noamani, B;Guo, M;Elston, S;Giesler, A;Hakgor, S;Jiang, H;McGaha, T;Brooks, DG;Haibe-Kains, B;Pugh, TJ;Ohashi, PS;Siu, LL;An interim report on the investigator-initiated phase 2 study of pembrolizumab immunological response evaluation (INSPIRE)J Immunother Cancer727130867072Immune checkpoint inhibitors (ICIs) demonstrate unprecedented efficacy in multiple malignancies; however, the mechanisms of sensitivity and resistance are poorly understood and predictive biomarkers are scarce. INSPIRE is a phase 2 basket study to evaluate the genomic and immune landscapes of peripheral blood and tumors following pembrolizumab treatment. Patients with incurable, locally advanced or metastatic solid tumors that have progressed on standard therapy, or for whom no standard therapy exists or standard therapy was not deemed appropriate, received 200 mg pembrolizumab intravenously every three weeks. Blood and tissue samples were collected at baseline, during treatment, and at progression. One core biopsy was used for immunohistochemistry and the remaining cores were pooled and divided for genomic and immune analyses. Univariable analysis of clinical, genomic, and immunophenotyping parameters was conducted to evaluate associations with treatment response in this exploratory analysis. Eighty patients were enrolled from March 21, 2016 to June 1, 2017, and 129 tumor and 382 blood samples were collected. Immune biomarkers were significantly different between the blood and tissue. T cell PD-1 was blocked (≥98%) in the blood of all patients by the third week of treatment. In the tumor, 5/11 (45%) and 11/14 (79%) patients had T cell surface PD-1 occupance at weeks six and nine, respectively. The proportion of genome copy number alterations and abundance of intratumoral 4-1BB+ PD-1+ CD8 T cells at baseline (P < 0.05), and fold-expansion of intratumoral CD8 T cells from baseline to cycle 2-3 (P < 0.05) were associated with treatment response. This study provides technical feasibility data for correlative studies. Tissue biopsies provide distinct data from the blood and may predict response to pembrolizumab.87,0IHC. For screening biopsies only, the FFPE blocks were used for PD-L1 IHC (clone 22C3) on 4-5 μm sections mounted on positively charged ProbeOn slides (QualTek, Goleta, CA). QualTek provided a modified proportion score ;from the same site as the baseline biopsy. IHC For screening biopsies only, the FFPE blocks were used for PD-L1 IHC (clone 22C3) on 4–5 μm sections mounted on positively charged ProbeOn slides (QualTek, Goleta, CA). QualTek provided a modified proportion score (MPS) indicating the proportion of PD-L1-expressing tumor cells and mononuclear inflammatory cells within tumor nests. For detailed ihttps://link.springer.com/article/10.1186/s40425-019-0541-0QualTek
2020Huetter, J;Gritzan, U;Gutcher, I;Doecke, WD;Luetke-Eversloh, MV;Golfier, S;Roider, HG;Frisk, AL;Hunter, J;Pow, A;Drake, A;Levine, Z;Levy, O;Azulay, M;Barbiro, I;Cojocaru, G;Vaknin, I;Kreft, B;Roese, L;Characterization of BAY 1905254, an immune checkpoint inhibitor targeting the immunoglobulin-like domain containing receptor 2 (ILDR2)Cancer Immunol Res32312711The immunoglobulin-like domain containing receptor 2 (ILDR2), a type I transmembrane protein belonging to the B7 family of immunomodulatory receptors, has been described to induce an immunosuppressive effect on T-cell responses. Besides its expression in several non-lymphoid tissue types, we found that ILDR2 was also expressed in fibroblastic reticular cells (FRC) in the stromal part of the lymph node. These immunoregulatory cells were located in the T-cell zone and were essential for the recruitment of naïve T cells and activated dendritic cells to the lymph nodes. Previously, it has been shown that an ILDR2-Fc fusion protein exhibits immunomodulatory effects in several models of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type I diabetes. Herein, we report the generation and characterization of a human/mouse/monkey cross-reactive anti-ILDR2 hIgG2 antibody, BAY 1905254, developed to block the immunosuppressive activity of ILDR2 for cancer immunotherapy. BAY 1905254 was shown to promote T-cell activation in vitro and enhance antigen-specific T-cell proliferation and cytotoxicity in vivo in mice. BAY 1905254 also showed potent efficacy in various syngeneic mouse cancer models, and the efficacy was found to correlate with increasing mutational load in the cancer models used. Additive or even synergistic antitumor effects were observed when BAY 1905254 was administered in combination with anti-PD-L1, an immunogenic cell death-inducing chemotherapeutic, or with tumor antigen immunization. Taken together, our data showed that BAY 1905254 is a potential drug candidate for cancer immunotherapy, supporting its further evaluation. Copyright ©2020, American Association for Cancer Research.86,0cancer patients, and commercially available cryopreserved human lymph node single- cell preparations from melanoma patients (Folio Conversant, USA) using the PrimeFlow™ RNA Assay technology (Invitrogen, CA, USA). Following enzymatichttps://cancerimmunolres.aacrjournals.org/content/early/2020/04/16/2326-6066.CIR-19-0321.abstract"Folio Conversant"
2001Afar, DE;Vivanco, I;Hubert, RS;Kuo, J;Chen, E;Saffran, DC;Raitano, AB;Jakobovits, A;Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epitheliaCancer Res.1686-169261411245484We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.84,0We thank Drs. Robert Eisenman, Owen Witte, and William Isaacs for helpful suggestions and critical reading of the manuscript; Frank Lynch and Page Erickson at QualTek Molecular Laboratories for their expertise in immunohistochemistry; and Celeste Jones for help in preparation of the manuscripthttps://cancerres.aacrjournals.org/content/61/4/1686.shortQualTek
2001Xu, J;Kalos, M;Stolk, JA;Zasloff, EJ;Zhang, X;Houghton, RL;Filho, AM;Nolasco, M;Badaró, R;Reed, SG;Identification and characterization of prostein, a novel prostate-specific proteinCancer Res.1563-156861411245466In this report, we describe the application of a systematic, genome-based approach to identify prostein, a novel prostate-specific protein expressed in normal and malignant prostate tissues. Characterization of the prostein gene shows that prostein cDNA encodes a 553-amino acid protein. The protein is predicted to be a type IIIa plasma membrane protein with a cleavable signal peptide and 11 transmembrane-spanning regions. The prostein gene is located on chromosome 1 at the WI-9641 locus between q32 and q42. Prostein mRNA is shown to be uniquely expressed in normal and cancerous prostate tissues using Northern blot, eDNA microarray, and real-time PCR analyses. Furthermore, prostein mRNA expression does not appear to be prostate tumor grade related and is restricted exclusively to prostate cell lines. Immunohistochemical staining using a mouse monoclonal antibody generated against prostein demonstrates that this protein is specifically detected in prostate tissues both at the plasma membrane and in the cytoplasm. Prostein expression is androgen responsive because treatment of LNCaP cells with androgen up-regulates prostein message and protein expression levels. These results validate prostein as a prostate-specific marker with potential utility in the diagnosis and treatment of prostate cancer.84,0Immunohistochemical Studies. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissues by QualTek Molecular Laboratories using the prostein-specific mouse monoclonal antibody 10E3- G4-D3. Chromosome Localization. The GeneBridge 4 Radiation Hybrid panel (Research Genetics) was used to determine the chromosomal location of prostein. Prostein primers 59-ACTATGGTCCAGAGGCTGTG-39 and 59- AGAGGCGGCACATAGGTGAT-39 were used in PCR reactions with DNA pools from the hybrid panel according to the manufacturer’s instructions. After 38 cycles of amplification, the reaction products were separated on a 1.2% agarose gel, and the results were analyzed through the Whitehead Institute/ Massachusetts Institute of Technology Center for Genome Research web server5 to determine the probable chromosomal locationhttps://cancerres.aacrjournals.org/content/61/4/1563.shortQualTek
2000Xu, J;Stolk, JA;Zhang, X;Silva, SJ;Houghton, RL;Matsumura, M;Vedvick, TS;Leslie, KB;Badaro, R;Reed, SG;Identification of differentially expressed genes in human prostate cancer using subtraction and microarrayCancer Res.1677-168260610749139We have identified human prostate cancer- and tissue-specific genes using cDNA library subtraction in conjunction with high throughput microarray screening. Subtracted cDNA libraries of prostate tumors and normal prostate tissue were generated. Characterization of subtracted libraries showed enrichment of both cancer- and tissue-specific genes. Highly redundant clones were eliminated by colony hybridization. The remaining clones were selected for microarray to determine gene expression levels in a variety of tumor and normal tissues. Clones showing overexpression in prostate tumors and/or normal prostate tissues were selected and sequenced. Here we report the identification of two genes, P503S and P504S, from subtracted libraries and a third gene, P510S, by subtraction followed by microarray screening. Their expression profiles were further confirmed by Northern blot, real-time PCR (TaqMan), and immunohistochemistry to be overexpressed in prostate tissues and/or prostate tumors. Full-length cDNA sequences were cloned, and their subcellular locations were predicted by a bioinformatic algorithm, PSORT, to be plasma membrane proteins. The genes identified through these approaches are potential candidates for cancer diagnosis and therapy.84,0Truncated P503S (amino acid 113–241) was cloned into pET32b (Novagen) and expressed in E. coli as thioredoxin fusion proteins with a histidine tag. P503S was purified by nickel chromatography, digested with thrombin, and further purified by reverse-phase chromatography. Full-length P504S was cloned into pTrcHisC (Invitrogen) and expressed in E. coli with a histidine tag. The protein was purified by nickel chromatography, followed by ion exchange chromatography. Rabbit mAbs were generated from rabbits immunized with P503S and P504S protein by ImmGenics using the previously published protocol (23). Immunohistochemistry was performed on formalinfixed, paraffin-embedded tissues by QualTek Molecular Laboratories using rabbit mAbs raised against P503S and P504S proteinhttps://cancerres.aacrjournals.org/content/60/6/1677.shortQualTek
2019Wu, T;Dejanovic, B;Gandham, VD;Gogineni, A;Edmonds, R;Schauer, S;Srinivasan, K;Huntley, MA;Wang, Y;Wang, TM;Hedehus, M;Barck, KH;Stark, M;Ngu, H;Foreman, O;Meilandt, WJ;Elstrott, J;Chang, MC;Hansen, DV;Carano, RAD;Sheng, M;Hanson, JE;Complement C3 Is Activated in Human AD Brain and Is Required for Neurodegeneration in Mouse Models of Amyloidosis and TauopathyCell Rep2111-2123.e628831433986Complement pathway overactivation can lead to neuronal damage in various neurological diseases. Although Alzheimer's disease (AD) is characterized by β-amyloid plaques and tau tangles, previous work examining complement has largely focused on amyloidosis models. We find that glial cells show increased expression of classical complement components and the central component C3 in mouse models of amyloidosis (PS2APP) and more extensively tauopathy (TauP301S). Blocking complement function by deleting C3 rescues plaque-associated synapse loss in PS2APP mice and ameliorates neuron loss and brain atrophy in TauP301S mice, improving neurophysiological and behavioral measurements. In addition, C3 protein is elevated in AD patient brains, including at synapses, and levels and processing of C3 are increased in AD patient CSF and correlate with tau. These results demonstrate that complement activation contributes to neurodegeneration caused by tau pathology and suggest that blocking C3 function might be protective in AD and other tauopathies. Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.78,0Human CSF cohort 1 Folio Biosciences n/a Human CSF cohort 2 PrecisionMed n/a Frozen human brain tissue Folio Biosciences n/https://www.sciencedirect.com/science/article/pii/S2211124719309647"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2013Soria, JC;Massard, C;Lazar, V;Ozoux, ML;Mery-Mignard, D;Deslandes, A;Tolcher, AW;A dose finding, safety and pharmacokinetic study of AVE1642, an anti-insulin-like growth factor-1 receptor (IGF-1R/CD221) monoclonal antibody, administered as a single agent and in combination with docetaxel in patients with advanced solid tumoursEur. J. Cancer1799-180749823485230AVE1642, a humanised mAb, binds the human IGF-1R specifically and with high affinity. This study aimed to select the dose of AVE1642 alone and then combined with docetaxel 75mg/m(2) (D). AVE1642 was administered alone at cycle (cy) 1 and then combined with D from cy2, q3w. A total of 27 patients received a median number of 5 cy (range, 1-10). The most common tumour types were sarcoma (18.5%), osseous tumours (11.1%) and colon cancer (11.1%). Two DLTs were reported in cy1 at dose level (DL) 18mg/kg and dose escalation was stopped. No major safety issue was observed. No anti-drug antibodies were detected. The Maximal Tolerated Dose of AVE1642 was 12mg/kg. The dose selected for further combinations is 6mg/kg, based on PK/PD data. Three objective responses, (two in sarcoma and one breast cancer) were observed but only one was confirmed. Eleven patients appeared to benefit from treatment with prolonged disease stabilisation ⩾4months. AVE1642 is well tolerated as a single agent and combined with D. The selected dose of AVE1642 combined with D is 6mg/kg. Promising activity was seen in sarcoma and breast cancer patients. Copyright © 2013 Elsevier Ltd. All rights reserved.67,0Tumour tissue: When possible, tumour tissue was collected before AVE1642 administration and at d21-d22 of cycle 1 to evaluate IGF-1R expression and signalling pathway markers, using validated immunohistochemistry assays, in Qualtek (USA)https://www.sciencedirect.com/science/article/pii/S0959804913000300QualTek
2001Wang, T;Fan, L;Watanabe, Y;McNeill, P;Fanger, GR;Persing, DH;Reed, SG;L552S, an alternatively spliced isoform of XAGE-1, is over-expressed in lung adenocarcinomaOncogene7699-7709205311753648Using a combination of cDNA subtraction and microarray analysis, we report here the identification and characterization of L552S, an over-expressed, alternatively spliced isoform of XAGE-1 in lung adenocarcinoma. Real-time RT-PCR analysis shows that L552S is expressed at levels greater than 10-fold in 12 of 25 lung adenocarcinoma tumors compared with the highest expression level found in all normal tissues tested. L552S is expressed in both early and late stages of lung adenocarcinoma, but it was not detected in large cell carcinoma, small cell carcinoma, or atypical lung neuroendocrine carcinoid. The full-length cDNA for L552S comprises 770 bp and encodes a polypeptide of 160 amino acids. C-terminal 94 amino acids of L552S are identical to a cancer testis antigen, XAGE-1, found in Ewing's sarcoma. Genomic sequence analysis has revealed that L552S and XAGE-1 are alternatively spliced isoforms, and expression of both L552S and XAGE-1 isoforms are present in lung adenocarcinoma. Immunohistochemistry analysis using affinity purified L552S polyclonal antibodies demonstrated specific nuclear staining in 10 of 12 lung adenocarcinoma samples. Furthermore, antibody responses to recombinant L552S protein were observed in seven of 17 lung pleural effusion fluids of lung cancer patients. These results strongly imply that L552S protein is immunogenic and suggest that it might have use as a vaccine target for lung cancer.66,0An affinity purified anti-L552S polyclonal antibody was subjected to immunohistochemistry analysis by QualTek Molecular Laboratories (Santa Barbara, CA, USA). In all cases, four micron sections of formalin fixed, paraffin-embedded tissues were used. Tissues were subjected to enzyme and steam heat induced epitope retrieval before being stained with 2.5 μg/ml of anti-L552S rabbit polyclonal antibody. Tissues were then incubated with a biotinylated anti-rabbit secondary antibody. After endogenous peroxidase blocking, the Avidin-Biotin Complex/HRP (ABC/HRP) was used along with DAB chromogen to visualize protein expressionhttps://www.nature.com/articles/1204939QualTek
2004Douglas, SA;Naselsky, D;Ao, Z;Disa, J;Herold, CL;Lynch, F;Aiyar, NV;Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell linesBr. J. Pharmacol.921-9321426152105731 In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0+/-11.8 pm, Bmax 9687+/-843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (< or =15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667+/-165 sites cell(-1), Kd 74+/-8 pm). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5+/-0.1-1.2+/-0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; Kis 0.4+/-0.1 and 864+/-193 nm, respectively). 5 U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9+/-2.2 nm) in SJRH30 cells. 6 The present study is the first to identify the presence of 'endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.66,0Search for more papers by this author. Frank Lynch QualTek Molecular Laboratories Inc., Newtown, PA 18940, USA Search for more papers by this author. Frank Lynch QualTek Molecular Laboratories Inc., Newtown, PA 18940, USA ;nch Frank 2 Aiyar Nambi V 1 11Department of Vascular Biology and Thrombosis, Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, PA 19406, U.S.A 22QualTek Molecular Laboratories Inc., Newtown, PA 18940, U.S.A. *Author for correspondence: steve.a.douglas@gsk.com 21 06 2004 15 07 2004 07 2004 142 6 921 932 21 11 2003 13 01 2004 13 02 2004 Copyrighhttps://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1038/sj.bjp.0705743QualTek;Frank Lynch[au]
2020Ulasov, I;Borovjagin, A;Fares, J;Yakushov, S;Malin, D;Timashev, P;Lesniak, MS;MicroRNA 345 (miR345) regulates KISS1-E-cadherin functional interaction in breast cancer brain metastasesCancer Lett.24-3148132246957Brain metastases manifest the advanced stage of breast cancer disease with poor prognosis for patient survival. Recent reports demonstrate that some therapeutic agents can activate the expression of several breast cancer-associated genes, whose products are involved in the onset and development of brain metastases. In this study, we discovered a functional link between KISS1 and E-cadherin that could be observed in both primary brain metastatic lesions and paired cell lines, such as parental CN34TGL and MDA-MB-231 and their respective brain metastatic subclones CN34Brm2Ctgl and MDA-MB-231Br. Remarkably, expression of KISS1 and E-cadherin genes consistently showed an inverse correlation in all of the above cell/tissue types. While E-cadherin expression was strongly upregulated in metastatic clones isolated from blood and brain, the levels of this protein in parental MDA-MB-231 cell line was low. Furthermore, E-cadherin upregulation can be artificially induced in MDA-MB-231Br and CN34Brm2Ctgl cell populations by knocking down KISS1 expression directly or through overexpressing the miR345 mimic. In the aggregate, our data suggest that the tumor microenvironment, which controls breast cancer spreading via miR345-regulated KISS1 expression, might modulate metastatic spreading by a mechanism(s) involving upregulation of E-cadherin production. Copyright © 2020 Elsevier B.V. All rights reserved.65,0One hundred and eleven tumor samples, representing invasive ductal carcinoma (IDC) were subjected for evaluation as a part of brain tissue microarrays presented by: US Biomax (BR812), Folio Biosciences (AY-HH0137) and biobank at the University of Chicago, and twentyhttps://www.sciencedirect.com/science/article/pii/S0304383520301580"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2019Kang, J;Yeom, G;Jang, H;Oh, J;Park, CJ;Kim, MG;Development of Replication Protein A-Conjugated Gold Nanoparticles for Highly Sensitive Detection of Disease BiomarkersAnal. Chem.10001-10007911531269392Paper-based lateral flow immunoassays (LFIAs) using conventional sandwich-type immunoassays are one of the most commonly used point-of-care (PoC) tests. However, the application of gold nanoparticles (AuNPs) in LFIAs does not meet sensitivity requirements for the detection of infectious diseases or biomarkers present at low concentrations in body fluids because of the limited number of AuNPs that can bind to the target. To overcome this problem, we first developed a single-stranded DNA binding protein (RPA70A, DNA binding domain A of human Replication Protein A 70 kDa) conjugated to AuNPs for a sandwich assay using a capture antibody immobilized in the LFIA and an aptamer as a detection probe, thus, enabling signal intensity enhancement by attaching several AuNPs per aptamer. We applied this method to detect the influenza nucleoprotein (NP) and cardiac troponin I (cTnI). We visually detected spiked targets at a low femtomolar range, with limits of detection for NP in human nasal fluid and for cTnI in serum of 0.26 and 0.23 pg·mL-1, respectively. This technique showed significantly higher sensitivity than conventional methods that are widely used in LFIAs involving antibody-conjugated AuNPs. These results suggest that the proposed method can be universally applied to the detection of substances requiring high sensitivity and can be used in the field of PoC testing for early disease diagnosis.63,0Patient samples for NP and cTnI were obtained from Discovery Life Sciences, Inc. The study protocol was thoroughly explained to the patients, and signed written informed consent forms were obtained in accordance with the approved guidelines and relevanthttps://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b01827"Discovery Life Sciences Inc"
2011Teichert, AE;Elalieh, H;Elias, PM;Welsh, J;Bikle, DD;Overexpression of hedgehog signaling is associated with epidermal tumor formation in vitamin D receptor-null miceJ. Invest. Dermatol.2289-22971311121814234The vitamin D receptor (VDR) ligand, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), reduces proliferation and enhances differentiation, and thus has been investigated for a role in preventing or treating cancer. Mice deficient for the VDR display a hyperproliferative response in the hair follicle and epidermis and decreased epidermal differentiation. Unlike their wild-type littermates, when treated with 7,12 dimethylbenzanthracene (DMBA) or UVB, they develop skin tumors, including some characteristic of overexpression of the hedgehog (Hh) pathway. Both the epidermis and utricles of the VDR-null animals overexpress elements of the Hh pathway (sonic hedgehog (Shh) 2.02-fold, patched1 1.58-fold, smoothened 3.54-fold, glioma-associated oncogene homolog (Gli)1 1.17-fold, and Gli2 1.66-fold). This overexpression occurs at an age (11 weeks) at which epidermal hyperproliferation is most visible and is spatially controlled in the epidermis. DMBA- or UVB-induced tumors in the VDR-null mice also overexpress elements of this pathway. Moreover, 1,25(OH)(2)D(3) downregulates the expression of some members of the Hh pathway in an epidermal explants culture system, suggesting a direct regulation by 1,25(OH)(2)D(3). Our results suggest that increased expression of Shh in the keratinocytes of the VDR-null animal activates the Hh pathway, predisposing the skin to the development of both malignant and benign epidermal neoplasms.63,0ffinity-purified, biotinylated goat anti-rabbit IgG, followed by ABC-peroxidase reagent, both purchased from Vector (Burlingame, CA). Peroxidase activity was detected with diaminobenzidine substrate (QualTek Laboratories, Santa Barbara, CA) followed by counterstaining with methyl green or hematoxylin. Omitting the first antibody resulted in no signal, indicating the specificity of the immunodetecthttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193543QualTek
2005De Vry, CG;Valdez, M;Lazarov, M;Muhr, E;Buelow, R;Fong, T;Iyer, S;Topical application of a novel immunomodulatory peptide, RDP58, reduces skin inflammation in the phorbol ester-induced dermatitis modelJ. Invest. Dermatol.473-481125316117788RDP58 is the first lead compound in a series of immunomodulating decapeptides discovered through activity-based screening and computer-aided, rational design. RDP58 disrupts cellular responses signaled through the Toll-like and tumor necrosis factor (TNF) receptor families and occludes important signal transduction pathways involved in inflammation, inhibiting the production of tumor necrosis factor alpha (TNFalpha), interferon-gamma, interleukin (IL)-2, IL-6, and IL-12. These pro-inflammatory cytokines are thought to be involved in the pathogenesis of several inflammatory and autoimmune diseases, including atopic dermatitis and psoriasis. The goal of this study was to determine the ability of RDP58 to inhibit skin inflammation following exposure to the well-characterized protein kinase C activator and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical application of RDP58 to the epidermis following TPA treatment resulted in the amelioration of the phorbol ester-induced irritant contact dermatitis. Substantial reductions were observed in skin thickness and tissue weight, neutrophil-mediated myeloperoxidase activity, inflammatory cytokine production, and various histopathological indicators. We also found RDP58 to be effective in reducing the compounding inflammatory damage brought on by chronic TPA exposure, and that it is capable of targeting inflammatory mediators specifically in the keratinocyte. These results demonstrate that topically applied RDP58 is an effective anti-inflammatory treatment in the phorbol ester-induced dermatitis model, and suggest that it may have therapeutic potential in a variety of immune-related cutaneous diseases.63,010% neutral-buffered formalin (Fisher Scientific). The preparation of tissues by embedding in paraffin, sectioning, and staining was carried out by QualTek Molecular Laboratories (Santa Barbara, California). A series of ear crosshttps://www.sciencedirect.com/science/article/pii/S0022202X15324416QualTek
2004Bikle, DD;Chang, S;Crumrine, D;Elalieh, H;Man, MQ;Choi, EH;Dardenne, O;Xie, Z;Arnaud, RS;Feingold, K;Elias, PM;25 Hydroxyvitamin D 1 alpha-hydroxylase is required for optimal epidermal differentiation and permeability barrier homeostasisJ. Invest. Dermatol.984-992122415102089Keratinocytes express high levels of 25OHD 1alpha-hydroxylase (1OHase). The product of this enzyme, 1,25-dihydroxyvitamin D (1,25(OH)(2)D), promotes the differentiation of keratinocytes in vitro suggesting an important role for this enzyme in epidermal differentiation. To test whether 1OHase activity is essential for keratinocyte differentiation in vivo we examined the differentiation process in mice null for the expression of the 1alphaOHase gene (1alphaOHase(-/-)). Heterozygotes for the null allele were bred, and the progeny genotyped by PCR. The epidermis of the 1alphaOHase(-/-) animals and their wild-type littermates (1alphaOHase(+/+)) were examined by histology at the light and electron microscopic level, by immunocytochemistry for markers of differentiation, and by function examining the permeability barrier using transepidermal water loss (TEWL). No gross epidermal phenotype was observed; however, immunocytochemical assessment of the epidermis revealed a reduction in involucrin, filaggrin, and loricrin-markers of differentiation in the keratinocyte and critical for the formation of the cornified envelope. These observations were confirmed at the electron microscopic level, which showed a reduction in the F (containing filaggrin) and L (containing loricrin) granules and a reduced calcium gradient. The functional significance of these observations was tested using TEWL to evaluate the permeability barrier function of the epidermis. Although TEWL was normal in the basal state, following disruption of the barrier using tape stripping, the 1alphaOHase(-/-) animals displayed a markedly delayed recovery of normal barrier function. This delay was associated with a reduction in lamellar body secretion and a failure to reform the epidermal calcium gradient. Thus, the 25OHD 1OHase is essential for normal epidermal differentiation, most likely by producing the vitamin D metabolite, 1,25(OH)(2)D, responsible for inducing the proteins regulating calcium levels in the epidermis that are critical for the generation and maintenance of the barrier.63,0Peroxidase activity was revealed with DAB substrate (QualTek Laboratories, Santa Barbara, California) followed by counterstaining with methyl green or hematoxylin. Omitting the first antibodies resulted in no signal, indicating the specificity of immunodetectionhttps://www.sciencedirect.com/science/article/pii/S0022202X15307545QualTek
2002Elias, PM;Ahn, SK;Denda, M;Brown, BE;Crumrine, D;Kimutai, LK;Kömüves, L;Lee, SH;Feingold, KR;Modulations in epidermal calcium regulate the expression of differentiation-specific markersJ. Invest. Dermatol.1128-1136119512445203Mammalian epidermis normally displays a distinctive calcium gradient, with low levels in the basal/spinous layers and high levels in the stratum granulosum. Although changes in stratum granulosum calcium regulate the lamellar body secretory response to permeability barrier alterations, whether modulations in calcium also regulate the expression of differentiation-specific proteins in vivo remains unknown. As acute barrier perturbations reduce calcium levels in stratum granulosum, we studied the regulation of murine epidermal differentiation after loss of calcium accompanying acute barrier disruption and by exposure of such acutely perturbed skin sites to either low (0.03 M) or high (1.8 M) calcium. Three hours after acute barrier disruption, coincident with reduced calcium and ultrastructural evidence of accelerated lamellar body secretion, both northern analyses and in situ hybridization revealed decreased mRNA levels for loricrin, profilaggrin, and involucrin in the outer epidermis, but protein levels did not change significantly. Moreover, exposure of acutely disrupted skin sites to low calcium solutions sustained the reduction in mRNA levels, whereas exposure to high calcium solutions restored normal mRNA levels (blocked by the L-type calcium channel inhibitor, nifedipine). Finally, with prolonged exposure to a low (<10% relative humidity) or high (>80% relative humidity) humidity, calcium levels increased and declined, respectively. Accordingly, mRNA and protein levels of the differentiation-specific markers increased and decreased at low and high relative humidity, respectively. These results provide direct evidence that acute and sustained fluctuations in epidermal calcium regulate expression of differentiation-specific proteins in vivo, and demonstrate that modulations in epidermal calcium coordinately regulate events late in epidermal differentiation that together form the barrier.63,0from Vector Laboratories (Burlingame, CA). Peroxidase activity was revealed with diaminobenzidine (QualTek Laboratories, Santa Barbara, CA), followed by counterstaining with methyl green. Omission of the first antibodieshttps://www.sciencedirect.com/science/article/pii/S0022202X15300427QualTek
2002Xie, Z;Komuves, L;Yu, QC;Elalieh, H;Ng, DC;Leary, C;Chang, S;Crumrine, D;Yoshizawa, T;Kato, S;Bikle, DD;Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growthJ. Invest. Dermatol.11-16118111851870The active vitamin D metabolite, 1,25-dihydroxyvitamin D, acting through the vitamin D receptor, regulates the expression of genes in a variety of vitamin D-responsive tissues, including the epidermis. To investigate the role of the vitamin D receptor in mediating epidermal differentiation, we examined the histomorphology and expression of differentiation markers in the epidermis of vitamin D receptor knockout mice generated by gene targeting. The homozygous knockout mouse displayed a phenotype that closely resembles vitamin D-dependent rickets type II in humans, including the development of rickets and alopecia. Hair loss developed by 3 mo after birth and gradually led to nearly total hair loss by 8 mo. Histologic analysis of the skin of homozygous knockout mice revealed dilation of the hair follicles with the formation of dermal cysts starting at the age of 3 wk. These cysts increased in size and number with age. Epidermal differentiation markers, including involucrin, profilaggrin, and loricrin, detected by immunostaining and in situ hybridization, showed decreased expression levels in homozygous knockout mice from birth until 3 wk, preceding the morphologic changes observed in the hair follicles. Keratin 10 levels, however, were not reduced. At the ultrastructural level, homozygous knockout mice showed increased numbers of small dense granules in the granular layer with few or no surrounding keratin bundles and a loss of keratohyalin granules. Thus, both the interfollicular epidermis and the hair follicle appear to require the vitamin D receptor for normal differentiation. The temporal abnormalities between the two processes reflect the apparent lack of requirement for the vitamin D receptor during the anagen phase of the first (developmental) hair cycle, but with earlier effects on the terminal differentiation of the interfollicular epidermis.63,0Peroxidase activity was revealed with diaminobenzidine substrate (QualTek Laboratories, Santa Barbara, CA) followed by counterstaining with methyl green or hematoxylin. Omitting the first antibodies resulted in no signal, indicating the specificity of the immunodetectionhttps://www.sciencedirect.com/science/article/pii/S0022202X15415246QualTek
2000Kömüves, LG;Hanley, K;Lefebvre, AM;Man, MQ;Ng, DC;Bikle, DD;Williams, ML;Elias, PM;Auwerx, J;Feingold, KR;Stimulation of PPARalpha promotes epidermal keratinocyte differentiation in vivoJ. Invest. Dermatol.353-360115310951268Our recent studies have demonstrated that PPARalpha activators stimulate differentiation and inhibit proliferation in cultured human keratinocytes and accelerate epidermal development and permeability barrier formation in fetal rat skin explants. As the role of PPARalpha activation in adult epidermis is not known, the aim of this study was to determine if topically applied PPARalpha ligands regulate keratinocyte differentiation in murine epidermis. Topical treatment with PPARalpha activators resulted in decreased epidermal thickness. Expression of structural proteins of the upper spinous/granular layers (involucrin, profilaggrin-filaggrin, loricrin) increased following topical treatment with PPARalpha activators. Furthermore, topically applied PPARalpha activators also increased apoptosis, decreased cell proliferation, and accelerated recovery of barrier function following acute barrier abrogation. Experiments with PPARalpha-/- knockout mice showed that these effects are specifically mediated via PPARalpha. Compared with the epidermis of PPARalpha+/+ mice, involucrin, profilaggrin-filaggrin, and loricrin expression were slightly decreased in PPARalpha-/- mice. Moreover, topical clofibrate treatment did not increase epidermal differentiation in PPARalpha-/- mice. Furthermore, in cultured human keratinocytes we have demonstrated that PPARalpha activators induce an increase in involucrin mRNA levels. We have also shown that this increase in gene expression requires an intact AP-1 response element at -2117 to -2111 bp. Thus, stimulation of PPARalpha stimulates keratinocyte/epidermal differentiation and inhibits proliferation.63,0purchased from Vector (Burlingame, CA). Peroxidase activity was revealed with diaminobenzidine (QualTek Laboratories, Santa Barbara, CA), followed by counterstaining with methyl green. Omitting the first antibodies resultedhttps://www.sciencedirect.com/science/article/pii/S0022202X15409790QualTek
1999Kömüves, LG;Hanley, K;Jiang, Y;Katagiri, C;Elias, PM;Williams, ML;Feingold, KR;Induction of selected lipid metabolic enzymes and differentiation-linked structural proteins by air exposure in fetal rat skin explantsJ. Invest. Dermatol.303-309112310084306The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants.63,0purchased from Vector (Burlingame, CA). Peroxidase activity was revealed with DAB substrate (QualTek Laboratories, Santa Barbara, CA) followed by counterstaining with methyl green. Omitting the first antibodies resultedhttps://www.sciencedirect.com/science/article/pii/S0022202X15404178QualTek
2019Planes-Laine, G;Rochigneux, P;Bertucci, F;Chrétien, AS;Viens, P;Sabatier, R;Gonçalves, A;PD-1/PD-L1 Targeting in Breast Cancer: The First Clinical Evidences Are Emerging. A Literature ReviewCancers (Basel)11731336685Recently, the development of immunotherapy through the immune checkpoint blockade led to long-lasting responses in several types of cancers that are refractory to conventional treatments, such as melanoma or non-small cell lung cancer. Immunotherapy has also demonstrated significant improvements in various other types of cancers. However, breast cancer remains one of the tumors that have not experienced the explosion of immunotherapy yet. Indeed, breast cancer was traditionally considered as being weakly immunogenic with a lower mutational load compared to other tumor types. In the last few years, anti-PD1/PD-L1 (Programmed death-ligand 1) agents have been evaluated in breast cancer, particularly in the triple negative subtype, with promising results observed when delivered as monotherapy or in combination with conventional treatments. In this review, we will report the results of the most recent studies evaluating immune checkpoint inhibitors in breast cancer. In addition, we will discuss the concomitant development of possible biomarkers, which is required for improving the selection of patients with the highest probability of benefiting from these agents.62,0ts included locally advanced or metastatic disease, after a failure of or inability to receive standard therapy, ECOG PS 0 or 1, ≥ 1 measurable lesion and PD-L1 positivity, using a prototype assay (QualTek Molecular Laboratories, Goleta, CA, USA) and the 22C3 antibody (Merck & Co., Kenilworth, NJ, USA). The specimen was considered to have a positive PD-L1 expression when CPS ≥ 1. Pembrolizumabhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679223QualTek
2020Mehnert, JM;Bergsland, E;O'Neil, BH;Santoro, A;Schellens, JHM;Cohen, RB;Doi, T;Ott, PA;Pishvaian, MJ;Puzanov, I;Aung, KL;Hsu, C;Le Tourneau, C;Hollebecque, A;Élez, E;Tamura, K;Gould, M;Yang, P;Stein, K;Piha-Paul, SA;Pembrolizumab for the treatment of programmed death-ligand 1-positive advanced carcinoid or pancreatic neuroendocrine tumors: Results from the KEYNOTE-028 studyCancer32320048Despite a protracted disease course and multiple available therapies, patients with well-differentiated neuroendocrine tumors (NETs) inevitably experience disease progression. Programmed death-ligand 1 (PD-L1) has been associated with NET progression and prognosis. The multicohort, phase 1 KEYNOTE-028 study (ClinicalTrials.gov identifier NCT02054806) evaluated the activity and safety of the anti-programmed cell death protein 1 immunotherapy pembrolizumab in patients with well-differentiated or moderately-differentiated NETs. Patients with PD-L1-positive, locally advanced or metastatic carcinoid or well-differentiated or moderately-differentiated pancreatic NETs (pNETs) were enrolled into separate cohorts and received pembrolizumab at a dose of 10 mg/kg every 2 weeks for up to 2 years. The objective response rate was the primary endpoint (as per Response Evaluation Criteria in Solid Tumors version 1.1, by investigator review). Safety was a secondary endpoint. Of 170 and 106 patients, respectively, who had evaluable samples among those screened for the carcinoid and pNET cohorts, 21% and 25%, respectively, had PD-L1-positive tumors; of these, 25 and 16 patients, respectively, were eligible and treated. The median follow-up was 20 months (range, 2-35 months) and 21 months (range, 5-32 months), respectively. The objective response rate was 12.0% (95% CI, 2.5%-31.2%) and 6.3% (95% CI, 0.2%-30.2%), respectively; 3 partial responses occurred among the carcinoid cohort and 1 among the pNET cohort. The median duration of response in the carcinoid cohort was 9.2 months (range, 6.9-11.1 months), and was not reached in the pNET cohort. No complete responses occurred. Treatment-related adverse events occurred in 68% and 69% of patients, respectively, most often diarrhea (7 patients in the carcinoid cohort and 4 patients in the pNET cohort) and fatigue (6 patients in each cohort). Hypothyroidism was the most common immune-mediated adverse event (5 patients in the carcinoid cohort and 2 patients in the pNET cohort). Pembrolizumab demonstrated antitumor activity in a subset of patients with NETs and was well-tolerated. © 2020 American Cancer Society.61,0PD was confirmed by repeat radiographic imaging ≥4 weeks after the initial scan. Investigators could opt to keep clinically stable patients on treatment until confirmed PD. Safety was monitored through 30 days after the last treatment dose (90 days for serious adverse events [AEs]). AE severity was graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0). Tumor PD‐L1 status was centrally evaluated in archived formalin‐fixed, paraffin‐embedded tumor samples or newly obtained core needle or excisional biopsies of previously nonirradiated lesions using a laboratory‐developed prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, California) with the 22C3 antibody clone (Merck & Company Inc, Kenilworth, New Jersey). PD‐L1 positivity was defined as partial or complete membrane staining on ≥1% of cells in tumor nests or positive bands in stroma.24https://acsjournals.onlinelibrary.wiley.com/doi/abs/10.1002/cncr.32883QualTek
2020Ho, AY;Barker, CA;Arnold, BB;Powell, SN;Hu, ZI;Gucalp, A;Lebron-Zapata, L;Wen, HY;Kallman, C;D'Agnolo, A;Zhang, Z;Flynn, J;Dunn, SA;McArthur, HL;A phase 2 clinical trial assessing the efficacy and safety of pembrolizumab and radiotherapy in patients with metastatic triple-negative breast cancerCancer850-860126431747077The current study was conducted to evaluate the efficacy and safety of pembrolizumab-mediated programmed cell death protein 1 inhibition plus radiotherapy (RT) in patients with metastatic triple-negative breast cancer who were unselected for programmed death-ligand 1 expression. The current study was a single-arm, Simon 2-stage, phase 2 clinical trial that enrolled a total of 17 patients with a median age of 52 years (range, 37-73 years). An RT dose of 3000 centigrays (cGy) was delivered in 5 daily fractions. Pembrolizumab was administered intravenously at a dose of 200 mg within 3 days of the first RT fraction, and then every 3 weeks ± 3 days until disease progression. The median follow-up was 34.5 weeks (range, 2.1-108.3 weeks). The primary endpoint of the current study was the overall response rate (ORR) at week 13 in patients with unirradiated lesions measured using Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1). Secondary endpoints included safety and progression-free survival. Exploratory objectives were to identify biomarkers predictive of ORR and progression-free survival. The ORR for the entire cohort was 17.6% (3 of 17 patients; 95% CI, 4.7%-44.2%), with 3 complete responses (CRs), 1 case of stable disease, and 13 cases of progressive disease. Eight patients died prior to week 13 due to disease progression. Among the 9 women assessed using RECIST version 1.1 at week 13, 3 (33%) achieved a CR, with a 100% reduction in tumor volume outside of the irradiated portal. The CRs were durable for 18 weeks, 20 weeks, and 108 weeks, respectively. The most common grade 1 to 2 toxicity (assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0) was dermatitis (29%). Four grade 3 adverse events were attributed to pembrolizumab: fatigue, lymphopenia, and infection. No were no grade 4 adverse events or treatment-related deaths reported. The combination of pembrolizumab and RT was found to be safe and demonstrated encouraging activity in patients with poor-prognosis, metastatic, triple-negative breast cancer who were unselected for programmed death-ligand 1 expression. Larger clinical trials of checkpoint blockade plus RT with predictive biomarkers of response are needed. © 2019 American Cancer Society.61,0PD‐L1 tumor expression was evaluated in prospectively collected samples of metastatic biopsies by a central laboratory (QualTek Molecular Laboratories, Goleta, California) using a previously described prototype assay using the 22C3 antibody (Merck and Companyhttps://acsjournals.onlinelibrary.wiley.com/doi/abs/10.1002/cncr.32599QualTek
2019Bernard-Gauthier, V;Mossine, AV;Knight, A;Patnaik, D;Zhao, WN;Cheng, C;Krishnan, HS;Xuan, LL;Chindavong, PS;Reis, SA;Chen, JM;Shao, X;Stauff, J;Arteaga, J;Sherman, P;Salem, N;Bonsall, D;Amaral, B;Varlow, C;Wells, L;Martarello, L;Patel, S;Liang, SH;Kurumbail, RG;Haggarty, SJ;Scott, PJH;Vasdev, N;Structural Basis for Achieving GSK-3β Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug DiscoveryJ. Med. Chem.9600-9617622131535859Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.61,0For autoradiography experiments, fresh-frozen postmortem human brain tissue blocks were acquired from Folio-Conversant and the University of California at San Francisco NDBB. In vitro autoradiography studies were performed as described in the Supporting Information Materials and Methodshttps://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.9b01030"Folio Conversant"
2020Gonzalez-Ericsson, PI;Stovgaard, ES;Sua, LF;Reisenbichler, E;Kos, Z;Carter, JM;Michiels, S;Le Quesne, J;Nielsen, TO;Laenkholm, AV;Fox, SB;Adam, J;Bartlett, JM;Rimm, DL;Quinn, C;Peeters, D;Dieci, MV;Vincent-Salomon, A;Cree, I;Hida, AI;Balko, JM;Haynes, HR;Frahm, I;Acosta-Haab, G;Balancin, M;Bellolio, E;Yang, W;Kirtani, P;Sugie, T;Ehinger, A;Castaneda, CA;Kok, M;McArthur, H;Siziopikou, K;Badve, S;Fineberg, S;Gown, A;Viale, G;Schnitt, SJ;Pruneri, G;Penault-Llorca, F;Hewitt, S;Thompson, EA;Allison, KH;Symmans, WF;Bellizzi, AM;Brogi, E;Moore, DA;Larsimont, D;Dillon, DA;Lazar, A;Lien, H;Goetz, MP;Broeckx, G;El Bairi, K;Harbeck, N;Cimino-Mathews, A;Sotiriou, C;Adams, S;Liu, SW;Loibl, S;Chen, IC;Lakhani, SR;Juco, JW;Denkert, C;Blackley, EF;Demaria, S;Leon-Ferre, R;Gluz, O;Zardavas, D;Emancipator, K;Ely, S;Loi, S;Salgado, R;Sanders, M;, ;The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practiceJ. Pathol.667-684250532129476Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.59,0PANACEA NCT02129556 58 Pembrolizumab + trastuzumab single arm phase Ib/II PreTx LAdv or mHER2+ BC Ib: PD‐L1+ (6) II: PD‐L1+ & PD‐L1‐ (52) PD‐L1 (QualTek/ 22C3) tested prospectively at BTx (58). TILs were evaluated retrospectively (48). II: Higher ORR (15 versus 0%) in PD‐L1+ (CPS ≥ 1). Longer OS for PD‐L1+ population. Higher TILs levels in objective responders (median ~25 versus 1.5% p = 0.006) and in PD‐L1+ population (p = 0.0004)https://onlinelibrary.wiley.com/doi/abs/10.1002/path.5406QualTek
2020Vijayvergia, N;Dasari, A;Deng, M;Litwin, S;Al-Toubah, T;Alpaugh, RK;Dotan, E;Hall, MJ;Ross, NM;Runyen, MM;Denlinger, CS;Halperin, DM;Cohen, SJ;Engstrom, PF;Strosberg, JR;Pembrolizumab monotherapy in patients with previously treated metastatic high-grade neuroendocrine neoplasms: joint analysis of two prospective, non-randomised trialsBr. J. Cancer1309-1314122932152503Metastatic high-grade neuroendocrine neoplasms (G3NENs) have limited treatment options after progression on platinum-based therapy. We addressed the role of Pembrolizumab in patients with previously treated metastatic G3NENs. Two open-label, phase 2 studies enrolled patients with G3NEN (Ki-67 > 20%) to receive Pembrolizumab at 200 mg I.V. every 3 weeks. Radiographic evaluation was conducted every 9 weeks with overall response rate as the primary endpoint. Between November 2016 and May 2018, 29 patients (13 males/16 females) with G3NENs were enrolled. One patient (3.4%) had an objective response and an additional six patients (20.7%) had stable disease, resulting in a disease control rate of 24.1%. Disease control rate (DCR) at 18 weeks was 10.3% (3/29). There was no difference in the DCR, PFS or OS between the PD-L1-negative and -positive groups (p 0.56, 0.88 and 0.55, respectively). Pembrolizumab was well tolerated with only 9 grade 3, and no grade 4 events considered drug-related. Pembrolizumab can be safely administered to patients with G3NENs but has limited activity as a single agent. Successful completion of our trials suggest studies in G3NENs are feasible and present an unmet need. Further research to identify active combination therapies should be considered. NCT02939651 (10/20/2016).54,0For patients enrolled into FC, PD-L1 immunohistochemistry (IHC) assay was performed at Qualtek Research Laboratories on formalin-fixed, paraffin-embedded tissue sections using anti-PD-L1 monoclonal antibody clone 22C3 [Merck Research Laboratories]https://www.nature.com/articles/s41416-020-0775-0QualTek
2018Maity, A;Mick, R;Huang, AC;George, SM;Farwell, MD;Lukens, JN;Berman, AT;Mitchell, TC;Bauml, J;Schuchter, LM;O'Hara, M;Lin, LL;Demichele, A;Christodouleas, JP;Haas, NB;Patsch, DM;Hahn, SM;Minn, AJ;Wherry, EJ;Vonderheide, RH;A phase I trial of pembrolizumab with hypofractionated radiotherapy in patients with metastatic solid tumoursBr. J. Cancer1200-12071191030318516We conducted a phase I trial evaluating pembrolizumab+hypofractionated radiotherapy (HFRT) for patients with metastatic cancers. There were two strata (12 patients each): (i) NSCLC/melanoma progressing on prior anti-PD-1 therapy, (ii) other cancer types; anti-PD-1-naive. Patients received 6 cycles of pembrolizumab, starting 1 week before HFRT. Patients had ≥2 lesions; only one was irradiated (8 Gy × 3 for first half; 17 Gy × 1 for second half in each stratum) and the other(s) followed for response. Of the 24 patients, 20 (83%) had treatment-related adverse events (AEs) (all grade 1 or 2). There were eight grade 3 AEs, none treatment related. There were no dose-limiting toxicities or grade 4/5 AEs. Stratum 1: two patients (of 12) with progression on prior PD-1 blockade experienced prolonged responses (9.2 and 28.1 months). Stratum 2: one patient experienced a complete response and two had prolonged stable disease (7.4 and 7.0 months). Immune profiling demonstrated that anti-PD-1 therapy and radiation induced a consistent increase in the proliferation marker Ki67 in PD-1-expressing CD8 T cells. HFRT was well tolerated with pembrolizumab, and in some patients with metastatic NSCLC or melanoma, it reinvigorated a systemic response despite previous progression on anti-PD-1 therapy. NCT02303990 ( www.clinicaltrials.gov ).54,0ange in volume of the irradiated (index) lesion was also calculated, but this was not used in the RECIST measurement. Immunohistochemical staining PD-L1 staining was performed by an outside company (QualTek Molecular Laboratories, Newtown, PA, USA) using the Dako 22-C3 antibody. PD-L1 staining was scored as 0, 1+, 2+ or 3+. H-score was calculated as ((1 × % cells with 1+ staining) + (2;ange in volume of the irradiated (index) lesion was also calculated, but this was not used in the RECIST measurement. Immunohistochemical staining PD-L1 staining was performed by an outside company (QualTek Molecular Laboratories, Newtown, PA, USA) using the Dako 22-C3 antibody. PD-L1 staining was scored as 0, 1+, 2+ or 3+. H-score was calculated as ((1 × % cells with 1+ staining) + (2https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251028QualTek
2018Mehra, R;Seiwert, TY;Gupta, S;Weiss, J;Gluck, I;Eder, JP;Burtness, B;Tahara, M;Keam, B;Kang, H;Muro, K;Geva, R;Chung, HC;Lin, CC;Aurora-Garg, D;Ray, A;Pathiraja, K;Cheng, J;Chow, LQM;Haddad, R;Efficacy and safety of pembrolizumab in recurrent/metastatic head and neck squamous cell carcinoma: pooled analyses after long-term follow-up in KEYNOTE-012Br. J. Cancer153-159119229955135Second-line treatment options for advanced head and neck squamous cell carcinoma (HNSCC) are limited. The phase Ib KEYNOTE-012 study evaluated the safety and the efficacy of pembrolizumab for the treatment of HNSCC after long-term follow-up. Multi-centre, non-randomised trial included two HNSCC cohorts (initial and expansion) in which 192 patients were eligible. Patients received pembrolizumab 10 mg/kg every 2 weeks (initial cohort; N = 60) or 200 mg every 3 weeks (expansion cohort; N = 132). Co-primary endpoints were safety and overall response rate (ORR; RECIST v1.1; central imaging vendor review). Median follow-up was 9 months (range, 0.2-32). Treatment-related adverse events (AEs) of any grade and grade 3/4 occurred in 123 (64%) and 24 (13%) patients, respectively. No deaths were attributed to treatment-related AEs. ORR was 18% (34/192; 95% CI, 13-24%). Median response duration was not reached (range, 2+ to 30+ months); 85% of responses lasted ≥6 months. Overall survival at 12 months was 38%. Some patients received 2 years of treatment and the responses were ongoing for more than 30 months; the durable anti-tumour activity and tolerable safety profile, observed with long-term follow-up, support the use of pembrolizumab as a treatment for recurrent/metastatic HNSCC.54,0PD-L1 expression status during screening was determined using a prototype PD-L1 immunohistochemical assay 18 performed at a laboratory site (QualTek) accredited by the College of American Pathologists and Clinical Laboratory Improvement Amendments, and used ;oropharynx were classified as non-HPV-associated disease. PD-L1 expression status during screening was determined using a prototype PD-L1 immunohistochemical assay18 performed at a laboratory site (QualTek) accredited by the College of American Pathologists and Clinical Laboratory Improvement Amendments, and used commercially available reagents from the EnVision FLEX+ HRP-Polymer kit (DAKO K8012https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048158QualTek
2018Wein, L;Luen, SJ;Savas, P;Salgado, R;Loi, S;Checkpoint blockade in the treatment of breast cancer: current status and future directionsBr. J. Cancer4-11119129808015There is now accumulating evidence that the host immune system plays an important role in influencing response to treatment and prognosis in breast cancer. Immunotherapy with immune checkpoint inhibitors is a promising and rapidly growing field of interest in many solid tumours, including breast cancer. Trials to date have largely focused on metastatic triple-negative disease, a genomically unstable subtype of breast cancer that is believed to be the most immunogenic and following the development of treatment resistance, has limited treatment options and a particularly poor prognosis. Both checkpoint inhibitor monotherapy and combinations with chemotherapy are being investigated. In this review, we discuss the current evidence for PD-1/PD-L1 blockade in metastatic triple-negative breast cancer (TNBC), HER2+ breast cancer and ER+ disease, as well as the emerging evidence for use in the early-stage (neoadjuvant) setting. We also propose potential ways of improving responses to checkpoint blockade in breast cancer.54,0Patients were required to be PD-L1 positive by immunohistochemistry (IHC), which was defined as PD-L1 positivity in the stroma or ≥1% tumour cells using their Qualtek assay.28 Most patients were heavily pre-treated; the median number of previous lines of systemic therapy ;stric cancer and head and neck cancer. Patients were required to be PD-L1 positive by immunohistochemistry (IHC), which was defined as PD-L1 positivity in the stroma or ≥1% tumour cells using their Qualtek assay.28 Most patients were heavily pre-treated; the median number of previous lines of systemic therapy for metastatic disease was two, with 46.9% of patients having received at least three lhttps://www.nature.com/articles/s41416-018-0126-6.pdf?origin=ppubQualTek
2003Wang, T;Fan, L;Watanabe, Y;McNeill, PD;Moulton, GG;Bangur, C;Fanger, GR;Okada, M;Inoue, Y;Persing, DH;Reed, SG;L523S, an RNA-binding protein as a potential therapeutic target for lung cancerBr. J. Cancer887-89488612644826Approaches to vaccine-based immunotherapy of human cancer may ultimately require targets that are both tumour-specific and immunogenic. In order to generate specific antitumour immune responses to lung cancer, we have sought lung cancer-specific proteins that can be targeted for adjuvant vaccine therapy. By using a combination of cDNA subtraction and microarray analysis, we previously reported the identification of an RNA-binding protein within the KOC family, L523S, to be overexpressed in squamous cell cancers of the lung. We show here that L523S exhibits significant potential for vaccine immunotherapy of lung cancer. As an oncofetal protein, L523S is normally expressed in early embryonic tissues, yet it is re-expressed in a high percentage of nonsmall cell lung carcinoma. The specificity of L523S expression in lung cancer was demonstrated by both mRNA and protein measurements using real-time PCR, Western blot, and immunohistochemistry analyses. Furthermore, we show that immunological tolerance of L523S is naturally broken in lung cancer patients, as evidenced by detectable antibody responses to recombinant L523S protein in eight of 17 lung pleural effusions from lung cancer patients. Collectively, our studies suggest that L523S may be an important marker of malignant progression in human lung cancer, and further suggest that treatment approaches based on L523S as an immunogenic target are worthy of pursuit.54,0ur studies indicate that L523S may be a valuable addition to the repertoire of cancer-specific targets for the development of new immunotherapeutic and perhaps other therapeutic approaches. We thank Qualtek for their expert IHC analysis. We are grateful to Dr Elizabeth Repasky for providing us with some of the lung cancer and normal tissues. Brinckerhoff LH Thompson LW Slingluff CL Jr Melanoma v;ur studies indicate that L523S may be a valuable addition to the repertoire of cancer-specific targets for the development of new immunotherapeutic and perhaps other therapeutic approaches. We thank Qualtek for their expert IHC analysis. We are grateful to Dr Elizabeth Repasky for providing us with some of the lung cancer and normal tissues. Brinckerhoff LH Thompson LW Slingluff CL Jr Melanoma vhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2377073QualTek
2020Schruf, E;Schroeder, V;Le, HQ;Schönberger, T;Raedel, D;Stewart, EL;Fundel-Clemens, K;Bluhmki, T;Weigle, S;Schuler, M;Thomas, MJ;Heilker, R;Webster, MJ;Dass, M;Frick, M;Stierstorfer, B;Quast, K;Garnett, JP;Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC-derived air-liquid interface modelFASEB J.32297676Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature, providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro. © 2020 Boehringer Ingelheim Pharma GmbH & Co. KG. The FASEB journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.54,0Formalin-fixed and paraffin embedded lung samples from human IPF patients were purchased from Folio Biosciences (Powell, OH, US) under the regulatory conditions of the Boehringer Ingelheim corporate policy regarding the acquisition and use of human biospecimen. Samples were reviewed internally by a trained pathologist and the initial diagnosis of IPF was confirmedhttps://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fj.201902926R"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2020Kang, J;Yeom, G;Jang, H;Park, C;Kim, M;Highly sensitive and universal detection strategy based on a colorimetric assay using target-specific heterogeneous sandwich DNA aptamerAnalytica Chimica ActaA simple, universal, and sensitive colorimetric biosensor for detecting of various biomarkers was devised using a target-specific DNA aptamer, as the recognition element, and engineered with streptavidin-fusion replication protein A 70kDa (RPA70A) linked to biotin-horseradish peroxidase, as the colorimetric element. To improve sensitivity and stability compared to other colorimetric detection techniques, we developed a novel detection strategy by integrating a newly selected heterogeneous sandwich DNA aptamer and protein engineering in this study. The proposed assay is based on a change in color from colorless to blue due to the interaction of the aptamer with RPA70A in the presence of the target; this color change could be observed by the naked eye or measured with a UV-vis spectrometer. We confirmed its high sensitivity and specificity for two model targets using their aptamers under optimal experimental conditions. In addition, the feasibility of the assay was tested in clinical samples containing NPs of influenza A or B virus. These results suggest that our detection system developed herein can be universally applied to the diagnosis of various diseases owing to its stability, sensitivity, and specificity.53,0All reagent concentrations were the same as those analyzed in the developed method. 2.8. Evaluation of clinical samples. We obtained patient samples containing NPs of influenza A and B viruses from Discovery Life Sciences, Inc (Huntsville, AL, USA)https://www.sciencedirect.com/science/article/pii/S0003267020305250"Discovery Life Sciences Inc"
2018Xu, Z;Wang, S;Li, Y;Zhu, F;Huang, J;PRIM: An Efficient Preconditioning Iterative Reweighted Least Squares Method for Parallel Brain MRI ReconstructionNeuroinformatics425-430163-429423650The most recent history of parallel Magnetic Resonance Imaging (pMRI) has in large part been devoted to finding ways to reduce acquisition time. While joint total variation (JTV) regularized model has been demonstrated as a powerful tool in increasing sampling speed for pMRI, however, the major bottleneck is the inefficiency of the optimization method. While all present state-of-the-art optimizations for the JTV model could only reach a sublinear convergence rate, in this paper, we squeeze the performance by proposing a linear-convergent optimization method for the JTV model. The proposed method is based on the Iterative Reweighted Least Squares algorithm. Due to the complexity of the tangled JTV objective, we design a novel preconditioner to further accelerate the proposed method. Extensive experiments demonstrate the superior performance of the proposed algorithm for pMRI regarding both accuracy and efficiency compared with state-of-the-art methods.51,0International Holdings Inc. Albin, Mary, Westinghouse Alexander, William, Qualtek Manufacturing, Inc. Alexander, Chuck, Solid Concepts, Inc. Alexopoulos, Nick, Broadcom Corporation Alford, Charles, UES, Inc. Alvarado, Igorhttps://peer.asee.org/the-proposed-design-national-network-for-manufacturing-innovation.pdfQualTek
2020Seyger, M;Abramovits, W;Liljedahl, M;Hoejen, MN;Teng, J;Safety and efficacy of fixed-dose combination calcipotriol (50 μg/g) and betamethasone dipropionate (0.5 mg/g) cutaneous foam in adolescent patients (aged 12 to <17 years) with plaque psoriasis: results of a phase II, open-label trialJ Eur Acad Dermatol Venereol32074665Fixed-dose combination of calcipotriol (50 μg/g; Cal) and betamethasone dipropionate (0.5 mg/g; BD) foam is approved for plaque psoriasis treatment in adults, with a paucity of data supporting use in adolescents. To evaluate safety of 4 weeks' treatment with Cal/BD foam in adolescent patients with psoriasis, and additional safety outcomes in patients with more severe disease (HPA-axis cohort). Primary objectives included treatment-emergent adverse events (TEAEs) and systemic calcium levels in the overall population, and HPA-axis function, change in calcium excretion and the calcium:creatinine ratio in the HPA-axis cohort. Secondary objectives included exploratory efficacy endpoints [treatment success: change in Psoriasis Area and Severity Index (PASI)]. Systemic exposure to Cal/BD was also assessed. A phase II, open-label, study (NCT02387853) in patients (12 to <17 years) with at least mild psoriasis, to evaluate Cal/BD foam applied once daily for ≤4 weeks. In patients assigned to treatment (n = 106), 32 TEAEs occurred in 22 patients (20.8%). All but two TEAEs were mild; none led to study withdrawal or death. Changes (0-4 weeks) in albumin-corrected serum calcium (overall population) and urinary calcium excretion (HPA-axis cohort) were small, transient and not considered clinically relevant. In the HPA-axis cohort, no change in urinary calcium:creatinine ratio was observed and responses to adrenocorticotropic-hormone (ACTH) challenge did not suggest disruption of the HPA-axis. Prespecified treatment success on the body and scalp was achieved by 71.8% and 75.7% of the overall population, respectively. Mean PASI decreased by 82.0% vs. baseline at Week 4. Systemic exposure to Cal/BD was minimal. Cal/BD foam was well tolerated in adolescent patients with body/scalp psoriasis. There was no evidence for dysregulation of the HPA-axis nor calcium homoeostasis in patients with more severe disease. Exploratory efficacy data in the overall population were encouraging. © 2020 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.51,0W. Abramovits has received honoraria or fees for serving on advisory boards as a speaker and as a consultant, and has received grants as an investigator from AbbVie, Akros, Allergan, Amgen, Anacor Pharm, Aqua Pharma, Celgene, Centocor, Conversant Bio, Dermavant, Elihttps://onlinelibrary.wiley.com/doi/abs/10.1111/jdv.16233"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2020Piha-Paul, SA;Oh, DY;Ueno, M;Malka, D;Chung, HC;Nagrial, A;Kelley, RK;Ros, W;Italiano, A;Nakagawa, K;Rugo, HS;de Braud, F;Varga, AI;Hansen, A;Wang, H;Krishnan, S;Norwood, KG;Doi, T;Efficacy and safety of pembrolizumab for the treatment of advanced biliary cancer: Results from the KEYNOTE-158 and KEYNOTE-028 studiesInt. J. Cancer32359091We present data from patients with advanced biliary tract cancer (BTC) receiving pembrolizumab in the KEYNOTE-158 (NCT02628067; phase 2) and KEYNOTE-028 (NCT02054806; phase 1b) studies. Eligible patients aged ≥18 years from both studies had histologically/cytologically confirmed incurable BTC that progressed after standard treatment regimen(s), measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, Eastern Cooperative Oncology Group performance status 0/1, and no prior immunotherapy. Programmed death ligand 1 (PD-L1)-positive tumors were required for eligibility in KEYNOTE-028 only. Patients received pembrolizumab 200 mg every three weeks (KEYNOTE-158) or 10 mg/kg every two weeks (KEYNOTE-028) for ≤2 years. Primary efficacy endpoint was objective response rate (ORR) by RECIST v1.1. Response assessed by independent central review is reported. KEYNOTE-158 enrolled 104 patients and KEYNOTE-028 enrolled 24 patients. Median (range) follow-up was 7.5 months (0.6-34.3) in KEYNOTE-158 and 5.7 months (0.6-55.4) in KEYNOTE-028. In KEYNOTE-158, ORR was 5.8% (6/104; 95% CI, 2.1%-12.1%); median duration of response (DOR) was not reached (NR) (range, 6.2-26.6+ months). Median (95% CI) OS and PFS were 7.4 (5.5-9.6) and 2.0 (1.9-2.1) months. Among PD-L1-expressers (n = 61) and PD-L1-nonexpressers (n = 34), respectively, ORR was 6.6% (4/61) and 2.9% (1/34). In KEYNOTE-028, ORR was 13.0% (3/23; 95% CI, 2.8%-33.6%); median DOR was NR (range, 21.5-53.2+ months). Median (95% CI) OS and PFS were 5.7 (3.1-9.8) and 1.8 (1.4-3.1) months. Grade 3 to 5 treatment-related adverse events occurred in 13.5% of patients in KEYNOTE-158 (no grade 4; grade 5 renal failure, n = 1) and 16.7% in KEYNOTE-028 (no grade 4/5). In summary, pembrolizumab provides durable antitumor activity in 6% to 13% of patients with advanced BTC, regardless of PD-L1 expression, and has manageable toxicity. © 2020 UICC.50,0In KEYNOTE‐028, a prototype immunohistochemistry assay (QualTek, Goleta, California) was used to prospectively detect PD‐L1 expression, with positivity defined as membranous PD‐L1 expression in ≥1% of tumor and associated inflammatory cells or positive staining inhttps://onlinelibrary.wiley.com/doi/abs/10.1002/ijc.33013QualTek
2009Fazzone, W;Wilson, PM;Labonte, MJ;Lenz, HJ;Ladner, RD;Histone deacetylase inhibitors suppress thymidylate synthase gene expression and synergize with the fluoropyrimidines in colon cancer cellsInt. J. Cancer463-473125219384949Despite recent therapeutic advances, the response rates to chemotherapy for patients with metastatic colon cancer remain at approximately 50% with the fluoropyrimidine, 5-fluorouracil (5-FU), continuing to serve as the foundation chemotherapeutic agent for the treatment of this disease. Previous studies have demonstrated that overexpression of thymidylate synthase (TS) is a key determinant of resistance to 5-FU-based chemotherapy. Therefore, there is a significant need to develop alternative therapeutic strategies to overcome TS-mediated resistance. In this study, we demonstrate that the histone deacetylase inhibitors (HDACi) vorinostat and LBH589 significantly downregulate TS gene expression in a panel of colon cancer cell lines. Downregulation of TS was independent of p53, p21 and HDAC2 expression and was achievable in vivo as demonstrated by mouse xenograft models. We provide evidence that HDACi treatment leads to a potent transcriptional repression of the TS gene. Combination of the fluoropyrimidines 5-FU or FUdR with both vorinostat and LBH589 enhanced cell cycle arrest and growth inhibition. Importantly, the downstream effects of TS inhibition were significantly enhanced by this combination including the inhibition of acute TS induction and the enhanced accumulation of the cytotoxic nucleotide intermediate dUTP. These data demonstrate that HDACi repress TS expression at the level of transcription and provides the first evidence suggesting a direct mechanistic link between TS downregulation and the synergistic interaction observed between HDACi and 5-FU. This study provides rationale for the continued clinical evaluation of HDACi in combination with 5-FU-based therapies as a strategy to overcome TS-mediated resistance. Copyright 2009 UICC.50,0liquid nitrogen or fixed in 10% formalin. Formalin‐fixed tumor specimens were paraffin‐embedded and subsequently analyzed by immunohistochemistry (IHC) (QualTek Laboratories, Newtown, PA). IHC for TS was conducted ashttps://onlinelibrary.wiley.com/doi/abs/10.1002/ijc.24403QualTek
2020Agnello, G;Alters, SE;Rowlinson, SW;Preclinical safety and antitumor activity of the arginine-degrading therapeutic enzyme pegzilarginase, a PEGylated, cobalt-substituted recombinant human arginase 1Transl Res11-2221731954097Metabolic remodeling contributes to the development and progression of some cancers and exposes them to vulnerabilities, including specific nutrient dependencies that can be targeted therapeutically. Arginine is a semiessential amino acid, and several cancers are unable to endogenously synthesize sufficient levels of arginine for survival and proliferation, most commonly due to reduced expression of argininosuccinate synthase (ASS1). Such cancers are dependent on arginine and they can be targeted via enzyme-mediated depletion of systemic arginine. We report the preclinical safety, antitumor efficacy, and immune-potentiating effects of pegzilarginase, a highly potent human arginine-degrading enzyme. Toxicology studies showed that pegzilarginase-mediated arginine depletion is well tolerated at therapeutic levels that elicit an antitumor growth effect. To determine which tumor types are best suited for clinical development, we profiled clinical tumor samples for ASS1 expression, which correlated with pegzilarginase sensitivity in vivo in patient-derived xenograft (PDx) models. Among the histologies tested, malignant melanoma, small cell lung cancer and Merkel cell carcinoma had the highest prevalence of low ASS1 expression, the highest proportion of PDx models responding to pegzilarginase, and the strongest correlation between low or no ASS1 expression and sensitivity to pegzilarginase. In an immune-competent syngeneic mouse model, pegzilarginase slowed tumor growth and promoted the recruitment of CD8+ tumor infiltrating lymphocytes. This is consistent with the known autophagy-inducing effects of arginine depletion, and the link between autophagy and major histocompatibility complex antigen presentation to T cells. Our work supports the ongoing clinical investigations of pegzilarginase in solid tumors and clinical combination of pegzilarginase with immune checkpoint inhibitors. Copyright © 2020 Elsevier Inc. All rights reserved.49,0CD8 IHC analysis of FFPE sections was performed by QualTek Molecular Laboratories (Goleta, Calif) and LC3B IHC analysis of FFPE sections was performed by GoPath Laboratories (Buffalo Grove, Ill). Statistical analyseshttps://www.sciencedirect.com/science/article/pii/S1931524419302592QualTek
2007Senzer, N;Nemunaitis, J;Nemunaitis, M;Lamont, J;Gore, M;Gabra, H;Eeles, R;Sodha, N;Lynch, FJ;Zumstein, LA;Menander, KB;Sobol, RE;Chada, S;p53 therapy in a patient with Li-Fraumeni syndromeMol. Cancer Ther.1478-14826517483435Li-Fraumeni syndrome is an autosomal dominant disorder that greatly increases the risk of developing multiple types of cancer. The majority of Li-Fraumeni syndrome families contain germ-line mutations in the p53 tumor suppressor gene. We describe treatment of a refractory, progressive Li-Fraumeni syndrome embryonal carcinoma with a p53 therapy (Advexin) targeted to the underlying molecular defect of this syndrome. p53 treatment resulted in complete and durable remission of the injected lesion by fluorodeoxyglucose-positron emission tomography scans with improvement of tumor-related symptoms. With respect to molecular markers, the patient's tumor had abnormal p53 and expressed coxsackie adenovirus receptors with a low HDM2 and bcl-2 profile conducive for adenoviral p53 activity. p53 treatment resulted in the induction of cell cycle arrest and apoptosis documented by p21 and cleaved caspase-3 detection. Increased adenoviral antibody titers after repeated therapy did not inhibit adenoviral p53 activity or result in pathologic sequelae. Relationships between these clinical, radiographic, and molecular markers may prove useful in guiding future application of p53 tumor suppressor therapy.49,0http://dx.doi.org/10.1158/1535-7163.MCT-07-0125Frank Lynch[au]
2006Evans, EE;Henn, AD;Jonason, A;Paris, MJ;Schiffhauer, LM;Borrello, MA;Smith, ES;Sahasrabudhe, DM;Zauderer, M;C35 (C17orf37) is a novel tumor biomarker abundantly expressed in breast cancerMol. Cancer Ther.2919-293051117121940Identification of shared tumor-specific targets is useful in developing broadly applicable therapies. In a study designed to identify genes up-regulated in breast cancer, a cDNA clone corresponding to a novel gene C35 (C17orf37) was selected by representational difference analysis of tumor and normal human mammary cell lines. Abundant expression of C35 transcript in tumors was confirmed by Northern blot and real-time PCR. The C35 gene is located on chromosome 17q12, 505 nucleotides from the 3' end of the ERBB2 oncogene, the antigenic target for trastuzumab (Herceptin) therapy. The chromosomal arrangement of the genes encoding C35 and ERBB2 is tail to tail. An open reading frame encodes a 12-kDa protein of unknown function. Immunohistochemical analysis detected robust and frequent expression of C35 protein, including 32% of grade 1 and 66% of grades 2 and 3 infiltrating ductal carcinomas of the breast (in contrast to 20% overexpressing HER-2/neu), 38% of infiltrating lobular carcinoma (typically HER-2/neu negative), as well as tumors arising in other tissues. C35 was not detected in 38 different normal human tissues, except Leydig cells in the testes and trace levels in a small percentage of normal breast tissue samples. The distinct and favorable expression profile of C35 spanning early through late stages of disease, including high frequency of overexpression in various breast carcinoma, abundant expression in distant metastases, and either absence or low level expression in normal human tissues, warrants further investigation of the relevance of C35 as a biomarker and/or a target for development of broadly applicable cancer-specific therapies.49,0Staining of formalin-fixed, paraffin-embedded breast carcinoma and normal tissue sections was done in part by QualTek Molecular Labs (Santa Barbara, CA). Ten lobular and 50 invasive ductal (grades 1, 2, and 3) carcinomas were selected from the QualTek tissue bank. Whenever possible, specimens were selected to include areas of ductal carcinoma in situ (DCIS) and/or normal-appearing adjacent tissue. Samples ranged in degree of lymph node involvement and in tumor size from 0.5 to 3.5 cm. Patient age range was 46 to 77 years, with a median of 57 years and mean age of 59 years. Specimens were qualified based on grade, pathologist’s assessment of morphologic criteria, and positive immunoreactivity to a positive control antibody specific for Ki-67. Infiltrating ductal breast carcinomas (IDC) were graded according to a modified Scarff-Bloom-Richardson system, according to histologic differentiation (tubule formation), the degree of nuclear atypia, and mitotic count (based on Ki-67 staining). Normal breast samples were derived from both cancer and noncancer biopsies. Samples of other normal human tissueshttps://mct.aacrjournals.org/content/5/11/2919.shortQualTek
2017Xu-Monette, ZY;Zhang, M;Li, J;Young, KH;PD-1/PD-L1 Blockade: Have We Found the Key to Unleash the Antitumor Immune Response?Front Immunol1597829255458PD-1-PD-L1 interaction is known to drive T cell dysfunction, which can be blocked by anti-PD-1/PD-L1 antibodies. However, studies have also shown that the function of the PD-1-PD-L1 axis is affected by the complex immunologic regulation network, and some CD8+ T cells can enter an irreversible dysfunctional state that cannot be rescued by PD-1/PD-L1 blockade. In most advanced cancers, except Hodgkin lymphoma (which has high PD-L1/L2 expression) and melanoma (which has high tumor mutational burden), the objective response rate with anti-PD-1/PD-L1 monotherapy is only ~20%, and immune-related toxicities and hyperprogression can occur in a small subset of patients during PD-1/PD-L1 blockade therapy. The lack of efficacy in up to 80% of patients was not necessarily associated with negative PD-1 and PD-L1 expression, suggesting that the roles of PD-1/PD-L1 in immune suppression and the mechanisms of action of antibodies remain to be better defined. In addition, important immune regulatory mechanisms within or outside of the PD-1/PD-L1 network need to be discovered and targeted to increase the response rate and to reduce the toxicities of immune checkpoint blockade therapies. This paper reviews the major functional and clinical studies of PD-1/PD-L1, including those with discrepancies in the pathologic and biomarker role of PD-1 and PD-L1 and the effectiveness of PD-1/PD-L1 blockade. The goal is to improve understanding of the efficacy of PD-1/PD-L1 blockade immunotherapy, as well as enhance the development of therapeutic strategies to overcome the resistance mechanisms and unleash the antitumor immune response to combat cancer.47,099.5%; 75.6% of patients had a response for ≥6 months Clinical activity was seen across all PD-L1 groups defined by PD-L1 intensity score, tumor-membrane staining score, and histiocyte score (QualTek IHC assay); 90.4% of patients had an intensity score of 3; 88.1% had 100% PD-L1+ membrane staining; 71.8% had a histiocyte score of 3 ------------------------- HNSCChttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723106QualTek
2019Xu, H;Muise, ES;Javaid, S;Chen, L;Cristescu, R;Mansueto, MS;Follmer, N;Cho, J;Kerr, K;Altura, R;Machacek, M;Nicholson, B;Addona, G;Kariv, I;Chen, H;Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia modelJ. Cell. Mol. Med.7063-7077231031449347This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML. © 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.47,02 METHODS. 2.1 Cells and reagents. Normal and AML patient BMMCs were purchased from Folio Conversant and ProteoGenex. Recombinant human fibronectin was purchased from Millipore. Rat tail collagen type 1 and matrigel were purchased from Corning Inc ;2.3 AML patient information. Acute myeloid leukaemia patient bone marrow mononuclear cells (BMMC) were purchased from Conversant Bio and ProteoGenex. A total of 49 AML BMMC samples were tested, and patient information is summarized in Table 1https://onlinelibrary.wiley.com/doi/abs/10.1111/jcmm.14608"Folio Conversant";"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2020Ross, AE;Hurley, PJ;Tran, PT;Rowe, SP;Benzon, B;Neal, TO;Chapman, C;Harb, R;Milman, Y;Trock, BJ;Drake, CG;Antonarakis, ES;A pilot trial of pembrolizumab plus prostatic cryotherapy for men with newly diagnosed oligometastatic hormone-sensitive prostate cancerProstate Cancer Prostatic Dis.184-19323131611635Monotherapy with immune checkpoint inhibitors has generally been unsuccessful in men with advanced prostate cancer. Preclinical data support the notion that cryotherapy may improve immune-mediated and anti-tumor responses. The objective of this study was to assess the safety and feasibility of whole-prostate gland cryotherapy combined with pembrolizumab and androgen deprivation in men with oligometastatic hormone-sensitive prostate cancer. This single-institution, pilot trial recruited 12 patients with newly diagnosed oligometastatic prostate cancer between 2015 and 2016. Patients underwent whole-prostate cryoablation combined with short-term androgen deprivation (eight months) and pembrolizumab (6 doses). The primary clinical endpoints were the number of patients with a PSA level of <0.6 ng/mL at one year and the frequency of adverse events. Other outcome measures included progression-free survival and systemic therapy-free survival. Exploratory analyses included PD-L1 protein expression. Forty two percent (5/12) of patients had a PSAs of <0.6 ng/mL at one year though only 2 of these patients had recovered their testosterone at this time point. Median progression-free survival was 14 months, and median systemic therapy-free survival was 17.5 months. PD-L1 expression was not detectable by IHC in patients with evaluable tissue. All adverse events were grade ≤2, and there were no apparent complications from cryotherapy. Whole-prostate cryoablation combined with short-term androgen deprivation and pembrolizumab treatment was well tolerated and no safety concerns were observed in men with oligometastatic prostate cancer. Though local disease appeared effectively treated in the majority of men, the regimen only infrequency led to sustained disease control following testosterone recovery.46,0pathologist. Post-treatment biopsies were evaluated for PD-L1, CD4, CD8, and FoxP3 proteins by single-stain semiquantitative immunohistochemistry performed by QualTek Molecular and Clinical Laboratories (Santa Barbara, CA) ;re assessed by a genitourinary clinical pathologist. Post-treatment biopsies were evaluated for PD-L1, CD4, CD8, and FoxP3 proteins by single-stain semi-quantitative immunohistochemistry performed by QualTek Molecular and Clinical Laboratories (Santa Barbara, CA). PD-L1 staining was performed using the monoclonal antibody 22C3 (Merck). In the majority of cases, viable tumor was not present on posthttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031012QualTek
2019Kim, YJ;Keam, B;Ock, CY;Song, S;Kim, M;Kim, SH;Kim, KH;Kim, JS;Kim, TM;Kim, DW;Lee, JS;Heo, DS;A phase II study of pembrolizumab and paclitaxel in patients with relapsed or refractory small-cell lung cancerLung Cancer122-12813631494530Patients with etoposide/platinum-refractory extensive disease (ED) small-cell lung cancer (SCLC) have a dismal prognosis. We aimed to evaluate the efficacy and safety of pembrolizumab and paclitaxel combination therapy in these patients. In this multi-center, phase II study, ED-SCLC patients who showed progression after etoposide/platinum chemotherapy received paclitaxel 175 mg/m2 every 3 weeks for up to six cycles. Pembrolizumab 200 mg was added from the second cycle and continued until disease progression or unacceptable toxicity. The primary endpoint was the objective response rate (ORR) and the secondary endpoints were progression-free survival (PFS), overall survival (OS), safety, and biomarker analyses including programmed death-ligand 1 (PD-L1) expression, next-generation sequencing, and flow cytometric analysis of peripheral blood cells. Of the 26 patients enrolled, the confirmed ORR was 23.1% (95%CI: 6.9%-39.3%); complete response: 3.9%, confirmed partial response [PR]: 19.2%, stable disease: 57.7%, progressive disease: 7.7%, and not evaluable: 11.5%. Including 4 cases of unconfirmed PRs, 38.5% of patients were responding and the disease control rate was 80.7%. The median PFS and OS were 5.0 months (95% CI: 2.7-6.7) and 9.1 months (95% CI: 6.5-15.0), respectively. The grade 3 or 4 adverse events observed included febrile neutropenia (7.7%), neutropenia (7.7%), asthenia (7.7%), hyponatremia (7.7%), and type I diabetes (7.7%). Targeted gene sequencing identified no specific genetic alterations correlated with the treatment, except for theMET copy number gain (PFS 10.5 versus 3.4 months, p = 0.019). Pembrolizumab and paclitaxel combination therapy showed a moderate activity with acceptable toxicity in patients with refractory ED-SCLC. Copyright © 2019 Elsevier B.V. All rights reserved.46,0Informed consent for specimens be obtained during screening for protocol enrollment from all subjects or legally acceptable representative, at a trial visit by the investigator or designate. Pretreatment blood sampling and tumor biopsies will be done. After one cycle of paclitaxel, blood sampling and tumor biopsies will be performed for translational research by investigator's discretion. Archival tissue obtained from the patients before the administration of study drug may also be used. Biomarker assessment before the administration of study drug will be performed at Seoul National University (SNU) Cancer Research Institute and QualTek, and later assessment will be performed at SNU Cancer Research Institute. Results of genetic test will be used for research purpose only, personal information will be protected. All specimens will be destroyed after storage for 5 years. Subjects may withdraw their consent and have their specimens and all derivatives destroyedhttps://www.clinicaltrials.gov/ProvidedDocs/32/NCT02551432/Prot_SAP_000.pdfQualTek
2019Yang, X;Saito, Y;Rao, A;Kim, HJ;Singh, P;Scott, E;Larson, M;Pan, W;Desai, M;Hubbell, E;Alignment-free filtering for cfNA fusion fragmentsBioinformaticsi225-i232351431510681Cell-free nucleic acid (cfNA) sequencing data require improvements to existing fusion detection methods along multiple axes: high depth of sequencing, low allele fractions, short fragment lengths and specialized barcodes, such as unique molecular identifiers. AF4 was developed to address these challenges. It uses a novel alignment-free kmer-based method to detect candidate fusion fragments with high sensitivity and orders of magnitude faster than existing tools. Candidate fragments are then filtered using a max-cover criterion that significantly reduces spurious matches while retaining authentic fusion fragments. This efficient first stage reduces the data sufficiently that commonly used criteria can process the remaining information, or sophisticated filtering policies that may not scale to the raw reads can be used. AF4 provides both targeted and de novo fusion detection modes. We demonstrate both modes in benchmark simulated and real RNA-seq data as well as clinical and cell-line cfNA data. AF4 is open sourced, licensed under Apache License 2.0, and is available at: https://github.com/grailbio/bio/tree/master/fusion. © The Author(s) 2019. Published by Oxford University Press.45,0We have obtained 40 ml of whole blood collected in Streck tubes, separated into plasma and extracted cfRNA samples from two Stage IV Prostate cancer patients and three healthy controls from Conversant Bio and generated cfRNA sequencing data with UMIs added. For the two cancer patients, it has been determined that TMPRSS2-ETV4 and TMPRSS2-ERG fusion were the most likely real fusion events, because TMPRSS2 fusion are detected in 50% of prostate cancer (Tomlins et al., 2008). The three healthy individuals had a primary diagnosis of normal, and therefore, should contain no real fusion events and serve as negative controlshttps://academic.oup.com/bioinformatics/article-abstract/35/14/i225/5529228"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2010Teichert, A;Elalieh, H;Bikle, D;Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout miceJ. Cell. Physiol.482-489225220458748Mice null for the Vitamin D receptor (VdrKO) have a disrupted first hair follicle cycle and aborted subsequent hair follicle cycling. We examined the expression of different markers and mediators of hair follicle cycling in the hair follicle of the VdrKO mouse during days 13-22 when the hair follicle normally initiates and completes the first catagen. We compared the expression of those genes in mice with a nonsense mutation in hairless (Rhino), which have a similar alopecia phenotype, and to Cyp27b1 null mice which are deficient in the production of 1,25(OH)2D3, the Vdr ligand, but display normal hair follicle cycling. Our results demonstrate the down regulation of hair follicle markers and the alteration of expression of hedgehog (Hh), Wnt, Fgf, and Tgfbeta pathways in VdrKO and Rhino mice, but not in Cyp27b1KO mice. Treatment of VdrKO mice with an agonist to the Hh pathway partially restored hair follicle cycling, suggesting a role of this pathway in the regulation of hair follicle cycling by VDR. These results suggest that Vdr regulates directly or indirectly the expression of genes required for hair follicle cycling, including Hh signaling, independent of 1,25(OH)2D3. (c) 2010 Wiley-Liss, Inc.45,0The binding of the antibodies was detected by biotinylated goat anti‐rabbit IgG, followed by ABC‐peroxidase reagent (Vector, Burlingame, CA) and revealed with diaminobenzidine substrate (QualTek Laboratories, Santa Barbara, CA) followed by hematoxylin counterstaininghttps://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.22227QualTek
2020Sebest, P;Fojt, L;Ostatna, V;Fojta, M;Danhel, A;Electrodeposited silver amalgam particles on pyrolytic graphite in (spectro)electrochemical detection of 4-nitrophenol, DNA and green fluorescent proteinBioelectrochemistry10743613231855832Catalytic properties and high adsorption affinity of nucleic acids and proteins to silver amalgam electrode surface make this kind of electrified interface perspective for bioanalytical and biomedical applications. For the first time, a basal-plane pyrolytic graphite electrode (bPGE) has been used as a substrate for electrodeposition of silver amalgam particles (AgAPs). Optimization of the resulting composition, surface morphology and electrochemical properties of the AgAPs was done by scanning electron microscopy with energy disperse X-ray spectroscopy, image processing software and voltammetric detection of electrochemically reducible model organic nitro compound, 4-nitrophenol. Spectro-electrochemical applicability of bPGE-AgAP has been demonstrated by electrolysis of 4-nitrophenol. Simultaneous UV-Vis-chronoamperometry provided information on the number of exchange electrons and the reduction rate constants. Preferential adsorption of the fluorescently labelled calf thymus DNA and the green fluorescent protein (GFP) on the surface of AgAPs was observed by fluorescence microscopy. In contrast to previously studied indium-tin oxide and vapour-deposited gold decorated by AgAPs, herein the presented bPGE-AgAP has provided sufficiently wide negative potential window allowing direct electroanalysis of non-labelled DNA and GFP using intrinsic electrochemical signals independently of the fluorescent labelling. The bPGE-AgAP can thus be expected to find application opportunities in protein electrochemistry, (bio)sensor development or in-situ spectro-electrochemical studies. Copyright © 2019 Elsevier B.V. All rights reserved.45,0electrode setup composed of: i) a bPGE or edge-plane PGE (ePGE, both Momentive) working electrode, together with ii) a refillable miniature Ag/AgCl/3M KCl reference electrode (eDAQ) placed into the salt bridge composed of a heat shrink Teflon tube (Qualtek) enclosed byhttps://www.sciencedirect.com/science/article/pii/S1567539419304098QualTek
2019Qi, Z;Wang, L;Desai, K;Cogswell, J;Stern, M;Lawson, B;Kerkar, SP;Vitazka, P;Reliable Gene Expression Profiling from Small and Hematoxylin and Eosin-Stained Clinical Formalin-Fixed, Paraffin-Embedded Specimens Using the HTG EdgeSeq PlatformJ Mol Diagn796-80721531255795Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (H&E)-stained, and immunohistochemistry-stained slides from patients with small-cell and non-small-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with H&E staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-γ signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with H&E-stained slides and a significant Spearman correlation (P = 0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm2 of a section (5 μm thick); sample equivalents ≥0.31 mm2 showed alignment rates >69% and pairwise Pearson correlation coefficients ≥0.87. EdgeSeq can, thus, be used to profile small and H&E-stained FFPE tumor specimens to obtain biomarker data from limited tissue in oncology clinical trials and enable research into tumor microenvironment and immune cell engagement with tumors at the locoregional level. Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.44,0Rockville, MD). Small-cell lung cancer (SCLC) samples (n = 4) were purchased from Conversant Biologics, Inc. (Huntsville, AL) or Proteogenex (Inglewood, CA). FFPE muscle tissue was obtained from Proteogenex. RNA sampleshttps://www.sciencedirect.com/science/article/pii/S1525157818303805"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2019Varga, A;Piha-Paul, S;Ott, PA;Mehnert, JM;Berton-Rigaud, D;Morosky, A;Yang, P;Ruman, J;Matei, D;Pembrolizumab in patients with programmed death ligand 1-positive advanced ovarian cancer: Analysis of KEYNOTE-028Gynecol. Oncol.243-250152230522700To evaluate safety, tolerability, and antitumor activity of pembrolizumab monotherapy in patients with programmed death ligand 1 (PD-L1)-expressing advanced ovarian cancer enrolled in the multicohort, phase Ib KEYNOTE-028 trial. Key inclusion criteria were age ≥18 years; advanced ovarian epithelial, fallopian tube, or primary peritoneal carcinoma; failure of previous therapy; and tumor PD-L1 positivity. Patients received pembrolizumab (10 mg/kg every 2 weeks) for ≤24 months or until disease progression/intolerable toxicity. Tumor response was assessed per RECIST v1.1 (investigator review). Adverse events (AEs) were graded using CTCAE version 4.0. Primary end point was confirmed objective response rate (ORR) per RECIST v1.1 (investigator review); data cutoff date was February 20, 2017. Twenty-six patients (median age, 57.5 years) with PD-L1-positive advanced metastatic ovarian cancer received pembrolizumab; 38.5% had metastatic disease, and 73.1% previously received ≥3 lines of therapy. Treatment-related AEs (TRAEs) occurred in 19 (73.1%) patients, most commonly arthralgia (19.2%), nausea (15.4%), and pruritus (15.4%). One grade 3 TRAE (increased plasma transaminase level) occurred. No deaths and no treatment discontinuations due to TRAEs occurred. After a median follow-up duration of 15.4 months, ORR was 11.5% (1 complete response, 2 partial responses); 7 patients (26.9%) achieved stable disease. Median progression-free and overall survival were 1.9 (95% CI, 1.8-3.5) and 13.8 (95% CI, 6.7-18.8) months, respectively. Pembrolizumab conferred durable antitumor activity with manageable safety and toxicity in patients with advanced PD-L1-positive ovarian cancer and is under further investigation in an ongoing phase II trial, KEYNOTE-100. Copyright © 2018. Published by Elsevier Inc.44,0disease based on Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1), PD-L1 positivity, determined immunohistochemically and defined as membranous staining on ≥1% modified proportion score or interface pattern (QualTek Molecular Laboratorieshttps://www.sciencedirect.com/science/article/pii/S0090825818314185QualTek
2020Grigorieva, J;Asmellash, S;Net, L;Tsypin, M;Roder, H;Roder, J;Mass Spectrometry-Based Multivariate Proteomic Tests for Prediction of Outcomes on Immune Checkpoint Blockade Therapy: The Modern Analytical ApproachInt J Mol Sci21332012941The remarkable success of immune checkpoint inhibitors (ICIs) has given hope of cure for some patients with advanced cancer; however, the fraction of responding patients is 15-35%, depending on tumor type, and the proportion of durable responses is even smaller. Identification of biomarkers with strong predictive potential remains a priority. Until now most of the efforts were focused on biomarkers associated with the assumed mechanism of action of ICIs, such as levels of expression of programmed death-ligand 1 (PD-L1) and mutation load in tumor tissue, as a proxy of immunogenicity; however, their performance is unsatisfactory. Several assays designed to capture the complexity of the disease by measuring the immune response in tumor microenvironment show promise but still need validation in independent studies. The circulating proteome contains an additional layer of information characterizing tumor-host interactions that can be integrated into multivariate tests using modern machine learning techniques. Here we describe several validated serum-based proteomic tests and their utility in the context of ICIs. We discuss test performances, demonstrate their independence from currently used biomarkers, and discuss various aspects of associated biological mechanisms. We propose that serum-based multivariate proteomic tests add a missing piece to the puzzle of predicting benefit from ICIs.42,050] PSEA reference set NSCLC patients; samples obtained from commercial biobanks Conversant Bio (Huntsville, AL) and Oncology Metrix (Fort Worth, TX) 100 Grigorieva et al., 2019 [51] 3. BDX008 Test and ICB Test BDX008https://www.mdpi.com/1422-0067/21/3/838"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2006Costello, C;Ward, M;Search, bioprospecting and biodiversity conservationJournal of Environmental Economics and Management615-62652342,0in a particular way. When executed in bulk, these tests cost between $4000 and $18,000 per species tested (eg Qualtek Molecular Laboratories, Santa Barbara, CA). We use a range for c of $4000-$18,000. • Discount rate (rhttps://www.sciencedirect.com/science/article/pii/S009506960600060XQualTek
2016Dolled-Filhart, M;Locke, D;Murphy, T;Lynch, F;Yearley, JH;Frisman, D;Pierce, R;Weiner, R;Wu, D;Emancipator, K;Development of a Prototype Immunohistochemistry Assay to Measure Programmed Death Ligand-1 Expression in Tumor TissueArch. Pathol. Lab. Med.1259-12661401127788043- With the abundance of therapeutics targeted against programmed death receptor-1 and its ligand (PD-L1) that are currently approved or in clinical development, there is interest in identifying those patients most likely to respond to these drugs. Expression of PD-L1 may be an indicator of an initial and robust inflammatory response to the presence of tumor cells. Therefore, tumors that express PD-L1 may be the most likely to respond to therapies that interrupt the negative feedback mechanism that leads to PD-L1 upregulation. - To develop a prototype immunohistochemistry assay using the anti-PD-L1 antibody clone 22C3. - The assay was developed and optimized using commercially available reagents and archival tumor-bank tissue. - The optimized immunohistochemistry method had high precision and reproducibility. Using the prototype assay in 142 non-small cell lung cancer and 79 melanoma archival tumor-bank tissue samples, PD-L1 staining was observed at the plasma membrane of nucleated tumor and nontumor cells and, in some cases, as a distinct lichenoid pattern at the tumor-stroma border. Using a preliminary scoring method, 56% (80 of 142) of non-small cell lung cancer and 53% (42 of 79) of melanoma samples were defined as PD-L1+ based on a modified H-score of 1 or more or the presence of a distinctive staining pattern at the tumor-stroma interface. - The immunohistochemistry assay using the anti-PD-L1 antibody 22C3 merits further investigation in clinical trials and prevalence assessments to further understand the prognostic and predictive value of PD-L1 expression in cancer.42,0Merck & Co, Inc, Kenilworth, New Jersey; the Departments of Early Clinical & Translational Research, Clinical Histochemistry (Dr Locke), Research and Clinical Client Services (Dr Murphy), and Operations and Client Services (Dr Lynch), QualTek Molecular Laboratorieshttp://www.archivesofpathology.org/doi/abs/10.5858/arpa.2015-0544-OAQualTek;Tiffany Murphy[au];Frank Lynch[au]
2016Dolled-Filhart, M;Roach, C;Toland, G;Stanforth, D;Jansson, M;Lubiniecki, GM;Ponto, G;Emancipator, K;Development of a Companion Diagnostic for Pembrolizumab in Non-Small Cell Lung Cancer Using Immunohistochemistry for Programmed Death Ligand-1Arch. Pathol. Lab. Med.1243-12491401127552095Context .- Programmed death ligand-1 (PD-L1) expression by tumors may enable them to avoid immunosurveillance. Objective .- To develop a PD-L1 immunohistochemical assay using the 22C3 anti-PD-L1 murine monoclonal antibody on the Dako platform as a possible companion diagnostic for pembrolizumab in patients with non-small cell lung cancer. Design .- Tumor samples from 146 patients with non-small cell lung cancer treated with pembrolizumab in KEYNOTE-001 and for whom response data were available were scored according to their staining intensity by a single pathologist using 4 methods: percentage of tumor cells staining at any intensity (PS1), moderate/strong intensity (PS2), strong intensity (PS3), and H-score (PS1 + PS2 + PS3). The cutoff score for predicting response to pembrolizumab was determined using receiver operating characteristic analysis. Progression-free and overall survival were assessed in patients with measurable disease per Response Evaluation Criteria in Solid Tumors, version 1.1 (n = 146). Results .- The 4 scoring methods assessed performed similarly; PS1 with a 50% cutoff score is the simplest and easiest method to implement in practice. Response to pembrolizumab was observed in 19 of 44 patients (43%) with a PS1 score of 50% or higher and 8 of 102 patients (8%) with PS1 lower than 50% (odds ratio, 8.93). Median progression-free and overall survival was 4.0 months and not yet reached, respectively, for patients with a PS1 of 50% or higher, and 2.1 and 6.1 months, respectively, for those with PS1 lower than 50%. Conclusion .- The PD-L1 immunohistochemical assay shows the potential for enrichment of trial populations and as a companion diagnostic tool in non-small cell lung cancer.42,0The prototype PD-L1 IHC assay was used to determine enrollment of patients in KEYNOTE-001 and was performed at a single laboratory site at QualTek (Goleta, California), which is a College of American Pathologists/Clinical Laboratory Improvement Amendments (CAP/CLIAhttps://www.archivesofpathology.org/doi/abs/10.5858/arpa.2015-0542-OAQualTek
2019Nicolini, F;Bocchini, M;Bronte, G;Delmonte, A;Guidoboni, M;Crinò, L;Mazza, M;Malignant Pleural Mesothelioma: State-of-the-Art on Current Therapies and Promises for the FutureFront Oncol1519932039010Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer of the pleural surface associated with asbestos exposure. The median survival of MPM patients is a mere 8-14 months, and there are few biomarkers and no cure available. It is hoped that, eventually, the incidence of MPM will drop and remain low and constant, given that most nations have banned the use of asbestos, but in the meantime, the incidence in Europe is still growing. The exact molecular mechanisms that explain the carcinogenicity of asbestos are not known. Standard therapeutic strategies for MPM include surgery, often coupled with chemotherapy and/or radiotherapy, in a small percentage of eligible patients and chemotherapy in tumors considered unresectable with or without adjuvant radiotherapy. In recent years, several new therapeutic avenues are being explored. These include angiogenesis inhibitors, synthetic lethal treatment, miRNA replacement, oncoviral therapies, and the fast-growing field of immunotherapy alone or in combination with chemotherapy. Of particular promise are the multiple options offered by immunotherapy: immune checkpoint inhibitors, tumor vaccines, and therapies taking advantage of tumor-specific antigens, such as specific therapeutic antibodies or advanced cell-based therapies exemplified by the CAR-T cells. This review comprehensively presents both old and new therapeutic options in MPM, focusing on the results of the numerous recent and on-going clinical trials in the field, including the latest data presented at international meetings (AACR, ASCO, and ESMO) this year, and concludes that more work has to be done in the framework of tailored therapies to identify reliable targets and novel biomarkers to impact MPM management. Copyright © 2020 Nicolini, Bocchini, Bronte, Delmonte, Guidoboni, Crinò and Mazza.41,0With a centrally-assessed 22C3 antibody-based immunohistochemistry assay (Qualtek laboratory), PDL1 expression was available in 62 patients: 45% were found negative (https://link.springer.com/chapter/10.1007/978-3-030-16884-1_20QualTek
2013Kinder, M;Greenplate, AR;Grugan, KD;Soring, KL;Heeringa, KA;McCarthy, SG;Bannish, G;Perpetua, M;Lynch, F;Jordan, RE;Strohl, WR;Brezski, RJ;Engineered protease-resistant antibodies with selectable cell-killing functionsJ. Biol. Chem.30843-308542884323986451Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.41,0Immunohistochemistry. All immunohistochemistry was performed by QualTek Molecular Laboratories (Newtown, PA). Four-micrometer sections were dewaxed through four changes of xylene (5 min each) followed by a graded alcohol series to distilled water ;. ‡ Brezski Randall J. ‡ 1 From ‡Biologics Research, Janssen Research and Development, LLC, Spring House, Pennsylvania 19477, §Huntingdon Life Sciences, East Millstone, New Jersey 08873, and ¶QualTek Molecular Laboratories, Newtown, Pennsylvania 18940 1 To whom correspondence should be addressed: Janssen Research and Development, LLC, 1400 McKean Rd., Spring House, PA 19477. Tel.: 215-628https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829399QualTek;Frank Lynch[au]
2001Lin, H;Ho, AS;Haley-Vicente, D;Zhang, J;Bernal-Fussell, J;Pace, AM;Hansen, D;Schweighofer, K;Mize, NK;Ford, JE;Cloning and characterization of IL-1HY2, a novel interleukin-1 family memberJ. Biol. Chem.20597-206022762311278614The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. The IL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of the IL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.41,0The anti-CD20 monoclonal antibody was purchased from DAKO (Carpenteria, CA), and the anti-Ki67 was obtained from Coulter Immunotech (Miami, FL). Immunohistochemistry was performed at QualTek Molecular Systems, Inchttp://www.jbc.org/content/276/23/20597.shortQualTek
2017Cho, JH;Sorensen, SF;Choi, YL;Feng, Y;Kim, TE;Choi, H;Georgsen, JB;Dolled-Filhart, M;Emancipator, K;Meldgaard, P;Sun, JM;Kim, HK;Choi, YS;Shim, YM;Zhou, W;Hager, H;Kim, J;Programmed Death Ligand 1 Expression in Paired Non-Small Cell Lung Cancer Tumor SamplesClin Lung Cancere473-e47918628669849Programmed death ligand 1 (PD-L1) expression may predict response to anti-programmed death 1 (anti-PD-1) or anti-PD-L1 treatment. There is limited information on changes in PD-L1 expression over time in patients with non-small cell lung cancer (NSCLC). Eligible patients with NSCLC who received surgery or underwent biopsy at Samsung Medical Center, Seoul, Republic of Korea, and Aarhus University Hospital, Aarhus, Denmark, between February 2004 and April 2012 were included. PD-L1 expression in paired tumor tissue samples from the same patients at different dates and lesions was measured using a laboratory-developed prototype immunohistochemistry assay (22C3 antibody). PD-L1 positivity was defined as tumor cell membrane positivity in ≥ 1% of tumor cells (proportion score). Concordance of PD-L1 expression was analyzed by treating proportion score as categoric or continuous variables. Ninety-one patients were included in the analysis. The median interval between the 2 tumor collection dates was 20 months, with 91% of paired samples collected > 3 months apart. The concordance rate for PD-L1 classification between paired samples was 67% (95% confidence interval, 57%-77%). When treating the immunohistochemistry proportional score as a continuous variable, a significant correlation of PD-L1 expression was observed between the paired samples (Pearson correlation coefficient, 0.61; P < .001). There are good correlations of PD-L1 expression from paired NSCLC samples. For patients whose PD-L1 status is negative, it may be valuable to obtain additional tissue samples for retesting PD-L1 expression when anti-PD-1 immunotherapy is considered. Copyright © 2017 Elsevier Inc. All rights reserved.41,0charged slides. The TechMate 500 instrument (QualTek, Goleta, CA) was used with the 22C3 antibody for automated staining. A membrane staining. All of the PD-L1 IHC assays were done at QualTek, as the central laboratory. PDhttps://www.sciencedirect.com/science/article/pii/S1525730417301134QualTek
2013North, GB;Lynch, FH;Maharaj, FD;Phillips, CA;Woodside, WT;Leaf hydraulic conductance for a tank bromeliad: axial and radial pathways for moving and conserving waterFront Plant Sci78423596446Epiphytic plants in the Bromeliaceae known as tank bromeliads essentially lack stems and absorptive roots and instead take up water from reservoirs formed by their overlapping leaf bases. For such plants, leaf hydraulic conductance is plant hydraulic conductance. Their simple strap-shaped leaves and parallel venation make them suitable for modeling leaf hydraulic conductance based on vasculature and other anatomical and morphological traits. Plants of the tank bromeliad Guzmania lingulata were investigated in a lowland tropical forest in Costa Rica and a shaded glasshouse in Los Angeles, CA, USA. Stomatal conductance to water vapor and leaf anatomical variables related to hydraulic conductance were measured for both groups. Tracheid diameters and numbers of vascular bundles (veins) were used with the Hagen-Poiseuille equation to calculate axial hydraulic conductance. Measurements of leaf hydraulic conductance using the evaporative flux method were also made for glasshouse plants. Values for axial conductance and leaf hydraulic conductance were used in a model based on leaky cable theory to estimate the conductance of the radial pathway from the vein to the leaf surface and to assess the relative contributions of both axial and radial pathways. In keeping with low stomatal conductance, low stomatal density, low vein density, and narrow tracheid diameters, leaf hydraulic conductance for G. lingulata was quite low in comparison with most other angiosperms. Using the predicted axial conductance in the leaky cable model, the radial resistance across the leaf mesophyll was predicted to predominate; lower, more realistic values of axial conductance resulted in predicted radial resistances that were closer to axial resistance in their impact on total leaf resistance. Tracer dyes suggested that water uptake through the tank region of the leaf was not limiting. Both dye movement and the leaky cable model indicated that the leaf blade of G. lingulata was structurally and hydraulically well-suited to conserve water.41,0http://dx.doi.org/10.3389/fpls.2013.00078Frank Lynch[au]
2017Sundahl, N;De Wolf, K;Rottey, S;Decaestecker, K;De Maeseneer, D;Meireson, A;Goetghebeur, E;Fonteyne, V;Verbeke, S;De Visschere, P;Reynders, D;Van Gele, M;Brochez, L;Ost, P;A phase I/II trial of fixed-dose stereotactic body radiotherapy with sequential or concurrent pembrolizumab in metastatic urothelial carcinoma: evaluation of safety and clinical and immunologic responseJ Transl Med15015128662677Current first-line standard of therapy for metastatic urothelial carcinoma is platinum-based combination chemotherapy. Pembrolizumab in phase III has demonstrated a promising overall response rate of 21.1% in patients with progression or recurrence after platinum-based chemotherapy. Preclinical and clinical evidence suggests that radiotherapy has a systemic anti-cancer immune effect and can increase the level of PD-L1 and tumor infiltrating lymphocytes in the tumor microenvironment. These findings gave rise to the hypothesis that the combination of radiotherapy with anti-PD1 treatment could lead to a synergistic effect, hereby enhancing response rates. The phase I part will assess the dose limiting toxicity of the combination treatment of stereotactic body radiotherapy (SBRT) with four cycles of pembrolizumab (200 mg intravenously, every 3 weeks) in patients with metastatic urothelial carcinoma. The dose of both pembrolizumab and SBRT will be fixed, yet the patients will be randomized to receive SBRT either before the first cycle of pembrolizumab or before the third cycle of pembrolizumab. SBRT will be delivered (24 Gy in 3 fractions every other day) to the largest metastatic lesion. Secondary objectives include response rate according to RECIST v1.1 and immune related response criteria, progression-free survival and overall survival. The systemic immune effect triggered by the combination therapy will be monitored on various time points during the trial. The PD-L1/TIL status of the tumors will be analyzed via immunohistochemistry and response rates in the subgroups will be analyzed separately. A Simon's two-stage optimum design is used to select the treatment arm associated with the best response rate and with acceptable toxicity to proceed to the phase II trial. In this phase, 13 additional patients will be accrued to receive study treatment. The progress made in the field of immunotherapy has lead to promising breakthroughs in various solid malignancies. Unfortunately, the majority of patients do not respond. The current trial will shed light on the toxicity and potential anti-tumor activity of the combination of radiotherapy with anti-PD1 treatment and may identify potential new markers for response and resistance to therapy. Trial registration this trial is registered on clinicaltrials.gov (NCT02826564).41,0Exploratory endpoints. Response rate according to expression of PD-L1. PD-L1 expression will be determined by immunohistochemistry in QualTek Molecular Laboratories on an archival or newly obtained tissue sample of the tumor or metastatic lesion ;. OS defined as the time from inclusion to death from any cause. Exploratory endpoints Response rate according to expression of PD-L1. PD-L1 expression will be determined by immunohistochemistry in QualTek Molecular Laboratories on an archival or newly obtained tissue sample of the tumor or metastatic lesion. Immunologic response in peripheral blood samples analyzed via fluorescence-activated chttps://translational-medicine.biomedcentral.com/articles/10.1186/s12967-017-1251-3QualTek
2013Park, S;Balasubramaniam, V;Sastry, S;Lee, J;Pressure–ohmic–thermal sterilization: A feasible approach for the inactivation of Bacillus amyloliquefaciens and Geobacillus stearothermophilus sporesInnovative Food Science & Emerging Technologies115-1231941,0Each soldered part was electrically insulated using heat shrink tubing (Q2-F-1/16-01-QB6IN- 36, Qualtek, Mentor, OH, USA) and epoxy (2-t ® epoxy clear, Devcon, Danvers, MA, USA) to prevent short circuits and current loss to the pressure-transmitting fluidhttps://www.sciencedirect.com/science/article/pii/S1466856413000453QualTek
2005Schirmer, A;Gadkari, R;Reeves, CD;Ibrahim, F;DeLong, EF;Hutchinson, CR;Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissolutaAppl. Environ. Microbiol.4840-484971816085882Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.41,0We thank Dan Morse (University of California, Santa Barbara) and David Myles for obtaining Discodermia dissoluta, Steve Bernstein (QualTek Molecular Laboratories, Santa Barbara, CA) for help with microscopy on sponge thin sections, and Ralph Reid for suggestions regarding the sequence analyses. We also thank Christian Siebel, David Hopwood, and Leonard Katz for critical comments during preparation of the manuscript. This study was supported in part by a Small Business Innovative Research grant from the National Institutes of Health (1R43CA97889-01)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183291QualTek
2020Awty-Carroll, D;Ravella, S;Clifton-Brown, J;Robson, P;Using a Taguchi DOE to investigate factors and interactions affecting germination in Miscanthus sinensisSci Rep160210132005862The Miscanthus genus of perennial grasses is grown for bioenergy and biorenewable feedstocks. Most Miscanthus crop is M × giganteus which is rhizome propagated and therefore difficult to multiply at large scale. Seed-based propagation of new hybrids is being developed, but Miscanthus is difficult to establish from seed especially in the field. Miscanthus is often grown on marginal land adding to the challenge of successfully establishing the crop. Improved understanding of the limits and biology of germination in Miscanthus species is needed. Seed germination is affected by physical and chemical factors that impact germination differently depending on level of exposure. In this investigation of Miscanthus germination, four hormones plus water stress were investigated and the range over which these factors affect germination was determined. An efficient Taguchi experimental design was used to assess the five factors in combination with the effects of light and seed priming. This determined an example of a set of optimum conditions for Miscanthus germination and demonstrated how this could change based on fixing one condition. The experiment showed how environmental stress impacted germination and how treatments such as gibberellic acid could be used to mitigate stress.40,0All dishes were prepared and tested at the same time to limit extraneous sources of variation. The Taguchi design of experiment (DOE) was produced and primarily analysed using Qualtek-4 (Nutek inc., Michigan, USA) with secondary analysis in R34 ;Full size table. The Taguchi design of experiment (DOE) was produced and primarily analysed using Qualtek-4 (Nutek inc., Michigan, USA) with secondary analysis in R 34 . The Qualtek-4 software calculated the percentage ;No 15 V-High High Low V-Low V-High High No 16 V-High V-High Low Low High Low Yes The Taguchi design of experiment (DOE) was produced and primarily analysed using Qualtek-4 (Nutek inc., Michigan, USA) with secondary analysis in R34. The Qualtek-4 software calculated the percentage effect of each treatment; this is the primary output of the Taguchi method. Thishttp://search.proquest.com/openview/3bfbceafe5482899d3ef67bba38572a1/1?pq-origsite=gscholar&cbl=2041939QualTek
2019Gani, AW;Wei, W;Shi, RZ;Ng, E;Nguyen, M;Chua, MS;So, S;Wang, SX;An Automated, Quantitative, and Multiplexed Assay Suitable for Point-of-Care Hepatitis B Virus DiagnosticsSci Rep156159131666635Hepatitis B virus (HBV) infection has a global reach with high prevalence in resource-limited areas like China and Africa. HBV patients in these areas have limited access to the currently used, costly HBV assays, which are performed in centralized clinical laboratories using single-plexed assays with bulky and expensive instruments. We aim to overcome these limitations by developing a simple and affordable HBV diagnostic platform to allow for timelier diagnosis and intervention of HBV infection. Using giant magnetoresistive (GMR) biosensor chips, we developed an automated and multiplexed quantitative platform for the measurement of a panel of HBV serology markers, including HBV "e" antigen (HBeAg), HBV surface antigen (HBsAg), and the antibody against HBsAg (anti-HBs). Our assay platform was able to detect each HBV marker with high specificity and sensitivity (with three orders of magnitude in dynamic range for each marker). Blinded analysis of HBV patient sera showed excellent correlation between our multiplexed quantitative HBsAg results and the qualitative results obtained using FDA-approved immunoassays, as well as those obtained using quantitative, single-plexed, enzyme-linked immunosorbent assays (ELISAs). The portable, automated, multiplexed, quantitative HBV serology assay platform we designed shows great promise as a more accessible alternative for HBV screening, diagnosis, and treatment monitoring.40,0HBV DNA levels). As controls, we recruited 5 healthy volunteers who had no prior history of HBV and who were vaccinated against HBV. Serum samples from healthy Asian donors were also purchased from Conversant Biologics (Huntsville, AL). Supplementary information Supplementary figures and legends Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in publish ... HBV DNA levels). As controls, we recruited 5 healthy volunteers who had no prior history of HBV and who were vaccinated against HBV. Serum samples from healthy Asian donors were also purchased from Conversant Biologics (Huntsville, AL). Supplementary information Supplementary figures and legends Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in publishhttps://www.nature.com/articles/s41598-019-52147-z"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2012Fedyk, ER;Wyant, T;Yang, LL;Csizmadia, V;Burke, K;Yang, H;Kadambi, VJ;Exclusive antagonism of the α4 β7 integrin by vedolizumab confirms the gut-selectivity of this pathway in primatesInflamm. Bowel Dis.2107-2119181122419649Biological therapies that antagonize specific molecules have demonstrated efficacy in inflammatory bowel diseases, but infections resulting from systemic immunosuppression underscore the need for safer therapies. The objective of this investigation was to determine if antagonism of the α(4) β(7) integrin would exclusively yield gut-selective antiinflammatory activity in primates. A series of experiments were conducted to investigate potential intra- and extraintestinal effects in healthy nonhuman primates dosed repeatedly with the α(4) β(7) -exclusive antagonist vedolizumab (former versions: MLN0002, MLN02, LDP-02) for 4, 13, and 26 weeks. No adverse clinical effects of vedolizumab were observed in healthy cynomolgus monkeys up to the highest doses tested (100 mg/kg). Histomorphologic analyses indicated a reduction in the frequency of leukocytes in gastrointestinal tissue, but not other organs. A significant (P < 0.05) decrease in the frequency of β 7+ lymphocytes in gastrointestinal tissues corresponded to a significant (P < 0.05) increase in α(4) β 7+ memory helper T lymphocytes in peripheral blood. This elevation was specific to α(4) β 7+ memory helper T lymphocytes; levels of other leukocyte subsets remained unaffected. Systemic opportunistic infections were not observed, and vedolizumab did not inhibit adaptive or innate immune responses systemically. These data demonstrate that blocking the α(4) β(7) integrin exclusively yields gut-selective antiinflammatory activity in primates. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.40,0Immunohistochemistry detecting the β7 integrin was conducted using a sheep antihuman β7 polyclonal antibody (R&D Systems, Minneapolis, MN) that does not compete with vedolizumab for binding to the α4β7 integrin expressed by leukocytes (data not shown). Immunohistochemistry was conducted by QualTek Molecular Laboratories (Goleta, CA), per testing facility SOP. The slides were imaged using the whole slide imaging device NanoZoomer (Hamamatsu Photonics, Hamamatsu City, Shizuoka, Japan) at 200× magnification. Individual tagged image file format (TIFF) images were processed with MetaMorph (MDS Analytical Technologies, Mississauga, Ontario, Canada) software (v. 7.6.3.0). A MetaMorph analysis journal was run to measure the total area of the crypt and the total area of the integrin β7-positive stain. The final analysis output was the % intraepithelial lymphocytes (IEL) or % lymphocyte area per crypt or total image area: % IEL or % lymphocyte = Size of area positive for integrin β7 / Total crypt or image area × 100%https://academic.oup.com/ibdjournal/article-abstract/18/11/2107/4608939QualTek
2014Hornby, PJ;Cooper, PR;Kliwinski, C;Ragwan, E;Mabus, JR;Harman, B;Thompson, S;Kauffman, AL;Yan, Z;Tam, SH;Dorai, H;Powers, GD;Giles-Komar, J;Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosisPharm. Res.908-92231424072267To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.39,0Paraffin-imbedded 5 μm sections were cut from full-thickness intestine harvested from monkey and human donors, and from human tissue bank (MTB-311 and MTB-316; Qualtek Molecular Labs, Goleta, CA) and dried on glass slides (60°C). Steam heat induced epitope ;Intestinal FcRn Immunohistochemistry Paraffin-imbedded 5 μm sections were cut from full-thickness intestine harvested from monkey and human donors, and from human tissue bank (MTB-311 and MTB-316; Qualtek Molecular Labs, Goleta, CA) and dried on glass slides (60°C). Steam heat induced epitope recovery (SHIER) system was used with pretreatment in the solutions in the capillary gap in the upperhttps://link.springer.com/article/10.1007/s11095-013-1212-3QualTek
2013Kauffman, AL;Gyurdieva, AV;Mabus, JR;Ferguson, C;Yan, Z;Hornby, PJ;Alternative functional in vitro models of human intestinal epitheliaFront Pharmacol79423847534Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.38,0n Majewski, Jason Ekert, and Kimberly Reese for help with iPSC maintenance culture. We also acknowledge the outstanding quality of immunohistochemistry performed by Steve Bernstein and Marko Sldanov, Qualtek Molecular Labs, Goleta, CA. References AldhousM. C.ShmakovA. N.BodeJ.GhoshS. (2001). Characterization of conditions for the primary culture of human small intestinal epithelial cells. Clin. E;n Majewski, Jason Ekert, and Kimberly Reese for help with iPSC maintenance culture. We also acknowledge the outstanding quality of immunohistochemistry performed by Steve Bernstein and Marko Sldanov, Qualtek Molecular Labs, Goleta, CA. References AldhousM. C.ShmakovA. N.BodeJ.GhoshS. (2001). Characterization of conditions for the primary culture of human small intestinal epithelial cells. Clin. E;n Majewski, Jason Ekert, and Kimberly Reese for help with iPSC maintenance culture. We also acknowledge the outstanding quality of immunohistochemistry performed by Steve Bernstein and Marko Sldanov, Qualtek Molecular Labs, Goleta, CA. References AldhousM. C.ShmakovA. N.BodeJ.GhoshS. (2001). Characterization of conditions for the primary culture of human small intestinal epithelial cells. Clin. Ehttp://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.933.9200&rep=rep1&type=pdfQualTek
2016Chisamore, MJ;Gentile, MA;Dillon, GM;Baran, M;Gambone, C;Riley, S;Schmidt, A;Flores, O;Wilkinson, H;Alves, SE;A novel selective androgen receptor modulator (SARM) MK-4541 exerts anti-androgenic activity in the prostate cancer xenograft R-3327G and anabolic activity on skeletal muscle mass & function in castrated miceJ. Steroid Biochem. Mol. Biol.88-9716327106747The androgen receptor (AR) is a member of the nuclear hormone receptor super family of transcription factors. Androgens play an essential role in the development, growth, and maintenance of male sex organs, as well as the musculoskeletal and central nervous systems. Yet with advancing age, androgens can drive the onset of prostate cancer, the second leading cause of cancer death in males within the United States. Androgen deprivation therapy (ADT) by pharmacologic and/or surgical castration induces apoptosis of prostate cells and subsequent shrinkage of the prostate and prostate tumors. However, ADT is associated with significant musculoskeletal and behavioral adverse effects. The unique pharmacological activity of selective androgen receptor modulator (SARM) MK-4541 recently has been reported as an AR antagonist with 5α-reductase inhibitor function. The molecule inhibits proliferation and induces apoptosis in AR positive, androgen dependent prostate cancer cells. Importantly, MK-4541 inhibited androgen-dependent prostate growth in male rats yet maintained lean body mass and bone formation following ovariectomy in female rats. In the present study, we evaluated the effects of SARM MK-4541 in the androgen-dependent Dunning R3327-G prostate carcinoma xenograft mouse model as well as on skeletal muscle mass and function, and AR-regulated behavior in mice. MK-4541 significantly inhibited the growth of R3327-G prostate tumors, exhibited anti-androgen effects on the seminal vesicles, reduced plasma testosterone concentrations in intact males, and inhibited Ki67 expression. MK-4541 treated xenografts appeared similar to xenografts in castrated mice. Importantly, we demonstrate that MK-4541 exhibited anabolic activity in androgen deficient conditions, increasing lean body mass and muscle function in adult castrated mice. Moreover, MK-4541 treatment restored general activity levels in castrated mice. Thus, MK-4541 exhibits an optimum profile as an adjuvant therapy to ADT which may provide potent anti-androgenic activity at the prostate yet protective activity on skeletal muscle and behavior in patients. Copyright © 2016 Elsevier Ltd. All rights reserved.38,0Excised tumor samples were immediately fixed with 10% buffered formalin, and sent to QualTek (Santa Barbara, CA) where tumors were embedded in paraffin, 4-μm thick sections were mounted on charged slides, dried at 60 °C for one hour, and deparaffinzed in xylene andhttps://www.sciencedirect.com/science/article/pii/S0960076016301030QualTek
2004Lynch, F;The use of an interactive computerized daily schedule in a busy interventional radiology practice increases efficiencyJ Am Coll Radiol965-97111217411739The effect on efficiency of patient care in a busy academic interventional radiology practice was studied by the analysis of procedure times both before and after the implementation of a computerized interactive daily schedule. Procedure start and end times were retrospectively collected from the department's quality assurance database for two identical 6-month periods, representing the time before and after the deployment of the software. The delay in the start of the first case, the time between cases, and the time required to complete each day's work were compared for the two periods. The average time of delay between cases was reduced after the implementation of the software (p < 0.025). More total cases were performed during the period of time after the implementation of the software, resulting in a greater work relative value unit production. Although the average number of cases performed per day was greater after the software was in use (p < 0.03), the average amount of time required to complete the day's work was not significantly changed (p = 0.08). There was no apparent effect on the average delay of the start of the day's first case (p = 0.34). The use of a computerized interactive daily schedule has a positive effect on departmental efficiency by allowing more cases to be performed without lengthening the workday.38,0http://dx.doi.org/10.1016/j.jacr.2004.06.026Frank Lynch[au]
2005Pyle-Chenault, RA;Stolk, JA;Molesh, DA;Boyle-Harlan, D;McNeill, PD;Repasky, EA;Jiang, Z;Fanger, GR;Xu, J;VSGP/F-spondin: a new ovarian cancer markerTumour Biol.245-25726516103746The discovery of genes that are overexpressed in ovarian cancers provides valuable insight into ovarian cancer biology and will lead to the development of more effective treatment strategies for combating this disease. To identify genes exhibiting ovarian- and ovarian cancer-specific expression, we generated four subtracted cDNA libraries from primary and metastatic ovarian adenocarcinoma tissues. 3,400 cDNA clones from these libraries were analyzed by microarray for tissue distribution and tumor specificity using 32 pairs of fluorophore-labeled cDNA samples from a variety of normal tissues and ovarian tumor tissues. cDNA clones showing elevated expression in ovarian tumors were identified by DNA sequencing with comparison to public databases, and the most promising candidates were further analyzed by quantitative real-time polymerase chain reaction and Northern blot. This systematic approach led to the identification of a number of genes including vascular smooth muscle growth-promoting factor (VSGP/F-spondin), a secreted protein previously identified and cloned from bovine and human ovary. VSGP/F-spondin protein was observed in ovarian carcinomas but not in normal ovarian epithelium by immunohistochemistry with a VSGP/F-spondin antibody. The expression profile of VSGP/F-spondin identifies this molecule as a potential diagnostic marker or target for developing therapeutic strategies to treat ovarian carcinoma. Copyright (c) 2005 S. Karger AG, Basel.36,0Rockford, Ill., USA). Immunohistochemistry was performed with affinity-purified rabbit polyclonal antibody on formalin-fixed, paraffin-embedded tissues by QualTek Molecular Laboratories (Santa Barbara, Calif., USA). Steam heathttps://www.karger.com/Article/PDF/87379QualTek
2002Fanger, GR;Houghton, RL;Retter, MW;Hendrickson, RC;Babcook, J;Dillon, DC;Durham, MD;Reynolds, LD;Johnson, JC;Carter, D;Fleming, TP;Roche, PC;Persing, DH;Reed, SG;Detection of mammaglobin in the sera of patients with breast cancerTumour Biol.212-22123412499777Current procedures for the diagnosis of breast cancer are cumbersome and invasive, making detection of this disease difficult. A rapid screening test for early detection of breast cancer would allow for better management of this deadly disease. In this report, we show that, with the exception of the skin, mammaglobin mRNA is specifically expressed in mammary tissue and commonly overexpressed in breast cancer. Mammaglobin is not expressed in other types of cancer including colon, lung, ovarian, and prostate cancer. Breast-specific expression of mammaglobin protein was shown using immunohistochemical methods. Mammaglobin is secreted from both established breast cancer cell lines and primary breast carcinoma cells cultured in vitro. Using a monoclonal antibody-based assay for monitoring the presence of mammaglobin in serum, elevated levels of mammaglobin were detected in sera of patients with breast cancer, but not in healthy women. Thus, mammaglobin, which is overexpressed and secreted from breast carcinoma cells, is detectable in sera of patients with breast cancer and may provide a rapid screening test for the diagnosis and management of breast cancer. Copyright 2002 S. Karger AG, Basel36,0Acknowledgments We thank Frank Lynch at Qualtek for immunohistochemistry efforts and expertise. Work was supported in part by USPHS grant CA76227 (TPF), and NIH grants CA-75794 (RLH) and CA- 86673 (DCD) References 1 Donegan W, Spratt J: Cancer of the breasthttps://www.karger.com/Article/Abstract/67251QualTek
2013Park, S;Balasubramaniam, V;Sastry, S;Estimating pressure induced changes in vegetable tissue using in situ electrical conductivity measurement and instrumental analysisJournal of Food Engineering47-56114136,0Pittsfield, MA, USA). The ends of the copper wires were soldered to the high pressure electrical feed through and electrically insulated using polyolefin heat shrink tubing (Q2-Z-1/8, Qualtek Electronics, Mentor, OH, USA). A typehttps://www.sciencedirect.com/science/article/pii/S0260877412003548QualTek
2016Aycock, KI;Campbell, RL;Lynch, FC;Manning, KB;Craven, BA;The Importance of Hemorheology and Patient Anatomy on the Hemodynamics in the Inferior Vena CavaAnn Biomed Eng3568-3582441227272211Inferior vena cava (IVC) filters have been used for nearly half a century to prevent pulmonary embolism in at-risk patients. However, complications with IVC filters remain common. In this study, we investigate the importance of considering the hemorheological and morphological effects on IVC hemodynamics by simulating Newtonian and non-Newtonian blood flow in three IVC models with varying levels of geometric idealization. Partial occlusion by an IVC filter and a thrombus is also considered. More than 99% of the infrarenal IVC volume is found to contain flow in the nonlinear region of the shear rate-viscosity curve for blood (less than 100 s-1) in the unoccluded IVCs. Newtonian simulations performed using the asymptotic viscosity for blood over-predict the non-Newtonian Reynolds numbers by more than a factor of two and under-predict the mean wall shear stress (WSS) by 28-54%. Agreement with the non-Newtonian simulations is better using a characteristic viscosity, but local WSS errors are still large (up to 50%) in the partially occluded cases. Secondary flow patterns in the IVC also depend on the viscosity model and IVC morphological complexity. Non-Newtonian simulations required only a marginal increase in computational expense compared with the Newtonian simulations. We recommend that future studies of IVC hemodynamics consider the effects of hemorheology and IVC morphology when accurate predictions of WSS and secondary flow features are desired.35,0http://dx.doi.org/10.1007/s10439-016-1663-xFrank Lynch[au]
2007Flores, J;Cepeda, IL;Cornfeldt, ML;O'Kusky, JR;Doudet, DJ;Characterization and survival of long-term implants of human retinal pigment epithelial cells attached to gelatin microcarriers in a model of Parkinson diseaseJ. Neuropathol. Exp. Neurol.585-59666717620984Previous studies have demonstrated that the intrastriatal implantation of human retinal pigment epithelial cells attached to gelatin microcarriers (hRPE-GM) ameliorates behavioral deficits in animal models of Parkinson disease. However, there are only sparse data on cell survival in the host. In this study, we characterized a variety of retinal pigment epithelial (RPE)-specific markers in vitro and used these markers to investigate the long-term survival of hRPE-GM implants. Sprague-Dawley rats (n = 22) were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and implanted with hRPE-GM without immunosuppression. Rats were euthanized at 48 hours, 7 days, 4 weeks, and 5 months postimplant and immunohistochemically processed using the following antibodies: 1) human-specific nuclear mitotic apparatus protein (NuMA-Ab2), 2) epithelial-specific extracellular matrix metalloproteinase inducer (EMMPRIN), 3) RPE cell-specific RPE65, and the inflammation markers 4) glial fibrillary acidic protein and 5) ED1 (rat CD68). Our analysis revealed NuMA-, EMMPRIN-, and RPE65-immunoreactive cells at different times postimplant. The morphologic features of hRPE cell implants (at 48 hours and 5 months) were confirmed by electron microscopy. Furthermore, despite evidence of chronic inflammation at the later time point, there is an appreciable number of surviving hRPE cells. This study suggests that hRPE-GM implants can survive in the absence of immunosuppression and can be potentially used as an alternative for treating Parkinson disease.35,0All primary antibodies were previously assessed for hRPE cell specificity in human RPE cultured monolayer and RPE-choroid eye tissue, and for nonspecificity to striatal rat tissue (Dr. S. Bernstein, QualTek Molecular Laboratories, Santa Barbara, CA, personal communicationhttps://academic.oup.com/jnen/article-abstract/66/7/585/2916816QualTek
2003DuHadaway, JB;Lynch, FJ;Brisbay, S;Bueso-Ramos, C;Troncoso, P;McDonnell, T;Prendergast, GC;Immunohistochemical analysis of Bin1/Amphiphysin II in human tissues: diverse sites of nuclear expression and losses in prostate cancerJ. Cell. Biochem.635-64288312532338The Bin1/Amphiphysin II gene encodes at least seven alternately spliced adapter proteins that have been implicated in membrane dynamics and nuclear processes. Nuclear localized Bin1 polypeptides have tumor suppressor and proapoptotic activities, suggesting that Bin1 may suppress cancer in tissues where nuclear expression may occur. One question is the extent to which human tissues express nuclear Bin1 isoforms. A secondary issue has been the need for a specific antibody that can detect all the splice isoforms expressed by the human, mouse, and rat Bin1 genes. Using a novel mouse monoclonal antibody with these characteristics, we performed an immunohistochemical analysis of Bin1 expression in a panel of normal human tissues. We also compared the expression profile of Bin1 in normal or malignant tissues derived from human prostate, where Bin1 is a candidate tumor suppressor gene. In brain, a distinct nuclear staining pattern overlapped with a cytosolic staining pattern present in certain layers of the cerebral cortex and cerebellum. Bone marrow cells displayed mainly nuclear localization whereas peripheral lymphoid cells exhibited mainly cytosolic localization. In several epithelial tissues, nuclear or nucleocytosolic staining patterns were displayed by basal cells in skin, breast, or prostate, whereas cytosolic or plasma membrane-associated staining patterns were noted in gastrointestinal cells. Interestingly, a striking gradient of expression was observed in gastrointestinal epithelia, particularly in the large intestine, with the strongest staining displayed by cells destined to undergo apoptosis at the villus tip. In prostate, Bin1 staining was frequently absent in cases of primary prostate adenocarcinoma. This study used a novel reagent to document the extent of expression of nuclear Bin1 isoforms, which exhibit cancer suppression and proapoptotic activity in human cells. Copyright 2002 Wiley-Liss, Inc.34,0Search for more papers by this author. Frank J. Lynch QualTek Molecular Laboratories, Newtown, Pennsylvania. Search for more papers by this author. Shawn Brisbay Search for more papers by this author. Frank J. Lynch QualTek Molecular Laboratories, Newtown, Pennsylvaniahttps://onlinelibrary.wiley.com/doi/abs/10.1002/jcb.10380QualTek;Frank Lynch[au]
2018Lin, EM;Gong, J;Klempner, SJ;Chao, J;Advances in immuno-oncology biomarkers for gastroesophageal cancer: Programmed death ligand 1, microsatellite instability, and beyondWorld J. Gastroenterol.2686-2697242529991874Blockade of the programmed death ligand 1 (PD-L1) and programmed cell death 1 (PD-1) receptor axis represents an effective form of cancer immunotherapy. Preclinical evidence initially suggested that gastric and gastroesophageal junction (GEJ) cancers are potentially immunotherapy-sensitive tumors. Early phase clinical trials have demonstrated promising antitumor activity with PD-1/PD-L1 blockade in advanced or metastatic gastric/GEJ cancer. Microsatellite instability (MSI) and PD-L1 expression have been shown to predict higher response to PD-1 inhibitors as highlighted by the recent approvals of pembrolizumab in treatment-refractory solid tumors with MSI status and the third-line or greater treatment of PD-L1 positive advanced gastric/GEJ cancers. However, predictive and prognostic biomarkers remain an ongoing need. In this review, we detail the preclinical evidence and early tissue biomarker analyses illustrating potential predictive biomarkers to PD-1/PD-L1 blockade in gastric/GEJ cancer. We also review the clinical development of PD-1/PD-L1 inhibitors in gastric/GEJ cancer and highlight several areas in need of future investigation in order to optimize the efficacy of PD-1/PD-L1 blockade in gastric/GEJ cancer.34,0Presence of tumor PD-L1 expression was a requirement for enrollment, with 40% of the patients screened demonstrating PD-L1-positive tumors. PD-L1 expression was detected using the 22C3 antibody with a prototype assay using QualTek or Dako platforms ;expression was a requirement for enrollment, with 40% of the patients screened demonstrating PD-L1-positive tumors. PD-L1 expression was detected using the 22C3 antibody with a prototype assay using QualTek or Dako platforms. Tumor positivity was based on a cutoff of at least 1% of scorable tumor cells or immune cells exhibiting membrane staining, or the presence of PD-L1-positive mononuclear infhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034145/QualTek
2019Patel, S;Knight, A;Krause, S;Teceno, T;Tresse, C;Li, S;Cai, Z;Gouasmat, A;Carroll, VM;Barret, O;Gottmukkala, V;Zhang, W;Xiang, X;Morley, T;Huang, Y;Passchier, J;Preclinical In Vitro and In Vivo Characterization of Synaptic Vesicle 2A-Targeting Compounds Amenable to F-18 Labeling as Potential PET Radioligands for Imaging of Synapse IntegrityMol Imaging Biol31728839Current synaptic vesicle 2A (SV2A) positron emission tomography (PET) imaging agents include the nanomolar affinity probes [11C]UCB-J and [18F]UCB-H derived from the anti-epileptic drug levitaracetam (Keppra®). An industry-utilized "de-risking" approach was used to carry out initial pharmacological characterization and to assess potential next-generation candidates amenable to F-18 radiolabeling for preliminary evaluation. Radioligand binding methods were employed in mammalian brain homogenates to determine the SV2A affinity (Kd) and maximal binding capacity (Bmax) of [3H]UCB-J. Novel leads were then screened to identify compounds minimally with comparable binding affinities with UCB-J in order to select a F-18-labeled candidate for subsequent in vivo assessment in rat. In parallel, mammalian brain tissue section autoradiography was performed to assess specific SV2A distribution. [3H]UCB-J bound with high affinity to a single population of sites in the rat brain (Kd = 2.6 ± 0.25 nM; Bmax = 810 ± 25 fmol/mg protein) and control human cortex (Kd = 2.9 ± 0.54 nM; Bmax = 10,000 ± 640 fmol/mg protein). Distribution of specific SV2A binding was shown to be homogeneous throughout the rodent brain and primarily in gray matter regions of rodent and human brain sections. Analog screening identified MNI-1038, MNI-1126/SDM-8, and SDM-2 as having comparable binding affinities with the currently available PET ligands. Subsequent [18F]MNI-1126/[18F]SDM-8 dynamic micro-PET imaging in rats revealed in vivo uptake and accumulation in the brain with favorable kinetics. Chase studies using 30 mg/kg levetiracetam confirmed that in vivo brain uptake of [18F]MNI-1126/[18F]SDM-8 was reversible. Taken together, these data suggest [18F]MNI-1126/[18F]SDM-8 (since renamed as [18F]SynVesT-1) characterized via an in vitro screening cascade provided a measurable in vivo SV2A specific signal in the rodent brain. This tracer as well as the close analog [18F]SDM-2 (since renamed as [18F]SynVesT-2) is currently undergoing further evaluation in preclinical and clinical studies.33,0The same procedure was used for human post-mortem tissue (Folio Biosciences, Miami Brain Bank, and UCSF Memory and Aging Center, USA). In Vitro Homogenate Binding. All homogenate studies were carried out in 96-well format as previously described [14]https://link.springer.com/article/10.1007/s11307-019-01428-0"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2003Tinkelenberg, BA;Fazzone, W;Lynch, FJ;Ladner, RD;Identification of sequence determinants of human nuclear dUTPase isoform localizationExp. Cell Res.39-46287112799180dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is the central regulator of cellular dUTP pools. Nuclear (DUT-N) and mitochondrial (DUT-M) isoforms of the protein have been identified in humans and arise from the same gene by the alternative use of 5' exons. Recently, it has been shown that these isoforms are aberrantly expressed in some cancers and overexpression of dUTPase in the nucleus is associated with resistance to chemotherapeutic agents that target thymidylate biosynthesis. In this study, we have examined the signals necessary for dUTPase isoform localization using green fluorescent protein fusion constructs. We report that the N-terminal 23 amino acids of DUT-N are required but not sufficient for complete nuclear localization. Within this region, we identified a small cluster of basic residues (K(14)R(15)R(17)) that resemble a classic monopartite nuclear localization signal (NLS). Mutation of these residues completely abolishes nuclear localization. In addition, phosphorylation of Ser11 near the putative NLS has no affect on DUT-N nuclear localization. Through deletion analysis we show improved sorting of DUT-N to the nucleus when most of the protein sequence is present. Therefore, we conclude that DUT-N may contain a complex NLS that is located throughout the entire protein.33,015 min), DUT415 primary antibody incubation (25 min), biotinylated goat anti-mouse IgG secondary antibody (25 min), peroxidase-conjugated ABC-Elite (25 min, Vector Labs, Burlingame, CA, USA), and DAB (diaminobenzidine) chromogen (15 min, QualTek Molecular Labs)https://www.sciencedirect.com/science/article/pii/S001448270300048XQualTek;Frank Lynch[au]
2020MacCarty, N;Bentson, S;Cushman, K;Au, J;Li, C;Murugan, G;Still, D;Stratification of particulate matter in a kitchen: A comparison of empirical to predicted concentrations and implications for cookstove emissions targetsEnergy for Sustainable Development14-2454Household air pollution continues to be a global public health burden as 2.8 billion people still rely on open fires or inefficient cookstoves. The majority of morbidity and mortality are related to inhalation of fine particulate matter (PM), and many initiatives have focused on reducing PM emission and resulting exposure. For this reason, targets for emission rates to meet air quality guidelines have been set by the World Health Organization and International Organization for Standardization. These targets are based on a single-zone box model that assumes released particles are distributed uniformly throughout the kitchen. However, particulate matter is known to stratify vertically and therefore personal exposure to the cook and others in the kitchen may not be predicted accurately by a single-zone model. Therefore, this study compares empirical measurements of PM stratification to box model predictions to determine if the single-zone model is appropriate for predicting PM exposure at the expected breathing zone of the inhabitants. A test kitchen with a fixed ventilation rate and dimensions common to kitchens in rural areas was built and fitted with thirty HAPEx PM Sensors were evenly distributed at 4 different heights within the kitchen. Seventy modified water boiling tests were performed with a common rocket stove operating at known firepower and emissions rates in two testing phases at 10, 15 and 20 air exchanges per hour. Results showed stratification was less pronounced with increasing ventilation and with decreasing firepower. Measured concentrations at 15 ACH were compared to the predicted value from the single-zone model, showing equilibrium at a height of approximately 1.6 m. The box model over predicted concentration at heights lower than this, including a 23–63% excess at the 1.3 m high breathing zone of a typical standing cook. This implies that the current emission rate targets may be overly conservative under these conditions, precluding use of some types of biomass cookstoves that may actually be able to meet air quality guidelines. Future modeling and empirical studies should be conducted to understand under what conditions the use of the box model is appropriate, and when it should be modified for more accurate incorporation of the effects of stratification.33,0the top perimeter (Fig. 3). Small computer fans (Qualtek FAD1-06020CSHW11 rated at 1.44 W @12 V and 16 CFM) covered the holes at the top perimeter to force air out of the test kitchen at a constant rate. In Phase 1 of thehttps://www.sciencedirect.com/science/article/pii/S0973082619307471QualTek
2011Weiss, L;Bernstein, S;Jones, R;Amunugama, R;Krizman, D;Jebailey, L;Almogi-Hazan, O;Hazan, O;Yekhtin, Z;Yachtin, J;Shiner, R;Reibstein, I;Triche, E;Slavin, S;Or, R;Barnea, ER;Preimplantation factor (PIF) analog prevents type I diabetes mellitus (TIDM) development by preserving pancreatic function in NOD miceEndocrine41-5440121424847Preimplantation factor (PIF) is a novel embryo-secreted immunomodulatory peptide. Its synthetic analog (sPIF) modulates maternal immunity without suppression. There is an urgent need to develop agents that could prevent the development of type 1 diabetes mellitus (TIDM). Herein, we examine sPIF's preventive effect on TIDM development by using acute adoptive-transfer (ATDM) and spontaneously developing (SDM) in non-obese diabetic (NOD) murine models. Diabetes was evaluated by urinary and plasma glucose, intraperitoneal glucose tolerance test (IPGTT), pancreatic islets insulin staining by immunohistochemistry and by pancreatic proteome evaluation using mass spectrometry, followed by signal pathway analysis. Continuous administration of sPIF for 4-weeks prevents diabetes development in ATDM model in >90% of recipients demonstrated by normal IPGTT, preserved islets architecture, number, and insulin staining. (P < 0.01). sPIF effect was specific; its protective effects are not replicated by scrambled PIF (χ(2) = 0.009) control. sPIF led also to increased circulating Th2 and Th1 cytokines. In SDM model, 4-week continuous sPIF administration prevented onset of diabetes for 21 weeks post-therapy (P < 0.01). Low-dose sPIF administration for 16 weeks prevented diabetes development up to 14 weeks post-therapy, evidenced by preserved islets architecture and insulin staining. In SDM model, pancreatic proteome pathway analysis demonstrated that sPIF regulates protein traffic, prevents protein misfolding and aggregation, and reduces oxidative stress and islets apoptosis, leading to preserved insulin staining. sPIF further increased insulin receptor expression and reduced actin and tubulin proteins, thereby blocking neutrophil invasion and inflammation. Exocrine pancreatic function was also preserved. sPIF administration results in marked prevention of spontaneous and induced adoptive-transfer diabetes suggesting its potential effectiveness in treating early-stage TIDM.33,0Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah University Hospital Ein Kerem, Hebrew University, Jerusalem, Israel Lola Weiss, Osnat Hazan, Janna Yachtin, Reut Shiner, Israel Reibstein & Reuven Or QualTek Molecular Laboratories, Santa Barbara, CA, USA Steve Bernsteinhttps://link.springer.com/article/10.1007/s12020-011-9438-5QualTek
2011Bunting, RA;Duffy, KE;Lamb, RJ;San Mateo, LR;Smalley, K;Raymond, H;Liu, X;Petley, T;Fisher, J;Beck, H;Flavell, RA;Alexopoulou, L;Ward, CK;Novel antagonist antibody to TLR3 blocks poly(I:C)-induced inflammation in vivo and in vitroCell. Immunol.9-16267121092943Toll-like receptor 3 (TLR3) binds and signals in response to dsRNA and poly(I:C), a synthetic double stranded RNA analog. Activation of TLR3 triggers innate responses that may play a protective or detrimental role in viral infections or in immune-mediated inflammatory diseases through amplification of inflammation. Two monoclonal antibodies, CNTO4685 (rat anti-mouse TLR3) and CNTO5429 (CDRs from CNTO4685 grafted onto a mouse IgG1 scaffold) were generated and characterized. These mAbs bind the extracellular domain of mouse TLR3, inhibit poly(I:C)-induced activation of HEK293T cells transfected with mTLR3, and reduce poly(I:C)-induced production of CCL2 and CXCL10 by primary mouse embryonic fibroblasts. CNTO5429 decreased serum IL-6 and TNFα levels post-intraperitoneal poly(I:C) administration, demonstrating in vivo activity. In summary, specific anti-mTLR3 mAbs have been generated to assess TLR3 antagonism in mouse models of inflammation. 2010 Elsevier Inc. All rights reserved.33,0Mouse tissues (pancreas, inguinal and mesenteric lymph node, brain, liver, heart, trachea, colon, kidneys, lungs, and spleen) from three C57BL/6 and three TLR3KO mice were fixed in 10% neutral buffered formalin and processed for immunohistochemistry (Qualtek Inc.)https://www.sciencedirect.com/science/article/pii/S0008874910002819QualTek
2020Sebest, P;Ostatna, V;Fojta, M;Danhel, A;Constant-current chronopotentiometric stripping detection of bovine serum albumin on silver amalgam particlesJournal of Electroanalytical Chemistry113854859Highly sensitive peak H registered by constant-current chronopotentiometric stripping (CPS) analysis relates due to the catalytic hydrogen evolution reaction of proteins adsorbed on mercury-based surfaces including recently introduced silver amalgam particles (AgAPs). Since the peak H allows a structure-sensitive analysis of proteins, including those important in biomedicine and cancer research, it has been systematically studied by CPS analysis using AgAPs in this work. Conditions of AgAPs' electrodeposition on basal plane pyrolytic graphite electrode (bPGE) were optimized towards CPS analysis of model protein (bovine serum albumin, BSA) using peak H. Optimized condition for bPGE-AgAP preparation allowed sensitive CPS detection of either native or denatured BSA down to 10 nM concentration, a discrimination between both forms, and registration of the BSA thermal denaturation kinetic. Obtained results have confirmed the applicability of AgAPs in protein electroanalysis and may contribute to the further development of novel (bio)sensors or (bio)electrochemical applications using micro-volume batch, flow-through or lab-on-chip detection system32,0in three-electrode setup composed of: i) base plane PGE (bPGE,od 3.0 mm, Momentive) as working electrode, together with ii) refillable miniature Ag/AgCl/3 M KCl reference electrode (eDAQ) placed into the salt bridge composed of heat shrink Teflon tube (Qualtek) enclosed byhttps://www.sciencedirect.com/science/article/pii/S1572665720300370QualTek
2011Trejos, AL;Jayaraman, S;Patel, RV;Naish, MD;Schlachta, CM;Force sensing in natural orifice transluminal endoscopic surgerySurg Endosc186-19225120559663Natural orifice transluminal endoscopic surgery (NOTES) may represent the next frontier for therapeutic minimally invasive surgery; however, its feasibility is currently limited by the lack of suitable instruments. Identifying the forces required to manipulate tissue during NOTES is a necessary first step in the development of better instrumentation. Sensorized instruments were used to measure the forces acting at the tip of the instruments during transgastric and transperineal NOTES procedures performed in two female pigs. The maximum and average forces when handling tissue were determined and compared. The results show that, for the transgastric approach, the average forces required are significantly less than in the transperineal approach (43% less), and that the maximum forces required are almost 8 and 16 N in the transgastric and transperineal approaches, respectively. The forces were higher than 5 N in 1.6% of the measurements in the transgastric approach and 2.9% in the transperineal approach. This study presents an experimental measurement of tissue manipulation forces in a NOTES procedure. This information may be valuable for research groups interested in developing NOTES instruments and devices. It is recommended that NOTES instruments be designed to easily handle forces as high as 16 N.32,0allowing the flexible shaft to bend freely. The shaft and the wires were covered with heat-shrink thin-wall tubing (0.26 mm wall thickness, model Q-150K 1/8-02-QB481N- 25; Qualtek Electronics). The 2.8-mm instruments with thehttps://link.springer.com/article/10.1007/s00464-010-1155-2QualTek
2016Sorensen, SF;Zhou, W;Dolled-Filhart, M;Georgsen, JB;Wang, Z;Emancipator, K;Wu, D;Busch-Sørensen, M;Meldgaard, P;Hager, H;PD-L1 Expression and Survival among Patients with Advanced Non-Small Cell Lung Cancer Treated with ChemotherapyTransl Oncol64-699126947883Recent clinical trial results have suggested that programmed cell death ligand 1 (PD-L1) expression measured by immunohistochemistry may predict response to anti-programmed cell death 1 (PD-1) therapy. Results on the association between PD-L1 expression and survival among patients with advanced non-small cell lung cancer (NSCLC) treated with chemotherapy are inconsistent. We evaluated the relationship between PD-L1 expression and overall survival (OS) among 204 patients with advanced NSCLC treated at Aarhus University Hospital, Aarhus, Denmark, from 2007 to 2012. PD-L1 expression was measured using a prototype immunohistochemistry assay with the anti-PD-L1 22C3 antibody (Merck). PD-L1 strong positivity and weak positivity were defined to be traceable to the clinical trial version of the assay. Twenty-five percent of patients had PD-L1 strong-positive tumors, and 50% had PD-L1 weak-positive tumors. No statistically significant association was found between PD-L1 expression and survival; adjusted hazard ratio of 1.34 (95% confidence interval, 0.88-2.03; median OS, 9.0 months) for the PD-L1 strong-positive group and 1.07 (0.74-1.55; median OS, 9.8 months) for the PD-L1 weak-positive group compared with the PD-L1-negative group (median OS, 7.5 months). No association was seen between PD-L1 expression and OS when PD-L1 expression levels were stratified by median or tertiles. In concordance with previous studies, we found PD-L1 measured by immunohistochemistry to be frequently expressed in patients with advanced NSCLC. However, PD-L1 expression is not a strong prognostic marker in patients with advanced NSCLC treated with chemotherapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.31,0gen retrieval, along with a 10-minute proteinase K incubation (#S3020, Dako) following a 1 × wash buffer rinse (#K8012 Envision FLEX + kit, Dako) as part of automated staining. Automated staining (QualTek TekMate 500, Newtown, PA) was followed by peroxidase blocking (#K8012, Dako). Slides were incubated overnight with the 22C3 antibody with a primary antibody diluent (#S0908 Primary Antibody Dihttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800057QualTek
2002Kumar, S;Hanning, CR;Brigham-Burke, MR;Rieman, DJ;Lehr, R;Khandekar, S;Kirkpatrick, RB;Scott, GF;Lee, JC;Lynch, FJ;Gao, W;Gambotto, A;Lotze, MT;Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma productionCytokine61-7118212096920We have recently reported the identification of four novel members of the interleukin-1 (IL-1) family which we designated as IL-1 homologue 1-4 (IL-1H1-4). These proteins exhibit significant sequence homology to other members of the IL-1 family. Of these homologues, only IL-1H4 (renamed IL-1F7b) was predicted to contain a propeptide domain and a caspase cleavage site. We now report that caspase-1 cleaves IL-1F7b at the predicted site to generate mature IL-1F7b. Caspase-4 was also able to process IL-1F7b, albeit inefficiently. Other caspases and Granzyme-B did not cleave IL-1F7b. Furthermore, adenovirus-mediated expression of IL-1F7b in HEK 293 cells led to in situ processing and secretion of mature IL-1F7b. In a screen to identify a potential receptor, both pro and mature IL-1F7b bound to the soluble IL-18 receptor alpha-Fc (IL-18Ralpha-Fc) but not to the soluble IL-1R-Fc or ST2R-Fc fusion proteins. Mature IL-1F7b bound to the IL-18Ralpha-Fc protein with higher affinity than the pro form, although the affinities for both proteins were significantly lower than that observed for IL-18. Consistent with this observation, only IL-18 and not IL-1F7b induced IFN-gamma production by KG1a cells. We also report that pro and mature IL-1F7b form homodimers with association constants of 4 microM and 5 nM, respectively, suggesting biological relevance to IL-1F7b processing. Finally, we have localized the expression of IL-1F7b protein in discrete cell populations including plasma cells and tumor cells. These data suggest that IL-1F7b may be involved in immune response, inflammatory diseases and/or cancer. Copyright 2002 Elsevier Science Ltd. All rights reserved.31,0http://dx.doi.org/10.1006/cyto.2002.0873Frank Lynch[au]
2020Bio, S;Nunes, B;Acute effects of diclofenac on zebrafish: Indications of oxidative effects and damages at environmentally realistic levels of exposureEnviron. Toxicol. Pharmacol.1033947832320907With the increasing awareness about the contamination of the aquatic environment by pharmaceuticals, there is a growing need to study their adverse effects on aquatic organisms. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), whose wide use contributes for its presence in freshwater ecosystems, increasing the probability of causing deleterious changes in aquatic biota. This study evaluated possible oxidative stress effects in Danio rerio embryos and larvae when exposed to a range of ecologically relevant concentrations of diclofenac. It was possible to conclude that diclofenac caused a scenario of oxidative stress, since all tested toxicological parameters were responsive to the drug. In general, diclofenac caused not only significant anti-oxidant adaptive responses for most levels of exposure, but also peroxidative damage. This work evidenced the responsiveness of D. rerio towards diclofenac in environmentally relevant concentrations, which shows that these organisms might face a scenario of oxidative stress in their natural habitat. Copyright © 2020. Published by Elsevier B.V.31,0With the increasing awareness about the contamination of the aquatic environment by pharmaceuticals, there is a growing need to study their adverse effects on aquatic organisms. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), whose wide usehttps://www.sciencedirect.com/science/article/pii/S1382668920300703"Sofia Bio"
2020Piedade, F;Bio, S;Nunes, B;Effects of common pharmaceutical drugs (paracetamol and acetylsalicylic acid) short term exposure on biomarkers of the mussel Mytilus sppEnviron. Toxicol. Pharmacol.1032767331704586Pharmaceutical drugs in the wild may pose significant risks to non-target exposed organisms. This situation is even more troublesome for coastal marine or estuarine environments, located in the vicinity of large human conglomerates, for which the putative number of pollutants is extremely high, and the regime by which wild organisms are exposed is continuous. In addition, the number of studies addressing this issue is still scarce, despite evidences that show the potential contamination profiles and adverse biological effects in organisms from such areas. In this study, the ecotoxicity of common pharmaceutical drugs (namely paracetamol and acetylsalicylic acid) was assessed, by studying the susceptibility of the mussel species Mytilus spp to oxidative stress after being exposed for 96 h to increasing but ecologically relevant concentrations of the two mentioned pharmaceuticals (paracetamol: 0, 0.5, 5, 50, and 500 μg/L; acetylsalicylic acid: 0, 0.1, 1, 10, and 100 μg/L). The oxidative status in exposed organisms was analyzed by measuring oxidative stress biomarkers, namely catalase (CAT), glutathione-S-transferases (GSTs), and lipoperoxidation (LPO) levels, whose alteration was indicative of chemical exposure, in both digestive gland and gills of the organisms. In addition, the food uptake and the nutritional reserve status of exposed organisms were also assessed, by measuring the consumption of ingested food, and levels of tissue reserves of glycogen in gills and digestive gland. No significant alterations were observed in the assessed oxidative stress parameters so it was possible to hypothesize that the studied drugs may have probably exerted a limited alteration of antioxidant defenses and damage, which was reverted by the activation of defensive adaptive mechanisms. This set of data evidenced that the pro-oxidative metabolism that was already described for both drugs in other animal models, was not fully established in the exposed mussels. On the contrary, glycogen reserves were substantially changed after exposure to both toxicants, being possible to observe opposite responses caused by both drugs. Food uptake was not altered following exposure to the drugs. Further evaluations are thus required to conclude about both drugs ecotoxicity and other parameters, namely seasonality, which should be considered when performing ecotoxicology tests, especially with the selected species. Copyright © 2019 Elsevier B.V. All rights reserved.31,0Pharmaceutical drugs in the wild may pose significant risks to non-target exposed organisms. This situation is even more troublesome for coastal marine or estuarine environments, located in the vicinity of large human conglomerates, for which the putativehttps://www.sciencedirect.com/science/article/pii/S1382668919301504"Sofia Bio"
2019Havird, JC;Meyer, E;Fujita, Y;Vaught, RC;Henry, RP;Santos, SR;Disparate responses to salinity across species and organizational levels in anchialine shrimpsJ. Exp. Biol.222Pt 2431727759Environmentally induced plasticity in gene expression is one of the underlying mechanisms of adaptation to habitats with variable environments. For example, euryhaline crustaceans show predictable changes in the expression of ion-transporter genes during salinity transfers, although studies have typically been limited to specific genes, taxa and ecosystems of interest. Here, we investigated responses to salinity change at multiple organizational levels in five species of shrimp representing at least three independent invasions of the anchialine ecosystem, defined as habitats with marine and freshwater influences with spatial and temporal fluctuations in salinity. Although all five species were generally strong osmoregulators, salinity-induced changes in gill physiology and gene expression were highly species specific. While some species exhibited patterns similar to those of previously studied euryhaline crustaceans, instances of distinct and atypical patterns were recovered from closely related species. Species-specific patterns were found when examining: (1) numbers and identities of differentially expressed genes, (2) salinity-induced expression of genes predicted a priori to play a role in osmoregulation, and (3) salinity-induced expression of orthologs shared among all species. Notably, ion transport genes were unchanged in the atyid Halocaridina rubra while genes normally associated with vision and light perception were among those most highly upregulated. Potential reasons for species-specific patterns are discussed, including variation among anchialine habitats in salinity regimes and divergent evolution in anchialine taxa. Underexplored mechanisms of osmoregulation in crustaceans revealed here by the application of transcriptomic approaches to ecologically and taxonomically understudied systems are also explored. © 2019. Published by The Company of Biologists Ltd.30,0Assembly of reference transcriptomes was previously described for H. rubra (Havird and Santos, 2016a) and the four species from Japan (Havird et al., 2014b). Briefly, total RNA was extracted from whole animals 1 month after preservation in RNAlater via beadbeating in Trizol and using an RNeasy Kit (Qiagen). Synthesis of cDNA libraries utilized a SMART cDNA construction kit (ClonTech) and libraries were sent to the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA) for indexing and subsequent sequencing on an Illumina HiSeq 2000, with 100 bp paired-end reads delivered in FASTQ format. Raw reads were digitally normalized and assembled using Trinity version Trinityrnaseq_r20131110 (Grabherr et al., 2011) with default parameters. The ‘completeness’ of the five reference transcriptomes was assessed with BUSCO version 3 to search for 978 single-copy orthologs conserved across metazoans (Simão et al., 2015). Transcriptomes are available from the Transcriptome Shotgun Assembly Sequence Database (NCBI TSA; accession numbers in Table S1 of figshare dataset – see ‘Data availability’ section, below) and Dryad (doi:10.5061/dryad.jn8t1) while raw reads are available from the Sequence Read Archive (NCBI SRA; accession numbers in Table S2 of figshare dataset – see ‘Data availability’ section, below)https://jeb.biologists.org/content/222/24/jeb211920.abstract"HudsonAlpha Genomic Services"
2020Lin, J;Song, AJ;Hoffman-Censits, J;Leiby, BE;Tuluc, M;Shaw, C;Harshyne, L;Kean, R;Bar-Ad, V;Den, RB;Hurwitz, MD;Louie, J;Philipose, S;Deshmukh, SP;Johnson, JM;Dicker, AP;Hooper, DC;Kelly, WK;Lu, B;A Pilot Study of Radiation Therapy in Combination With Pembrolizumab in Patients With Metastatic Renal Cell CancerAm. J. Clin. Oncol.82-8643231693508There is no study published regarding the benefit of radiation therapy (RT) in combination with immune checkpoint inhibitors (ICIs) for the treatment of metastatic renal cell cancer (mRCC). This report is part of an exploratory study aiming to determine the immunomodulatory activity of RT alone or in combination with pembrolizumab in solid tumors. mRCC patients were treated with a combination of RT (8 Gy×1 or 4 Gy×5) followed by pembrolizumab with or without lead-in dose of pembrolizumab. Treatment response was measured based on the modified Response Evaluation Criteria in Solid Tumors criteria. Adverse events were monitored and graded. Pre-RT and post-RT tumor biopsies were obtained to evaluate programmed death-ligand 1 expression. Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. Twelve mRCC patients who progressed on prior antiangiogenic therapy were enrolled. Half had 2 lines of prior therapy. Two patients (16.7%) had partial responses and were on study for 12.4 and 14.5 months. Three patients had stable disease for a period ranging from 4.2 to 10.4 months, whereas 7 patients had progressive disease. Median progression-free survival was 8.6 months and median overall survival was 32.3 months. Three patients had grade ≥3 events (hyperglycemia, thrombocytopenia, transaminitis). Biopsied tissue programmed death-ligand 1 expression and tumor-infiltrating lymphocytes were numerically higher in responders comparing to nonresponders (Modified Proportion Score 45% vs. 30.45%; tumor-infiltrating lymphocytes odds ratio 4.92). Combining RT with pembrolizumab in pretreated mRCC is well-tolerated and appears to have comparable efficacy with single-agent nivolumab.30,0When possible preradiation and post- radiation biopsy material was compared. Biopsy samples were analyzed by a QualTek pathologist (King of Prussia, PA) and Merck 22C3 antibody was used to score PD-L1 membrane expressionhttps://www.ingentaconnect.com/content/wk/ajco/2020/00000043/00000002/art00002QualTek
2018Cohen, RB;Delord, JP;Doi, T;Piha-Paul, SA;Liu, SV;Gilbert, J;Algazi, AP;Damian, S;Hong, RL;Le Tourneau, C;Day, D;Varga, A;Elez, E;Wallmark, J;Saraf, S;Thanigaimani, P;Cheng, J;Keam, B;Pembrolizumab for the Treatment of Advanced Salivary Gland Carcinoma: Findings of the Phase 1b KEYNOTE-028 StudyAm. J. Clin. Oncol.29462123Treatment options for patients with unresectable or metastatic salivary gland carcinoma (SGC) are limited. Safety and efficacy of pembrolizumab for SGC expressing programmed death ligand 1 (PD-L1) were explored. A cohort of patients with advanced, PD-L1-positive SGC was enrolled in the nonrandomized, multicohort, phase Ib trial of pembrolizumab in patients with PD-L1-positive advanced solid tumors (KEYNOTE-028; NCT02054806). Key inclusion criteria included recurrent or metastatic disease, failure of prior systemic therapy, and PD-L1 expression on ≥1% of tumor or stroma cells (per a prototype immunohistochemistry assay). Patients received pembrolizumab 10 mg/kg every 2 weeks for ≥2 years or until confirmed disease progression or unacceptable toxicity. Primary end point was objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 by investigator review. Twenty-six patients with PD-L1-positive SGC were enrolled and treated; median age was 57 years, 88% were men, and 74% had received prior therapy for recurrent/metastatic disease. Confirmed objective response rate after median follow-up of 20 months was 12% (95% confidence interval, 2%-30%), with 3 patients achieving partial response; there were no complete responses. Median duration of response was 4 months (range, 4 to 21 mo). Treatment-related adverse events occurred in 22 patients (85%), resulting in discontinuation in 2 patients and death in 1 (interstitial lung disease); those occurring in ≥15% of patients were diarrhea, decreased appetite, pruritus, and fatigue. Pembrolizumab demonstrated promising antitumor activity and a manageable safety profile in patients with advanced, PD-L1-positive SGC.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.30,0Tumor PD-L1 positivity was determined at a central laboratory using formalin-fixed archived or newly obtained biopsy samples and a prototype immunohistochemical assay (QualTek Molecular Laboratories, Goleta, CA),17 based on the 22C3 antibody (Merck & Co ;tribution by the investigator. Tumor PD-L1 positivity was determined at a central laboratory using formalin-fixed archived or newly obtained biopsy samples and a prototype immunohistochemical assay (QualTek Molecular Laboratories, Goleta, CA),17 based on the 22C3 antibody (Merck & Co. Inc., Kenilworth, NJ). Positivity was defined as membranous staining on ≥1% modified proportion score or interfhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211783/QualTek
2000Johnson, LV;Ozaki, S;Staples, MK;Erickson, PA;Anderson, DH;A potential role for immune complex pathogenesis in drusen formationExp. Eye Res.441-44970410865992Drusen are abnormal extracellular deposits that accumulate between the retinal pigmented epithelium and Bruch's membrane and are commonly associated with age-related macular degeneration. Our recent work has identified a number of plasma proteins as molecular components of drusen. Of interest is the fact that many of these drusen-associated molecules are acute phase reactant proteins and some have established roles in mediating immune responsiveness. As immune and inflammatory responses appear to play a role in the formation of other pathologic age-related deposits, we examined the distribution of immunoglobulin molecules and terminal complement complexes at sites of drusen deposition. Here, we report that concentrations of immunoglobulin G and terminal C5b-9 complement complexes are present in drusen. In addition, we observe that retinal pigmented epithelial cells overlying or directly adjacent to drusen, as well as some within apparently normal epithelia, exhibit cytoplasmic immunoreactivity for immunoglobulin and the C5 component of complement. Taken together, these results suggest that drusen biogenesis may be a byproduct of immune responsiveness, and they implicate immune complex-mediated pathogenesis involving retinal pigmented epithelial cells as an initiating event in drusen formation.30,0ANDERSONa a Center for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA, b QualTek Molecular Laboratories, Santa Barbara, CA 93111, USA (Receivedhttps://www.sciencedirect.com/science/article/pii/S0014483599907984QualTek
2000Yeung, G;Mulero, JJ;McGowan, DW;Bajwa, SS;Ford, JE;CD39L2, a gene encoding a human nucleoside diphosphatase, predominantly expressed in the heartBiochemistry12916-12923394211041856E-NTPDases are extracellular enzymes that hydrolyze nucleotides. The human E-NTPDase gene family currently consists of five reported members (CD39, CD39L1, CD39L2, CD39L3, and CD39L4). Both membrane-bound and secreted family members have been predicted by encoded transmembrane and leader peptide motifs. In this report, we demonstrate that the human CD39L2 gene is expressed predominantly in the heart. In situ hybridization results from heart indicate that the CD39L2 message is expressed in muscle and capillary endothelial cells. We also show that the CD39L2 gene encodes an extracellular E-NTPDase. Flow cytometric experiments show that transiently expressed CD39L2 is present on the surface of COS-7 cells. Transfected cells also produce recombinant glycosylated protein in the medium, and this process can be blocked by brefeldin A, an inhibitor of the mammalian secretory pathway. The enzymology of CD39L2 shows characteristic features of a typical E-NTPDase, but with a much higher degree of specificity for NDPs over NTPs as enzymatic substrates. The kinetics of the ADPase activity exhibit positive cooperativity. The predominance of CD39L2 expression in the heart supports a functional role in regulating platelet activation and recruitment in this organ.30,0the manufacturer. Automated in situ hybridization was performed by QualTek Molecular labs (Santa Barbara, CA) using a modified version of a previously published procedure (17). The Ventana Medical Systems, Inc. (Tucsonhttps://pubs.acs.org/doi/abs/10.1021/bi000959zQualTek
2019Mehnert, JM;Varga, A;Brose, MS;Aggarwal, RR;Lin, CC;Prawira, A;de Braud, F;Tamura, K;Doi, T;Piha-Paul, SA;Gilbert, J;Saraf, S;Thanigaimani, P;Cheng, JD;Keam, B;Safety and antitumor activity of the anti-PD-1 antibody pembrolizumab in patients with advanced, PD-L1-positive papillary or follicular thyroid cancerBMC Cancer19619130832606Treatment options for advanced thyroid cancer refractory to standard therapies are limited. The safety and efficacy of pembrolizumab were evaluated in patients with advanced differentiated thyroid cancer expressing programmed death ligand 1 (PD-L1). Patients with advanced thyroid cancer were enrolled in the nonrandomized, phase Ib KEYNOTE-028 trial conducted to evaluate safety and antitumor activity of the anti-programmed death 1 (PD-1) antibody pembrolizumab in advanced solid tumors. Key eligibility criteria were advanced papillary or follicular thyroid cancer, failure of standard therapy, and PD-L1 expression in tumor or stroma cells (assessed by immunohistochemistry). Pembrolizumab 10 mg/kg was administered every 2 weeks up to 24 months or until confirmed progression or intolerable toxicity. The primary endpoint was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors, version 1.1. Twenty-two patients were enrolled: median age was 61 years; 59% were women; and 68% had papillary carcinoma. Median follow-up was 31 months (range, 7-34 months). Treatment-related adverse events were observed in 18 (82%) patients; those occurring in ≥15% of patients were diarrhea (n = 7) and fatigue (n = 4). One grade ≥ 3 treatment-related adverse event occurred (colitis, grade 3); no treatment-related discontinuations or deaths occurred. Two patients had confirmed partial response, for an ORR of 9% (95% confidence interval [CI], 1-29%); response duration was 8 and 20 months. Median progression-free survival was 7 months (95% CI, 2-14 months); median overall survival was not reached (95% CI, 22 months to not reached). Results of this phase Ib proof-of-concept study suggest that pembrolizumab has a manageable safety profile and demonstrate evidence of antitumor activity in advanced differentiated thyroid cancer in a minority of patients treated. Further analyses are necessary to confirm these findings. Clinicaltrials.gov identifier: NCT02054806 . Registered 4 February 2014.29,0biopsy sample. PD-L1 expression was assessed using a prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, California) [22] and the 22C3 antibody (Merck & Co., Inc., Kenilworth, New Jersey). PD ;screening period using either an archived formalin-fixed, paraffin-embedded tumor sample or a newly obtained biopsy sample. PD-L1 expression was assessed using a prototype immunohistochemistry assay (QualTek Molecular Laboratories, Goleta, California) [22] and the 22C3 antibody (Merck & Co., Inc., Kenilworth, New Jersey). PD-L1 positivity was defined as membranous staining on ≥1% modified proporhttps://link.springer.com/article/10.1186/s12885-019-5380-3QualTek
2011Karkera, J;Steiner, H;Li, W;Skradski, V;Moser, PL;Riethdorf, S;Reddy, M;Puchalski, T;Safer, K;Prabhakar, U;Pantel, K;Qi, M;Culig, Z;The anti-interleukin-6 antibody siltuximab down-regulates genes implicated in tumorigenesis in prostate cancer patients from a phase I studyProstate1455-1465711321321981Interleukin-6 (IL-6) is associated with prostate cancer morbidity. In several experimental models, IL-6 has been reported to have anti-apoptotic and pro-angiogenic effects. Siltuximab (CNTO 328) is a monoclonal anti-IL-6 antibody which has been successfully applied in several models representing prostate cancer. This study was designed to assess preliminary safety of siltuximab in patients with early prostate cancer. Twenty patients scheduled to undergo radical prostatectomy received either no drug or siltuximab (6 mg/kg, five patients per group with administration once, two times, and three times prior to surgery). Blood samples were collected for pharmacokinetic and pharmacodynamic analyses. Expression of elements of IL-6 signaling pathways was analyzed in tumor tissue by immunohistochemistry. Gene analysis in tumor specimens was performed with the DASL array. No adverse events related to siltuximab were observed. Patients treated with siltuximab presented with higher levels of proliferation and apoptosis markers. Following a single dose, serum concentrations of siltuximab declined in a biexponential manner. This study revealed a decrease in phosphorylation of Stat3 and p44/p42 mitogen-activated protein kinases. In addition, gene expression analyses indicate down-regulation of genes immediately downstream of the IL-6 signaling pathway and key enzymes of the androgen signaling pathway. Preliminary safety of siltuximab is favorable. Future studies in which siltuximab could be combined with androgen-deprivation therapy and experimental therapies in advanced prostate cancer are justified. Copyright © 2011 Wiley-Liss, Inc.29,0PSA, IL‐6R, alpha‐methylacyl‐CoA racemase (AMACR), AR, and Chromogranin A were tested in tumor tissue samples at Qualtek Laboratories (Goleta, CA). Steam heat‐induced epitope retrieval (SHIER) method was used and immunohistochemical procedure was performedhttps://onlinelibrary.wiley.com/doi/abs/10.1002/pros.21362QualTek
2004Kalos, M;Askaa, J;Hylander, BL;Repasky, EA;Cai, F;Vedvick, T;Reed, SG;Wright, GL;Fanger, GR;Prostein expression is highly restricted to normal and malignant prostate tissuesProstate246-25660315176054Prostein is a recently described molecule expressed at the mRNA level in a prostate-specific manner. A murine monoclonal antibody was developed, characterized, and used to evaluate the expression of prostein protein in prostatic, other normal tissue and tumor samples. The murine anti-prostein monoclonal antibody 10E3-G4-D3 was generated using recombinant prostein. ELISA, FACS, and Western analyzes were used to characterize 10E3-G4-D3. Immunohistochemistry was used to characterize the expression of prostein in tissues. 10E3-G4-D3 specifically recognizes a linear intracellular epitope of prostein. IHC analysis demonstrates that prostein is expressed in the vast majority of normal and malignant prostatic tissues, regardless of grade and metastatic status. No protein expression is detected in a panel of approximately 4,700 normal and malignant tissue samples representing the great majority of essential tissues and tumors. Prostein is exquisitely specific for prostate tissues, indicating a potential clinical utility of 10E3-G4-D3 as a diagnostic biomarker, and support the use of prostein as a novel target for development of prostein-specific antibody and T-cell based therapeutic strategies for prostate cancer. Copyright 2004 Wiley-Liss, Inc.29,0We thank Patrick Roche at the Mayo Clinic and Frank Lynch at Qualtek for performing immunohistochemistry experiments and Masazumi Matsamura for initial work on the generation of the 10E3-G4-D3 monoclonal antibodyhttps://onlinelibrary.wiley.com/doi/abs/10.1002/pros.20043QualTek
2017Aycock, KI;Campbell, RL;Lynch, FC;Manning, KB;Craven, BA;Computational predictions of the embolus-trapping performance of an IVC filter in patient-specific and idealized IVC geometriesBiomech Model Mechanobiol1957-196916628656515Embolus transport simulations are performed to investigate the dependence of inferior vena cava (IVC) filter embolus-trapping performance on IVC anatomy. Simulations are performed using a resolved two-way coupled computational fluid dynamics/six-degree-of-freedom approach. Three IVC geometries are studied: a straight-tube IVC, a patient-averaged IVC, and a patient-specific IVC reconstructed from medical imaging data. Additionally, two sizes of spherical emboli (3 and 5 mm in diameter) and two IVC orientations (supine and upright) are considered. The embolus-trapping efficiency of the IVC filter is quantified for each combination of IVC geometry, embolus size, and IVC orientation by performing 2560 individual simulations. The predicted embolus-trapping efficiencies of the IVC filter range from 10 to 100%, and IVC anatomy is found to have a significant influence on the efficiency results ([Formula: see text]). In the upright IVC orientation, greater secondary flow in the patient-specific IVC geometry decreases the filter embolus-trapping efficiency by 22-30 percentage points compared with the efficiencies predicted in the idealized straight-tube or patient-averaged IVCs. In a supine orientation, the embolus-trapping efficiency of the filter in the idealized IVCs decreases by 21-90 percentage points compared with the upright orientation. In contrast, the embolus-trapping efficiency is insensitive to IVC orientation in the patient-specific IVC. In summary, simulations predict that anatomical features of the IVC that are often neglected in the idealized models used for benchtop testing, such as iliac vein compression and anteroposterior curvature, generate secondary flow and mixing in the IVC and influence the embolus-trapping efficiency of IVC filters. Accordingly, inter-subject variability studies and additional embolus transport investigations that consider patient-specific IVC anatomy are recommended for future work.28,0http://dx.doi.org/10.1007/s10237-017-0931-5Frank Lynch[au]
2016Stavropoulos, SW;Chen, JX;Sing, RF;Elmasri, F;Silver, MJ;Powell, A;Lynch, FC;Abdel Aal, AK;Lansky, A;Muhs, BE;, ;Analysis of the Final DENALI Trial Data: A Prospective, Multicenter Study of the Denali Inferior Vena Cava FilterJ Vasc Interv Radiol1531-1538.e1271027569678To report the final 2-year data on the efficacy and safety of a nitinol retrievable inferior vena cava (IVC) filter for protection against pulmonary embolism (PE). This was a prospective multicenter trial of 200 patients with temporary indications for caval filtration who underwent implantation of the Denali IVC filter. After filter placement, all patients were followed for 2 years after placement or 30 days after filter retrieval. The primary endpoints were technical success of filter implantation in the intended location and clinical success of filter placement and retrieval. Secondary endpoints were incidence of clinically symptomatic recurrent PE, new or propagating deep vein thrombosis (DVT), and filter-related complications including migration, fracture, penetration, and tilt. Filter placement was technically successful in 199 patients (99.5%). Filters were clinically successful in 190 patients (95%). The rate of PE was 3% (n = 6), with 5 patients having a small subsegmental PE and 1 having a lobar PE. New or worsening DVT was noted in 26 patients (13%). Filter retrieval was attempted 125 times in 124 patients and was technically successful in 121 patients (97.6%). The mean filter dwell time at retrieval was 200.8 days (range, 5-736 d). There were no instances of filter fracture, migration, or tilt greater than 15° at the time of filter retrieval or during follow-up. The Denali IVC filter exhibited high success rates for filter placement and retrieval while maintaining a low complication rate in this clinical trial. Copyright © 2016 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2016.06.028Frank Lynch[au]
2014Stavropoulos, SW;Sing, RF;Elmasri, F;Silver, MJ;Powell, A;Lynch, FC;Aal, AK;Lansky, AJ;Settlage, RA;Muhs, BE;, ;The DENALI Trial: an interim analysis of a prospective, multicenter study of the Denali retrievable inferior vena cava filterJ Vasc Interv Radiol1497-505, 1505.e1251025066514To assess safety and effectiveness of a nitinol retrievable inferior vena cava (IVC) filter in patients who require caval interruption to protect against pulmonary embolism (PE). Two hundred patients with temporary indications for an IVC filter were enrolled in this prospective, multicenter clinical study. Patients undergoing filter implantation were to be followed for 2 years or for 30 days after filter retrieval. At the time of the present interim report, all 200 patients had been enrolled in the study, and 160 had undergone a retrieval attempt or been followed to 6 months with their filter in place. Primary study endpoints included technical and clinical success of filter placement and retrieval. Patients were also evaluated for recurrent PE, new or worsening deep vein thrombosis, and filter migration, fracture, penetration, and tilt. Clinical success of placement was achieved in 94.5% of patients (172 of 182), with a one-sided lower limit of the 95% confidence interval of 90.1%. Technical success rate of filter placement was 99.5%. Technical success rate of retrieval was 97.3%; 108 filters were retrieved in 111 attempts. In two cases, the filter apex could not be engaged with a snare, and one device was engaged but could not be removed. Filter retrievals occurred at a mean indwell time of 165 days (range, 5-632 d). There were no instances of filter fracture, migration, or tilt greater than 15° at the time of retrieval or 6-month follow-up. In this interim report, the nitinol retrievable IVC filter provided protection against pulmonary embolism, and the device could be retrieved with a low rate of complications. Copyright © 2014 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2014.07.001Frank Lynch[au]
2012Hughes, JA;Lynch, FC;Transhepatic removal of an inferior vena cava filterJ Vasc Interv Radiol983-9852372272089928,0http://dx.doi.org/10.1016/j.jvir.2012.04.006Frank Lynch[au]
2012Lynch, FC;Modified loop snare technique for the removal of bard recovery, G2, G2 express, and eclipse inferior vena cava filtersJ Vasc Interv Radiol687-69023522525025The present work describes the preliminary results of the use of a novel technique for the removal of tilted and apex-embedded Recovery, G2, G2 Express, and Eclipse inferior vena cava filters. A retrospective review was performed of 33 filters removed in 32 patients by using the described modified loop snare technique. All filters were successfully removed with the use of the technique. The average duration of filter implantation for the devices removed with the technique was 556 days (range, 11-2,437 d; median, 268 d). No filter fractures occurred related to the removal technique. No procedure-related complications occurred. Copyright © 2012 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2012.01.060Frank Lynch[au]
2012Vijay, K;Hughes, JA;Burdette, AS;Scorza, LB;Singh, H;Waybill, PN;Lynch, FC;Fractured Bard Recovery, G2, and G2 express inferior vena cava filters: incidence, clinical consequences, and outcomes of removal attemptsJ Vasc Interv Radiol188-19423222173108To increase the understanding of risks of inferior vena cava (IVC) filter fracture and embolization and the safety of removing fractured filters via retrospective review of a prospectively collected database of fractured IVC filters. A total of 63 fractured IVC filters were discovered among 548 patients presenting for retrievable filter removal between April 2004 and November 2010 at a single institution. Device type, duration of implantation, component fracture, and embolization events were recorded. Success rates and techniques for removal of components were recorded. A total of 63 fractured Recovery, G2, and G2 Express IVC filters were identified, for an overall fracture rate of 12%. Excluding foot process fractures, the fracture rate for only filter arms and/or legs was 6%. The incidence of fracture increased with longer filter dwell times. Success rates for removal of the nonfractured component (ie, main body) and fractured components (ie, arm or leg) were 98.4% and 53.4%, respectively. The distal embolization rate of fractured filter components was 13%. There were no immediate clinically significant complications associated with fracture component embolization or filter removal. A single patient was encountered with symptoms related to their fractured filter. IVC filter fracture rates increase with longer dwell times; however, removal of fractured filters and fractured components (ie, arms and legs) can be achieved safely and effectively. Clinically significant complications of IVC filter fracture are rare, and there were no immediate clinical sequelae related to embolization of fracture components. Copyright © 2012 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2011.10.005Frank Lynch[au]
2011Lynch, FC;A method for following patients with retrievable inferior vena cava filters: results and lessons learned from the first 1,100 patientsJ Vasc Interv Radiol1507-1512221121903414Patients who have undergone implantation of a retrievable inferior vena cava (IVC) filter require continued follow-up to have the device removed when clinically appropriate and in a timely fashion to avoid potential long-term filter-related complications. The efficacy of a method for patient follow-up was evaluated based on a retrospective review of a single-institutional retrievable IVC filter experience. Patients with retrievable IVC filters were tracked via a prospectively collected database designed specifically for patient follow-up. Follow-up consisted of periodic review of the electronic medical record. Patients were contacted by mail (at regular intervals one or more times) when removal of the filter was deemed appropriate. A retrospective review of the ultimate fate of the first 1,127 retrievable IVC filters placed at a single institution was performed. Retrieval rates were compared with those seen in the initial experience, during which no structured follow-up was performed. Of 1,127 filters placed, 658 (58.4%) were removed. Filter removal or declaration of the device as permanent was achieved in 860 patients (76.3%). Filter removal, declaration of the device as permanent, or establishment of the need for continued follow-up was achieved in 941 patients (83.5%). Only 186 patients (16.5%) were lost to follow-up. The follow-up method described in the present study resulted in a statistically significant difference (P < .001) in the likelihood of a patient returning for IVC filter removal compared with a lack of follow-up (59% vs 24%). Copyright © 2011 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2011.07.019Frank Lynch[au]
2011Shaw, CM;Scorza, LB;Waybill, PN;Singh, H;Lynch, FC;Optional vena cava filter use in the elderly populationJ Vasc Interv Radiol824-82822621530308To review utility, safety, and efficacy of optional inferior vena cava (IVC) filters in patients 65 years or older at a single institution over a 6-year period. Retrospective review of permanent and optional IVC filters placed in elderly patients was performed. Older and younger groups were compared based on technical success of filter placement and clinical success measured by recurrent pulmonary embolism (PE) or thrombotic complications. The rate of successful filter removal was compared with that in the cohort of patients of all ages who received optional filters. Fifty-three patients received an optional filter and 445 received a permanent filter. Technical success rates for filter placement in the permanent and optional filter groups were 99.8% (447 of 448) and 98.1% (53 of 54), respectively (P = .51). Rates of PE after filter placement were 0% and 1.4% (five of 359) in the optional and permanent filter groups, respectively (P = .87). Incidences of deep vein thrombosis were 12% (six of 50) and 4.5% (16 of 359) in optional and permanent filter recipients, respectively (P = .06). Filter retrieval was attempted in 55.6% of optional filter recipients (30 of 54), similar to that seen in patients of any age with optional filters. Retrieval was unsuccessful in one patient in whom a suprarenal IVC filter was placed. Optional filters are safe and effective in patients aged 65 years or older. Age alone is a poor predictor of a clinical opportunity to remove a filter. With appropriate patient selection and aggressive follow-up, retrieval rates comparable with those in younger populations can be achieved. Copyright © 2011 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2011.03.008Frank Lynch[au]
2011Lynch, FC;Removal of a Günther Tulip filter after 3,006 daysJ Vasc Interv Radiol337-34022321277802Patients may be denied the opportunity to have their inferior vena cava (IVC) filters removed because of a perception that retrievable filters that have been in place for a long period of time may be more technically difficult or hazardous to remove. A case report on the removal of a Günther Tulip filter that was implanted for a total of 3,006 days is presented. This case report adds to the literature that suggests that no time limits may exist after which many retrievable IVC filters can no longer be safely removed. Copyright © 2011 SIR. Published by Elsevier Inc. All rights reserved.28,0http://dx.doi.org/10.1016/j.jvir.2010.11.016Frank Lynch[au]
2009Binkert, CA;Drooz, AT;Caridi, JG;Sands, MJ;Bjarnason, H;Lynch, FC;Rilling, WS;Zambuto, DA;Stavropoulos, SW;Venbrux, AC;Kaufman, JA;Technical success and safety of retrieval of the G2 filter in a prospective, multicenter studyJ Vasc Interv Radiol1449-1453201119875062To assess the technical success and safety for retrieval of the G2 filter. The authors performed a prospective, multicenter study of 100 patients with temporary indication for caval interruption. Patients were enrolled consecutively between December 2005 and July 2006. There were 67 men and 33 women with a mean age of 52.1 years (range, 19-82 years). Indications for filter placement were trauma (n = 56), perioperative risk (n = 16), and medical indications (n = 28). Forty-two patients had venous thromboembolism at filter placement. Fifty-eight filters were placed prophylactically. Retrieval was attempted in 61 patients. Fifty-eight of the 61 filters (95%) were successfully retrieved after a mean dwell time of 140 days (range, 5-300 days). In all failed retrievals, the filter tip was against the caval wall. There was no difference in dwell times between successful and unsuccessful retrievals. Although there were no cases of cranial migration, caudal migrations were observed in 12% of cases (10 of 85 patients with a complete data set). Other device-related complications included filter fracture (1/85, 1.2%), filter tilt of more than 15 degrees (15/85, 18%), and leg penetration (16/61, 26%). The recurrent pulmonary embolism (PE) rate was 2%, with no PE in the 30-day period after filter retrieval. Retrieval of the Recovery G2 filter was safe and successful in most patients. Caudal migration was observed as an unexpected phenomenon.28,0http://dx.doi.org/10.1016/j.jvir.2009.08.007Frank Lynch[au]
2009Lynch, FC;Balloon-assisted removal of tilted inferior vena cava filters with embedded tipsJ Vasc Interv Radiol1210-121420919729132The successful removal of most retrievable inferior vena cava (IVC) filters requires the capture of the filter apex. The severely tilted filter with an apex in contact with the caval wall and covered by an endothelial cap represents a major technical challenge to removal. While a variety of techniques to deal with this problem have been reported, most require complex wire manipulations or the use of rigid endobronchial forceps. This article describes the successful use of a standard angioplasty balloon to free the apex of severely tilted filters with endothelial apical caps. A retrospective review of those patients who presented for removal of their Bard Recovery or G2 IVC filter between June 2005 and August 2008 was performed. Imaging studies and medical records were reviewed for those patients who had their IVC filters removed using the balloon-assisted technique. The presence of filter tilt and movement as well as the outcome of the technique was recorded for each case. Forty-eight Recovery and 209 G2 filters presented for removal. Ten of these 257 filters (3.6%) were found to be severely tilted with filter apex embedded into the wall of the cava. Eight of these filters were successfully removed using the balloon-assisted technique. No complications resulting from the technique occurred. Balloon-assisted removal of severely tilted embedded G2 and Recovery filters is an effective technique that can be performed using tools commonly available and familiar to most interventionists.28,0http://dx.doi.org/10.1016/j.jvir.2009.06.022Frank Lynch[au]
2009Lynch, FC;Kekulawela, S;Removal of the G2 filter: differences between implantation times greater and less than 180 daysJ Vasc Interv Radiol1200-120920919640738To investigate whether filters implanted for longer periods are more difficult or hazardous to remove. A retrospective review of G2 inferior vena cava filter removals was performed. Objective measures reflecting the difficulty of the removal procedure were evaluated for differences required to remove a filter with an implantation period greater or less than 180 days. One hundred seventy of 174 G2 filters were successfully removed (97.7% success rate). There was no significant difference in the success rate (P = .86), total procedure time (P = .87), fluoroscopy time (P = .13), or contrast medium use (P = .22) required to remove filters implanted for more than 180 days compared to those implanted for a shorter period of time. There was no significant difference in the frequency of filter movement (P = .90), tilt (P = .87), and caval penetration (P = .41) between the two groups. Six filter fractures were observed, all with implantation times greater than 180 days. The removal of a G2 filter that has been in place for more than 180 days can be performed as easily, as safely, and with a similar degree of success as one that has been in place for less time. Movement, tilt, and penetration are early events after implantation that may have an effect on successful filter removal.28,0http://dx.doi.org/10.1016/j.jvir.2009.05.039Frank Lynch[au]
2009Cantwell, CP;Pennypacker, J;Singh, H;Scorza, LB;Waybill, PN;Lynch, FC;Comparison of the recovery and G2 filter as retrievable inferior vena cava filtersJ Vasc Interv Radiol1193-119920919640733To compare the technical success of the Recovery and G2 filters as retrievable inferior vena cava (IVC) filters. Recovery (n = 128) and G2 (n = 113) filters were placed in the IVCs of 241 patients with the intent of retrieval. The referring physician and/or patient were contacted at 6-month intervals to ensure filter retrieval when indicated. The Recovery and G2 filter groups were compared regarding technical success of filter placement, technical success of attempted retrieval, filter tilt, filter migration, filter fracture, and filter efficacy. Filter placement was technically successful in 95% of Recovery filters (n = 122) and 100% of G2 filters (n = 113). Recovery filter retrieval was attempted in 55% of patients (n = 71) at a mean of 228 days (range, 0-838 d) after filter placement. G2 filter retrieval was attempted in 55% of patients (n = 62) at a mean of 230 days (range, 7-617 d) after filter placement. Technical success rates of filter retrieval were 94% (n = 67) and 97% (n = 60) in the Recovery and G2 filter groups, respectively. The G2 filter group had significantly fewer cases of (i) filter tilt at placement, (ii) filter tilt at attempted retrieval, and (iii) filter fracture than the Recovery filter group. In the G2 filter group, there was a significantly higher technical success rate of filter placement and there were more cases of caudal filter migration than in the Recovery filter group. Compared with the Recovery filter, the G2 filter is associated with significantly less filter fracture and tilt, greater technical success of filter placement, and more caudal filter migration.28,0http://dx.doi.org/10.1016/j.jvir.2009.05.037Frank Lynch[au]
2007Lynch, FC;Use of a covered stent for percutaneous transrenal occlusion of the ureterJ Vasc Interv Radiol1456-145818111800400128,0http://dx.doi.org/10.1016/j.jvir.2007.08.005Frank Lynch[au]
2007Cantwell, CP;Lynch, FC;Caudal migration of a G2 filter during carbon dioxide cavographyJ Vasc Interv Radiol814-8151861753815228,0http://dx.doi.org/10.1016/j.jvir.2007.02.038Frank Lynch[au]
2006Cantwell, CP;Lynch, FC;Ureterocutaneous fistula and urostomy exclusion with use of a covered wallstentJ Vasc Interv Radiol733-73517416614159The present report describes a case of urostomy breakdown and failed urinary diversion with bilateral nephrostomy drainage treated with transrenal placement of a covered stent. Covered stents can be used successfully for the exclusion of ureterocutaneous fistulas and urostomies with the potential for ureteric occlusion.28,0http://dx.doi.org/10.1097/01.RVI.0000199405.43063.ADFrank Lynch[au]
2019Tsypin, M;Asmellash, S;Meyer, K;Touchet, B;Roder, H;Extending the information content of the MALDI analysis of biological fluids via multi-million shot analysisPLoS ONEe0226012141231815946Reliable measurements of the protein content of biological fluids like serum or plasma can provide valuable input for the development of personalized medicine tests. Standard MALDI analysis typically only shows high abundance proteins, which limits its utility for test development. It also exhibits reproducibility issues with respect to quantitative measurements. In this paper we show how the sensitivity of MALDI profiling of intact proteins in unfractionated human serum can be substantially increased by exposing a sample to many more laser shots than are commonly used. Analytical reproducibility is also improved. To assess what is theoretically achievable we utilized spectra from the same samples obtained over many years and combined them to generate MALDI spectral averages of up to 100,000,000 shots for a single sample, and up to 8,000,000 shots for a set of 40 different serum samples. Spectral attributes, such as number of peaks and spectral noise of such averaged spectra were investigated together with analytical reproducibility as a function of the number of shots. We confirmed that results were similar on MALDI instruments from different manufacturers. We observed an expected decrease of noise, roughly proportional to the square root of the number of shots, over the whole investigated range of the number of shots (5 orders of magnitude), resulting in an increase in the number of reliably detected peaks. The reproducibility of the amplitude of these peaks, measured by CV and concordance analysis also improves with very similar dependence on shot number, reaching median CVs below 2% for shot numbers > 4 million. Measures of analytical information content and association with biological processes increase with increasing number of shots. We demonstrate that substantially increasing the number of laser shots in a MALDI-TOF analysis leads to more informative and reliable data on the protein content of unfractionated serum. This approach has already been used in the development of clinical tests in oncology.28,0100 serum samples were purchased from the commercial biobanks Conversant Bio (Huntsville, AL) and Oncology Metrics (Fort Worth, TX). Samples were collected under ethics-approved protocols according to the requirements of Conversant Bio and Oncology Metricshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901224/"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2018Gallery, M;Zhang, J;Bradley, DP;Brauer, P;Cvet, D;Estevam, J;Danaee, H;Greenfield, E;Li, P;Manfredi, M;Loke, HK;Rabino, C;Stringer, B;Williamson, M;Wyant, T;Yang, J;Zhu, Q;Abu-Yousif, A;Veiby, OP;A monomethyl auristatin E-conjugated antibody to guanylyl cyclase C is cytotoxic to target-expressing cells in vitro and in vivoPLoS ONEe019104613129370189Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.28,0scanning system. Colon tissue microarrays (US Biomax) were stained and scored by two independent pathologists following a protocol developed at Qualtek Molecular Laboratories on their Tek-Mate automated stainer. Briefly ;ith hematoxylin and imaged using the Aperio whole slide scanning system. Colon tissue microarrays (US Biomax) were stained and scored by two independent pathologists following a protocol developed at Qualtek Molecular Laboratories on their Tek-Mate automated stainer. Briefly, a composite scoring system (0, 1, 2, or 3) was established that captures both staining intensity (negative, weak, moderate,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784926/QualTek
2017O'Neil, BH;Wallmark, JM;Lorente, D;Elez, E;Raimbourg, J;Gomez-Roca, C;Ejadi, S;Piha-Paul, SA;Stein, MN;Abdul Razak, AR;Dotti, K;Santoro, A;Cohen, RB;Gould, M;Saraf, S;Stein, K;Han, SW;Safety and antitumor activity of the anti-PD-1 antibody pembrolizumab in patients with advanced colorectal carcinomaPLoS ONEe0189848121229284010Colorectal cancers (CRCs) expressing programmed death ligand 1 (PD-L1) have poor prognosis. In the multicohort KEYNOTE-028 trial, the anti-PD-1 antibody pembrolizumab was evaluated in 20 PD-L1-positive advanced solid tumors. Herein, we report results for the advanced CRC cohort. Patients with advanced, treatment-resistant PD-L1-positive carcinoma of the colon or rectum were enrolled, regardless of microsatellite instability (MSI) status. Pembrolizumab 10 mg/kg was administered every 2 weeks for up to 2 years or until disease progression/unacceptable toxicity. Response was assessed every 8 weeks for the first 6 months and every 12 weeks thereafter. Primary end points were safety and overall response rate by investigator review per Response Evaluation Criteria in Solid Tumors version 1.1. Data cutoff was June 20, 2016. Of 137 patients with CRC and samples evaluable for PD-L1 expression, 33 (24%) had PD-L1-positive tumors, of which 23 were enrolled. Median follow-up was 5.3 months, and 8 patients (35%) reported treatment-related adverse events (AEs), most commonly fatigue (n = 3, 13%), stomatitis (n = 2, 9%), and asthenia (n = 2, 9%). One patient (4%) experienced grade 4 treatment-related increased blood bilirubin. No grade 3 AEs, discontinuations, or deaths were attributed to treatment. Most patients (n = 15, 65%) experienced progressive disease. One partial response occurred in a patient (4%) with MSI-high CRC. Pembrolizumab demonstrated a favorable safety profile in advanced PD-L1-positive CRC. Antitumor activity was observed in a single patient with MSI-high CRC, warranting further evaluation in this patient population. (Clinicaltrials.gov registration: NCT02054806).28,0mbedded tumor sample or a newly obtained biopsy specimen was assessed at a central laboratory for PD-L1 expression at screening with a laboratory-developed prototype immunohistochemistry (IHC) assay (QualTek Molecular Laboratories, Goleta, CA, USA) [13] using the 22C3 antibody (Merck & Co., Inc., Kenilworth, NJ, USA). PD-L1 positivity was defined as membrane staining in ≥1% of scorable cells orhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746232QualTek
2017Danaee, H;Kalebic, T;Wyant, T;Fassan, M;Mescoli, C;Gao, F;Trepicchio, WL;Rugge, M;Consistent expression of guanylyl cyclase-C in primary and metastatic gastrointestinal cancersPLoS ONEe0189953121229261789The transmembrane receptor guanylate cyclase-C (GCC) has been found to be expressed in colorectal cancers. However, limited data are available on GCC protein expression in non-colorectal gastrointestinal tumors and few studies have reported whether GCC protein expression was consistently preserved in synchronous primary and metastatic cancer tissues. GCC protein status was assessed by immunohistochemistry in tumor specimens from individuals (n = 627) with gastrointestinal tumors, including esophageal (n = 130), gastric (n = 276), pancreatic (n = 136), and colorectal (n = 85) primary and metastatic tumors. Tissue specimens consisted of tissue microarrays containing esophageal, gastric, pancreatic tumors, and whole-slide tissue sections from colorectal cancer patients with matching primary and metastatic tumors. Among the evaluated esophageal, gastric, and pancreatic tumors, the frequency of GCC positivity at the protein level ranged from 59% to 68%. GCC was consistently expressed in primary and matched/synchronous metastatic lesions of colorectal cancer tissues derived from the same patients. This observational study demonstrated the protein expression of GCC across various gastrointestinal malignancies. In all cancer histotypes, GCC protein localization was observed predominantly in the cytoplasm compared to the membrane region of tumor cells. Consistent immunohistochemistry detection of GCC protein expression in primary colorectal cancers and in their matched liver metastases suggests that the expression of GCC is maintained throughout the process of tumor progression and formation of metastatic disease.28,0GCC IHC analysis and pathology assessment. GCC staining was performed on the Techmate Automated IHC Platform (BioTek Solutions/Ventana Medical Systems, Tucson, AZ, USA; QualTek Laboratories, Goleta, CA, USA) ;two independent pathologists. GCC IHC analysis and pathology assessment GCC staining was performed on the Techmate Automated IHC Platform (BioTek Solutions/Ventana Medical Systems, Tucson, AZ, USA; QualTek Laboratories, Goleta, CA, USA). In brief, 4 μm sections of tissue samples were dewaxed through xylene and a series of alcohol washes and finally placed into water. Antigen retrieval was perfohttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736218/QualTek
2017O'Neil, BH;Wallmark, JM;Lorente, D;Elez, E;Safety and antitumor activity of the anti–PD-1 antibody pembrolizumab in patients with advanced colorectal carcinomaPloS oneIn contrast with the other studies mentioned herein, all patients enrolled in the CRC cohort of KEYNOTE-028 had PD-L1–expressing tumors and response was still limited to a single patient with known MSI-H disease and a _BRAF_V600E mutation. _BRAF_ mutations in CRC are associated with poor prognosis [20,21], and combined MSI-H/_BRAF_ mutant status has been shown to have adverse prognostic significance [22]. MSI-H CRC tumors also seem to have greater PD-L1 expression than their MSS counterparts, possibly suggesting that PD-1 checkpoint blockade may be particularly beneficial in the management of MSI-H CRC [10]. In the current study, antitumor activity was observed in MSI-H CRC (_n_ = 1/1) but not in MSS CRC (_n_ = 22/23), even when patients were preselected for PD-L1 expression, albeit not for equivalent levels of PD-L1 expression. These results are consistent with those of the phase II KEYNOTE-016 study of pembrolizumab in patients with MSI-H CRC and non-CRC tumors [23,24]. In KEYNOTE-016, the ORR of the CRC and non-CRC MMR-deficient (MSI-H) arms were 57% (95% CI, 39%–73%) and 53% (95% CI, 36%–70%), respectively, whereas that in the MSS CRC arm was 0% (95% CI, 0%–13%) [23,24]. Furthermore, antitumor activity was observed in only the 2 MRR-deficient arms (in which the median PFS and OS were not reached) and not in the MMR-proficient arm (median PFS of 2.3 months, median OS of 6.0 months) [23,24]. Based on these results, the US Food and Drug Administration has granted breakthrough therapy designation to pembrolizumab for the treatment of patients with MSI-H metastatic CRC.28,0archived formalin-fixed, paraffin-embedded tumor sample or a newly obtained biopsy specimen was assessed at a central laboratory for PD-L1 expression at screening with a laboratory- developed prototype immunohistochemistry (IHC) assay (QualTek Molecular Laboratorieshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746232/QualTek
2020Barroso-Sousa, R;Krop, IE;Trippa, L;Tan-Wasielewski, Z;Li, T;Osmani, W;Andrews, C;Dillon, D;Richardson, ET;Pastorello, RG;Winer, EP;Mittendorf, EA;Bellon, JR;Schoenfeld, JD;Tolaney, SM;A Phase II Study of Pembrolizumab in Combination With Palliative Radiotherapy for Hormone Receptor-positive Metastatic Breast CancerClin. Breast Cancer32113750The purpose of this study was to investigate whether combining pembrolizumab with palliative radiation therapy (RT) improves outcomes in patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC). Eligible patients had HR+/human epidermal growth factor receptor 2-negative MBC; were candidates for RT to ≥ 1 bone, soft tissue, or lymph node lesion; and had ≥ 1 lesion outside the RT field. Patients received 200 mg pembrolizumab intravenously 2 to 7 days prior to RT and on day 1 of repeating 21-day cycles. RT was delivered to a previously unirradiated area in 5 treatments each of 4 Gy. The primary endpoint was objective response rate. The study used a 2-stage design: 8 women were enrolled into the first stage, and if at least 1 of 8 patients experienced an objective response, 19 more would be enrolled. Secondary endpoints included progression-free survival, overall survival, and safety. Exploratory endpoints included association of overall response rate with programmed death-ligand 1 status and tumor-infiltrating lymphocytes. Eight patients were enrolled in stage 1. The median age was 59 years, and the median prior lines of chemotherapy for metastatic disease was 2. There were no objective responses, and the study was closed to further accrual. The median progression-free survival was 1.4 months (95% confidence interval, 0.4-2.1 months), and the median overall survival was 2.9 months (95% confidence interval, 0.9-3.6 months). All-cause adverse events occurred in 87.5% of patients, including just 1 grade 3 event (elevation of aspartate aminotransferase). RT combined with pembrolizumab did not produce an objective response in patients with heavily pre-treated HR+ MBC. Future studies should consider alternative radiation dosing and fractionation in patients with less heavily pre-treated HR+ MBC. Copyright © 2020 Elsevier Inc. All rights reserved.28,0Clinical benefit is defined as complete response, partial response, or stable disease ≥ 24 weeks according to RECIST 1.1. Exploratory Biomarkers. Five patients had PD-L1 testing assessed centrally by QualTek using the 22C3 antibodyhttps://www.sciencedirect.com/science/article/pii/S1526820920300288QualTek
2006Kutty, RK;Chen, S;Samuel, W;Vijayasarathy, C;Duncan, T;Tsai, JY;Fariss, RN;Carper, D;Jaworski, C;Wiggert, B;Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) proteinBiochem. Biophys. Res. Commun.1333-1341345416729964NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P(270)KKRKAP(276)) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.27,0Immunohistochemistry. Immunohistochemistry was performed by QualTek Molecular Laboratories. Briefly, four-micron human placenta sections were deparaffinized, subjected to 20 min of steam heat induced epitope recovery, and then treated with anti-NORPEG antibodyhttps://www.sciencedirect.com/science/article/pii/S0006291X06010333QualTek
2001Taylor, RE;Shows, KH;Zhao, Y;Pittler, SJ;A PDE6A promoter fragment directs transcription predominantly in the photoreceptorBiochem. Biophys. Res. Commun.543-547282211401494Rod photoreceptor cGMP phosphodiesterase (PDE6) is a key enzyme in the phototransduction cascade. Lines of transgenic mice were established to determine the spatial expression pattern directed by an upstream fragment of the PDE6A gene. RT-PCR analysis showed that three of four lines analyzed transcribed the transgene predominantly in the retina and weakly in brain. The line showing no transgene transcription did not contain an intact transgene. Transcription of the transgene in the three lines was found in retina and weakly in brain, but not in heart, kidney, liver, or lung. Transcripts were most predominant in the photoreceptors of the retina. These results demonstrate that a short segment of the upstream region of the PDE6A gene comprises a functional promoter that is most active in photoreceptors. Copyright 2001 Academic Press.27,0Mannheim). An outside commercial company (Qualtek Inc., CA) confirmed results obtained in-house using the same exper- imental probes as above with proprietary in situ hybridization re- agents and the following conditionshttps://www.sciencedirect.com/science/article/pii/S0006291X01946054QualTek
2016Shimizu, T;Seto, T;Hirai, F;Takenoyama, M;Nosaki, K;Tsurutani, J;Kaneda, H;Iwasa, T;Kawakami, H;Noguchi, K;Shimamoto, T;Nakagawa, K;Phase 1 study of pembrolizumab (MK-3475; anti-PD-1 monoclonal antibody) in Japanese patients with advanced solid tumorsInvest New Drugs347-35434327000274Background This phase I study evaluated the safety and tolerability, pharmacokinetics and pharmacodynamics, immunogenicity, and antitumor activity of pembrolizumab in Japanese patients with advanced solid tumors. Methods Following an initial dose and a 28-day rest (cycle 1), pembrolizumab was administered as an intravenous infusion at escalating doses (2 or 10 mg/kg) every 2 weeks (Q2W) until disease progression or unacceptable toxicity. Adverse events (AEs) were assessed using CTCAE v4.0, and tumor response was assessed using both RECIST v1.1 and immune-related response criteria (irRC). Full pharmacokinetic sampling was performed during cycle 1. Results Three patients received pembrolizumab at 2.0 mg/kg and seven at 10 mg/kg. No dose-limiting toxicities were observed during cycle 1. Eighty percent of patients experienced drug-related AEs (mostly grade 1 or 2); the most common drug-related AEs were nausea, malaise, pyrexia, and aspartate aminotransferase/alanine transaminase (AST/ALT) elevations (n = 2 each). No drug-related grade 4 or 5 AEs occurred. Immune-related AEs comprised grade 3 ALT elevation (n = 1), grade 3 AST elevation (n = 1), grade 1 pneumonitis (n = 1), and grade 1 thyroid-stimulating hormone elevation (n = 1). The safety and pharmacokinetic profiles of Japanese patients were similar to those previously reported for Caucasian patients. A partial tumor response was observed in one patient with non-small-cell lung cancer (NSCLC) and in one patient with melanoma. Conclusions Pembrolizumab at both 2 and 10 mg/kg Q2W was well tolerated in Japanese patients with advanced solid tumors and showed encouraging anti-tumor activity against melanoma and NSCLC.27,0on the confirmation test. PD-L1 expression. PD-L1 expression was measured using immunohistochemistry performed on formalin-fixed, paraffin-embedded tissue sections at QualTek Clinical Laboratories. PD-L1 positivity was ;or postdose sample was positive on the confirmation test. PD-L1 expression PD-L1 expression was measured using immunohistochemistry performed on formalin-fixed, paraffin-embedded tissue sections at QualTek Clinical Laboratories. PD-L1 positivity was defined as staining in ≥1 % of the tumor cells (modified proportion score) or stroma using immunohistochemistry with the PD-L1 22C3 antibody. Rehttps://link.springer.com/article/10.1007/s10637-016-0347-6QualTek
2002Mulero, JJ;Boyle, BJ;Bradley, S;Bright, JM;Nelken, ST;Ho, TT;Mize, NK;Childs, JD;Ballinger, DG;Ford, JE;Rupp, F;Three new human members of the lipid transfer/lipopolysaccharide binding protein family (LT/LBP)Immunogenetics293-30054512185532We have identified three novel, rarely expressed human genes that encode new members of the lipid transfer/lipopolysaccharide binding protein (LT/LBP) gene family based on sequence homology. BPI and other members of the LT/LBP family are structurally related proteins capable of binding phospholipids and lipopolysaccharides. Real-time PCR studies indicate that BPIL1 and BPIL3 are highly expressed in hypertrophic tonsils. In situ hybridization analysis of BPIL2 shows prominent expression in skin specimens from psoriasis patients. BPIL1 and BPIL3 map to Chromosome 20q11; thus, these novel genes form a cluster with BPI and two other members of the LT/LBP gene family on the long arm of human Chr 20. BPIL2maps to Chr 22q13. The exon/intron organization of all three genes is highly conserved with that of BPI, suggesting evolution from a common ancestor.25,0The BPIL2 probe was derived from a 414-nt coding sequence between the primers 5'-CATTATTGCAAGTGAAGTCAAAGC-3' and 5'-ACCATGA- AGGGCTGGGACAAGATG-3'. QualTek Molecular labs (Santa Barbara, Calif.) performed the automated in situ hybridization us- ing ahttps://link.springer.com/article/10.1007/s00251-002-0467-3QualTek
2019Burbelo, PD;Chaturvedi, A;Notkins, AL;Gunti, S;Luciferase-Based Detection of Antibodies for the Diagnosis of HPV-Associated Head and Neck Squamous Cell CarcinomaDiagnostics (Basel)9331390810Point-of-care tests are needed for the screening of head and neck squamous cell carcinoma (HNSCC) and other malignancies. Luciferase immunoprecipitation systems (LIPS), employing light-emitting proteins, were used to examine serum antibodies against several cancer-associated targets in blood donor controls and subjects with colon cancer (CC) and HNSCC. The assessment of antibodies against the wild type p53 tumor antigen showed that approximately 25% of the CC and 20% of the HNSCC patients were seropositive. In addition, humoral responses against two p53 mutants, p53-R175H and p53-R273H, generally tracked the antibody responses seen against wild type p53. Analysis of antibodies against highly specific biomarkers of HPV-16-associated malignancy, E2, E6, and E7 oncoproteins, revealed no seropositivity in blood donors and CC patients. However, 45% (9/20) of the HNSCC patients showed E6 seropositivity, which overlapped all the detectable E2 (40%; 8/20) and E7 seropositive subjects (35%; 7/20). Using neodymium magnets, ultrarapid LIPSTICKS testing of HPV-16 E6 antibodies in <60 s per HNSCC sample demonstrated almost the same diagnostic performance (40% sensitivity and 100% specificity) as LIPS testing in 2.5 h. While additional improvements and standardization are needed, these results highlight the possibility of using these approaches for the diagnosis of HPV-16-associated HNSCC.25,0The serum samples from colon cancer (CC), systemic lupus erythematosus (SLE), and HNSCC patients used in this report were obtained from Conversant Bio (601 Genome Way Suite 1200, Huntsville, Alabama 35806). Subjectshttps://www.mdpi.com/2075-4418/9/3/89"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2020Patel, JJ;Levy, DA;Nguyen, SA;Knochelmann, HM;Day, TA;Impact of PD-L1 expression and human papillomavirus status in anti-PD1/PDL1 immunotherapy for head and neck squamous cell carcinoma-Systematic review and meta-analysisHead Neck774-78642431762164Programmed cell death-1 (PD-1) pathway inhibition in head and neck squamous cell carcinoma (HNSCC) has demonstrated inconsistent efficacy regarding human papillomavirus (HPV) status and PD-L1 expression. This study compared outcomes in HNSCC in the context of PD-L1 and HPV expression. Outcomes: PD-L1 and HPV expression; overall survival (OS), and tumor response (ORR). 1088 patients received PD-1/L1 inhibitors. Four methodologies were identified in determining PD-L1 expression, most commonly using the Dako PD-L1 IHC 22C3 pharmaDx assay. Using a 1% threshold, ORR was greater for PD-L1 expressers vs non-expressers (18.9%, CI 16.1-21.8 v 8.8% CI 5.3-13.7, P = 0.009), as was OS at 6 months (60.6%, CI 49.2-71.4 v 49.0%, CI 39.1-59.0, P = 0.04) but not at 12 or 18 months. No advantages were identified for HPV expressers. Patients expressing PD-L1 may have a better tumor response and OS. No impact on survival or response was observed based on HPV status. © 2019 Wiley Periodicals, Inc.24,0Oro Valley, Arizona). One study28 did not specify the assay utilized for PD‐L1 expression, but rather reported the laboratory where analysis occurred (QualTek Molecular Laboratories, Goleta, California). This lab confirmed thehttps://onlinelibrary.wiley.com/doi/abs/10.1002/hed.26036QualTek
2020Adekanmbi, EO;Giduthuri, AT;Srivastava, SK;Dielectric Characterization and Separation Optimization of Infiltrating Ductal Adenocarcinoma via Insulator-DielectrophoresisMicromachines (Basel)11432218322The dielectrophoretic separation of infiltrating ductal adenocarcinoma cells (ADCs) from isolated peripheral blood mononuclear cells (PBMCs) in a ~1.4 mm long Y-shaped microfluidic channel with semi-circular insulating constrictions is numerically investigated. In this work, ADCs (breast cancer cells) and PBMCs' electrophysiological properties were iteratively extracted through the fitting of a single-shell model with the frequency-conductivity data obtained from AC microwell experiments. In the numerical computation, the gradient of the electric field required to generate the necessary dielectrophoretic force within the constriction zone was provided through the application of electric potential across the whole fluidic channel. By adjusting the difference in potentials between the global inlet and outlet of the fluidic device, the minimum (effective) potential difference with the optimum particle transmission probability for ADCs was found. The radius of the semi-circular constrictions at which the effective potential difference was swept to obtain the optimum constriction size was also obtained. Independent particle discretization analysis was also conducted to underscore the accuracy of the numerical solution. The numerical results, which were obtained by the integration of fluid flow, electric current, and particle tracing module in COMSOL v5.3, reveal that PBMCs can be maximally separated from ADCs using a DC power source of 50 V. The article also discusses recirculation or wake formation behavior at high DC voltages (>100 V) even when sorting of cells are achieved. This result is the first step towards the production of a supplementary or confirmatory test device to detect early breast cancer non-invasively.24,0The DEP suspending medium (dextrose solution) was prepared and characterized as described in our previous article [38]. The prepared 100 mL suspending medium was divided into five separate beakers. Into each beaker, except the first, calculated volume of phosphate buffer saline (PBS) was added to successively change the conductivity of the DEP suspending medium to obtain the following conductivities (in mS/m): 50, 60, 74, 88 and 97. Female normal peripheral mononuclear cells (PBMCs) and infiltrating ductal adenocarcinoma cells (ADCs) with no identifiable angiolymphatic invasion were obtained from Conversant Bio, Huntsville, AL, USA. Also, the cells obtained did not have any identifiable information about the patient itself except their gender and age (Institutional Review Board - IRB exempt). The cells were prepared for experiment according to the supplier’s instructions. Thereafter, a known number of cells were transferred into each of the five DEP suspending medium solution where they were washed twice and diluted in 1:400 cell: suspending medium ratio before being pipetted into the DEP microwell for experimenthttps://www.mdpi.com/2072-666X/11/4/340"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2013Macias, JD;Gerkin, RD;Locke, D;Macias, MP;Differential gene expression in cholesteatoma by DNA chip analysisLaryngoscopeS1-2112323670528In contrast to normal epithelium, the desquamating stratified squamous epithelium of temporal bone cholesteatoma characteristically exhibits sustained hyperproliferative growth and a capacity for bone erosion. We conducted genome-wide microarray analyses to determine the molecular nature of cholesteatoma's biological processes and identify disease-associated, altered gene activity. We tested the hypothesis that genes contributing to the pathophysiology of cholesteatoma are differentially expressed compared to control tissue. Prospective experimental analysis. Using new, enhanced microarray platforms and well-annotated human transcriptome probes, we measured global gene expression levels in surgical specimens of cholesteatoma and in the corresponding normal postauricular skin in four patients. Genes of interest were verified by quantitative real time reverse transcriptase polymerase chain reaction analyses using cholesteatoma and postauricular sample pairs (n = 13). External auditory canal skin from six additional patients was also evaluated as a normal control. Immunohistochemistry detected protein expression in tissue sections and the cells involved. DNA chip analyses identified 282 differentially expressed genes in cholesteatoma compared to control samples. Of these, 104 genes were upregulated and 178 were downregulated. Ontological classifications indicate relationships to cellular processes including receptor binding, cell communication and motion, vitamin metabolism, and cytokine-mediated inflammation. Based on potential involvement in disease pathology, 10 genes were selected and independently verified by quantitative polymerase chain reaction. Immunohistochemical detection of transcobalamin-1 and CCL27 implicates cholesteatoma keratinocytes and dermal endothelial cells as contributors in disease processes. We present a comprehensive, human genome-wide survey of disease-associated gene expression that extends the public database and provides new evidence for molecular mechanisms involved in cholesteatoma pathology. Laryngoscope, 123:S1-S21, 2013. Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.23,0From the Macias Otology (J.D.M.), Biomedical Research Program, the EAR Foundation of Arizona (J.D.M., M.P.M.), and Scientific Services, Banner Health Research Institute (R.D.G.), Phoenix, Arizona, USA; and QualTek Molecular Laboratories (D.L.), Newton, Pennsylvaniahttps://onlinelibrary.wiley.com/doi/abs/10.1002/lary.24176QualTek
2009Pfeffer, BA;Bernstein, SA;Bartels, SP;Preservation of structure and immunoreactivity at the vitreoretinal interface of the rabbit eyeGraefes Arch. Clin. Exp. Ophthalmol.193-205247219020891The vitreous body is implicated in the etiology and pathology of a variety of retinal conditions. Many such conditions are treated surgically to remove the posterior vitreous from the inner limiting lamina (ILL) of the retina, and there is current interest in the adjunct use of enzymes for this purpose. To evaluate the efficacy of these agents in future preclinical studies, improved preservation and assessment methods were developed to establish a baseline histological profile of the vitreous and retina of the rabbit, to identify and distinguish artifactual vitreoretinal separation from authentic posterior vitreous detachment, and to preserve structural integrity while maintaining antigenicity for immunohistochemical analysis. Two pigmented rabbits each underwent perfusion with one of three fixatives, either: (1) 10% neutral buffered formalin + cetylpyridinium chloride (NBF/CPC), (2) acid-ethanolic formalin + CPC (AEF/CPC), or (3) formaldehyde-glutaraldehyde + CPC (FG/CPC). An eye fixed in NBF/CPC by immersion, from an additional rabbit, was also included for comparison. Eyes were processed whole through paraffin infiltration. Treatments were assessed by immunohistochemical labeling for retinal and cortical vitreous (CV) markers. In contrast to immersion fixation, perfusion with either NBF/CPC or AEF/CPC maintained vitreous adherence to the ILL during histological processing. NBF/CPC proved best for highlighting intralaminar structure and for labeling type II collagen in the CV, particularly with antigen retrieval. AEF/CPC caused condensation of fibrillar elements in the CV. Collagen XVIII in the ILL was observed with AEF/CPC exclusively. Only retinal vessels near the optic nerve head were labeled for type IV collagen. The labeling of glia was useful for distinguishing between cellular and extracellular elements. GF/CPC hindered detection of collagen II and disrupted posterior segment structure. Expression of type II collagen extended from the ONH directly to CV affiliated with the central canal of Cloquet, a feature characteristic of rabbit eyes. Careful tissue preservation and processing techniques minimize artifactual separation of the CV from the ILL. By optimizing the tissue architecture and antigenicity of the vitreoretinal complex, CV may be distinguished from the ILL immunohistochemically. The techniques described may be used to evaluate more effectively the utility of pharmacologic vitreolysis, using experimental animal models.22,0Presented in part at the 2006 meeting of The Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida. This study was supported by Bausch & Lomb, Inc., with whom QualTek Molecular Laboratories has a contractual relationshiphttps://link.springer.com/article/10.1007/s00417-008-0991-4QualTek
2017Block, TS;Murphy, TI;Munster, PN;Nguyen, DP;Lynch, FJ;Glucocorticoid receptor expression in 20 solid tumor types using immunohistochemistry assayCancer Manag Res65-72928293120Glucocorticoid receptor (GR) activity plays a role in many aspects of human physiology and may play a crucial role in chemotherapy resistance in a wide variety of solid tumors. A novel immunohistochemistry (IHC) based assay has been previously developed and validated in order to assess GR immunoreactivity in triple-negative breast cancer. The current study investigates the standardized use of this validated assay to assess GR expression in a broad range of solid tumor malignancies. Archived formalin-fixed paraffin-embedded tumor bank samples (n=236) from 20 different solid tumor types were analyzed immunohistochemically. Nuclear staining was reported based on the H-score method using differential intensity scores (0, 1+, 2+, or 3+) with the percent stained (out of at least 100 carcinoma cells) recorded at each intensity. GR was expressed in all tumor types that had been evaluated. Renal cell carcinoma, sarcoma, cervical cancer, and melanoma were those with the highest mean H-scores, indicating high levels of GR expression. Colon, endometrial, and gastric cancers had lower GR staining percentages and intensities, resulting in the lowest mean H-scores. A validated IHC assay revealed GR immunoreactivity in all solid tumor types studied and allowed for standardized comparison of reactivity among the different malignancies. Baseline expression levels of GR may be a useful biomarker when pharmaceutically targeting GR in research or clinical setting.22,0Tiffany I Murphy. 3 QualTek Molecular Laboratories, Newtown, PA. Find articles by Tiffany I Murphy. Pamela N Munster Dat P Nguyen. 1 Corcept Therapeutics, Inc, Menlo Park, CA. Find articles by Dat P Nguyen. Frank J Lynch. 3 QualTek Molecular Laboratories, Newtown, PA ;Corcept Therapeutics, Inc, Menlo Park, CA; Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA.++QualTek Molecular Laboratories, Newtown, PA.++Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.++Corcept Therapeutics, Inc, Menlo Park, CA.++QualTek Molecular Laboratories, Newtown, PAhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345989/QualTek;Tiffany Murphy[au];Frank Lynch[au]
2015Baker, GM;Murphy, T;Block, T;Nguyen, D;Lynch, FJ;Development and validation of an immunohistochemistry assay to assess glucocorticoid receptor expression for clinical trials of mifepristone in breast cancerCancer Manag Res361-368726673410Glucocorticoid receptor (GR) activity has been associated with chemotherapy resistance and poor outcomes in patients with triple negative breast cancer (TNBC). The aim of this study was to develop an immunohistochemistry (IHC) assay to assess GR expression in archival formalin-fixed, paraffin-embedded human invasive breast carcinoma samples. An optimized GR assay protocol was developed using rabbit monoclonal antibody to GR clone D8H2. Precision and reproducibility of the GR IHC assay was determined by conducting multiple staining runs of four invasive breast carcinoma samples using replicate serial sections. Assay sensitivity was examined in 50 TNBC samples (>10 mm) obtained from a tumor bank, and 43 paired TNBC samples from a tissue microarray (TMA) (1.5 mm). GR positivity was assessed using a percent scoring approach with a ≥10% cutoff for nuclear staining of tumor cells at any intensity. Analysis of the paired TMA cores was performed by averaging the scores of the two cores for each case. Equivalent cellular patterns of GR reactivity were observed in all replicates from the multiple staining runs; coefficients of variation did not exceed 4.7% for average H-scores greater than 3.4, thus meeting the criteria for assay precision and reproducibility (coefficient of variation ≤20%). GR expression in TNBC single-tissue samples and TMA cores was characterized as mostly nuclear, with some concurrent cytoplasmic reactivity. Eighty-four percent of the 49 evaluable TNBC samples and 60% of the 42 evaluable paired TMA samples were positive for GR expression. A robust and reproducible GR IHC assay was successfully developed for use in invasive breast carcinoma tissues. Differences in GR expression between larger single tissues and smaller TMA cores illustrate the heterogeneity of the disease, as well as potential intra-tumoral heterogeneity. This assay is currently being utilized in clinical trials of mifepristone, a GR antagonist, in patients with TNBC.22,0Tiffany Murphy. 2 QualTek Molecular Laboratories, Newtown, PA, USA. Find articles by Tiffany Murphy. Thaddeus Block Dat Nguyen. 3 Corcept Therapeutics, Menlo Park, CA, USA. Find articles by Dat Nguyen. Frank J Lynch. 2 QualTek Molecular Laboratories, Newtown, PA, USA ;Department of Pathology, The University of Chicago, Chicago, IL, USA.++QualTek Molecular Laboratories, Newtown, PA, USA.++Corcept Therapeutics, Menlo Park, CA, USA ; Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.++Corcept Therapeutics, Menlo Park, CA, USA.++QualTek Molecular Laboratories, Newtown, PA, USAhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675647/QualTek;Tiffany Murphy[au];Frank Lynch[au]
2017Coletti, M;Hultquist, C;Kennedy, WG;Cervone, G;Validating Safecast data by comparisons to a U. S. Department of Energy Fukushima Prefecture aerial surveyJ Environ Radioact9-2017128167372Safecast is a volunteered geographic information (VGI) project where the lay public uses hand-held sensors to collect radiation measurements that are then made freely available under the Creative Commons CC0 license. However, Safecast data fidelity is uncertain given the sensor kits are hand assembled with various levels of technical proficiency, and the sensors may not be properly deployed. Our objective was to validate Safecast data by comparing Safecast data with authoritative data collected by the U. S. Department of Energy (DOE) and the U. S. National Nuclear Security Administration (NNSA) gathered in the Fukushima Prefecture shortly after the Daiichi nuclear power plant catastrophe. We found that the two data sets were highly correlated, though the DOE/NNSA observations were generally higher than the Safecast measurements. We concluded that this high correlation alone makes Safecast a viable data source for detecting and monitoring radiation. Copyright © 2017 Elsevier Ltd. All rights reserved.22,0Second, units are randomly selected, assembled, and undergo calibration tests at the Julich Research Centre in Germany, QualTek in the US, and the International Atomic Energy Agency (IAEA) testing laboratory in Seibersdorf, Austriahttps://www.sciencedirect.com/science/article/pii/S0265931X17300218QualTek
1999Widder, E;Johnsen, S;Bernstein, S;Case, J;Neilson, D;Thin layers of bioluminescent copepods found at density discontinuities in the water columnMarine Biology429-4371343To learn how organisms apportion space in the open ocean, biological oceanographers have sought to improve temporal and spatial resolution of ocean sampling systems. Their objectives are to simultaneously measure physical, chemical and biological structure in the water column in order to find significant correlations that may reveal underlying processes. Here we report one such correlation between intense peaks of bioluminescence and density discontinuities in the water column. Intensified video recordings made in these bioluminescent “hot spots” were analyzed with a computer image-recognition program that identifies organisms based on the temporal and spatial characteristics of their luminescent displays. Based on this analysis, the source of the “hot spots” was found to be very thin layers (0.5 m) of the bioluminescent copepod Metridia lucens present at from 5 to 100 times average background concentrations. Given the recent discovery that the vertical distribution of marine snow is also strongly correlated with density discontinuities in the water column, we suggest that this finding may provide a possible explanation for the disparity between estimated energy requirements of marine copepods and measurements of average in situ food concentrations. The energy costs associated with locating food-rich micro-patches is greatly reduced if those patches are spread out into very thin layers, because the search strategy can be reduced from three dimensions to one.21,0Communicated by NH Marcus, Tallahassee EA Widder (8) a S. Johnsen Harbor Branch Oceanographic Institution, Fort Pierce, Florida 34946, USA Fax: 001 (0)561 468-0757 e-mail: widder@hboi.edu SA Bernstein QualTek, Santa Barbara, California 93111, USA JF Casehttps://link.springer.com/article/10.1007/s002270050559QualTek
2014Aycock, KI;Campbell, RL;Manning, KB;Sastry, SP;Shontz, SM;Lynch, FC;Craven, BA;A computational method for predicting inferior vena cava filter performance on a patient-specific basisJ Biomech Eng136824805200A computational methodology for simulating virtual inferior vena cava (IVC) filter placement and IVC hemodynamics was developed and demonstrated in two patient-specific IVC geometries: a left-sided IVC and an IVC with a retroaortic left renal vein. An inverse analysis was performed to obtain the approximate in vivo stress state for each patient vein using nonlinear finite element analysis (FEA). Contact modeling was then used to simulate IVC filter placement. Contact area, contact normal force, and maximum vein displacements were higher in the retroaortic IVC than in the left-sided IVC (144 mm(2), 0.47 N, and 1.49 mm versus 68 mm(2), 0.22 N, and 1.01 mm, respectively). Hemodynamics were simulated using computational fluid dynamics (CFD), with four cases for each patient-specific vein: (1) IVC only, (2) IVC with a placed filter, (3) IVC with a placed filter and model embolus, all at resting flow conditions, and (4) IVC with a placed filter and model embolus at exercise flow conditions. Significant hemodynamic differences were observed between the two patient IVCs, with the development of a right-sided jet, larger flow recirculation regions, and lower maximum flow velocities in the left-sided IVC. These results support further investigation of IVC filter placement and hemodynamics on a patient-specific basis.20,0http://dx.doi.org/10.1115/1.4027612Frank Lynch[au]
2008Gallagher, J;Lynch, FW;Barragry, J;A prolactinoma masked by a herbal remedyEur. J. Obstet. Gynecol. Reprod. Biol.257-25813721729886320,0http://dx.doi.org/10.1016/j.ejogrb.2006.12.020Frank Lynch[au]
2019Montazeri, M;Marbut, C;Huitink, D;Interconnect Fatigue Failure Parameter Isolation for Power Device Reliability Prediction in Alternative Accelerated Mechanical Cycling TestJournal of Electronic Packaging1413In this work, a rapid and low-cost accelerated reliability test methodology which was designed to simulate mechanical stresses induced in flip–chip bonded devices during the thermal cycling reliability test under isothermal conditions, is introduced and demonstrated using power device analogous test chips. By stressing these devices in a controlled environment, mechanical stresses become decoupled from the design and temperature, such that useful lifetimes can be predictable. Mechanical shear stress was cyclically applied directly to device relevant, flip–chip solder interconnects while monitoring for failure. Herein, finite element analysis (FEA) is used to extract various damage metrics of different solder materials, including PbSn37/63, SAC305, and nanosilver, in both thermal operation and the introduced alternative mechanical testing conditions. Plastic work density and strain are calculated in the critical solder interconnects as factors that indicate the amount of the damage accumulation per cycle during the mechanical cycling, thermal cycling, and power cycling tests. The number of cycles to failure for each test was calculated using the fatigue life model developed by Darveaux for eutectic PbSn solder, while for SAC305 Syed's method was used, and for nanosilver, the Knoerr et al. equations are applied. The effects of environmental temperature and shearing force frequency were studied for the mechanical cycling reliability test, where a modified Norris–Landzberg equation for mechanical cycling tests was explored using the simulation results. Finally, comparing the mechanical cycling with the equivalent thermal cycling and power cycling demonstrated a significant reduction in required test duration to achieve a reliability estimation.20,0IEEE Forth Workshop on Wide Bandgap Power Devices and Applications ( WiPDA), Fayetteville, AR, Nov. 7-9, pp. 194– 199. 19. Qualitek, 2014, “Technical Data Sheet: Sn42/Bi58 Solder Wire,” Qualtek Electronics Corporation, Addison, IL, Accessed May 13, 2019, https://www.qualitek.com/sn42_bi58_solder_wire_tech_data.pdf 20. Qualitek, 2014, “Technical Data Sheet: Sn95/Sb5 Solder Wire,https://asmedigitalcollection.asme.org/electronicpackaging/article-abstract/141/3/031011/726619QualTek
2003Gibbs, WW;Untangling the roots of cancerSci. Am.56-6528911284094719,0SCIENCE PHOTO LIBRARY; FRANK LYNCH QualTek Molecular Laboratories; ANDREJS LIEPINS/SPL; CNRI/SPL; SPL 1. GROWTH EVEN IN THE ABSENCE OF NORMAL "GO" SIGNALS Most normal cells wait for an external message before dividinghttps://www.jstor.org/stable/26060363QualTek
2019Mohan, S;Lawton, R;Palmer, C;Rojas, AC;Competitive ELISA method for novel estrogen-negative breast cancer biomarker quantitationJ. Immunol. Methods11267147431533022Estrogen-negative (ER-) breast cancer, is recognized as an aggressive subtype, more difficult to treat, with poor survival and prognosis. They are hormonally unresponsive, with no readily effective and specific target therapy. We have previously identified Nw-hydroxy L-Arginine (NOHA) as a blood-based biomarker to distinguish between ER- and ER+ breast cancer tumors based upon disease burden, progression and molecular phenotype (U.S. Utility Patent 10,073,099). In this study we have demonstrated a competitive ELISA based assay for NOHA measurement using a proprietary monoclonal antibody (mAb) specific for NOHA (U.S. provisional patent 62/754,053). The ELISA assay was evaluated on sensitivity, selectivity, precision, dilution linearity and percent recovery parameters. The assay showed sensitivity at ≥60 pg/ml NOHA antigen with 1 ng/ml NOHA mAb, and maintained NOHA antigen specificity even in the presence of other closely related cationic amino acids (i.e. L-Arginine, D-Arginine, l-Lysine, d-Lysine, L-Ornithine, and L-Citrulline). The reliability of the ELISA protocol was confirmed with the low percent-covariance, for all tested parameters of sensitivity (≤8.2%), selectivity (≤8.6%), precision (≤12.6%), dilution linearity (≤11.2%) and recovery (≤6.7%). Additionally, we can demonstrate NOHA quantification by this ELISA assay to complement the sensitivity achievable with LC-MS (in both assay buffer and with patient plasma samples), thus suggesting it's utility as a simple yet sensitive methodology that might help in ER- breast cancer prognosis, and disease progression monitoring without the need for expensive analytical equipment (such as LC-MS), large lab space, or specialized technical training. Copyright © 2019 Elsevier B.V. All rights reserved.19,0system) (Rakha et al., 2008), and categorized based on estrogen-hormone-receptor orientation (viz., ER − versus ER + , n = 50), were obtained from cooperative human tissue network (Philadelphia, PA), Tissue-for-Research (Atlanta, GA), and Conversant Bio (Huntsville, AL)https://www.sciencedirect.com/science/article/pii/S002217591930211X"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2005Wang, B;Berger, M;Masters, G;Albone, E;Yang, Q;Sheedy, J;Kirksey, Y;Grimm, L;Wang, B;Singleton, J;Soltis, D;Radiotherapy of human xenograft NSCLC tumors in nude mice with a 90Y-labeled anti-tissue factor antibodyCancer Biother. Radiopharm.300-30920315989475Tissue factor (TF) is a type I transmembrane protein and the initiator of the extrinsic blood coagulation pathway. TF plays a critical role in tumor development and its overexpression is observed in many tumors. To understand the prevalence and relative level of TF expression in non-small-cell lung cancer (NSCLC), we analyzed 50 NSCLC tumors by immunohistochemical staining and found that 88% of human NSCLC tumors overexpressed TF. We then generated a high affinity anti-TF antibody, TF278, which specifically binds TF on the surface of cells and is internalized upon binding. An 111In-labeled TF278 demonstrated favorable tumor accumulation in an SW-900 xenograft tumor model with a maximum mean percent of injected dose per gram of tissue (%ID/g) of 73.1% at 96 hours postinjection. In addition, we labeled the antibody with 90Y and tested its ability to inhibit the growth of tumors in an SW-900 xenograft tumor model in immunocompromised mice. The 90Y-TF278 slowed the growth of SW-900 tumors at a 50 microCi dose and completely regressed SW-900 tumors at a 150 microCi dose with little toxicity.19,0An IHC study of TF expression in human tissues was performed at QualTek Labs (Newton, PA). Briefly, formalin-fixed, paraffin-embedded sec- tions from human NSCLC tumors and different normal human tissues were taken from QualTek's human tissue bankhttps://www.liebertpub.com/doi/abs/10.1089/cbr.2005.20.300QualTek
2001Ladner, RD;The role of dUTPase and uracil-DNA repair in cancer chemotherapyCurr. Protein Pept. Sci.361-3702412374095Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the molecular mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion observed following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metabolism as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metabolism. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clinical studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer.19,0await further investigation. ACKNOWLEDGEMENTS Special thanks is extended to Dr. Frank J. Lynch at QualTek Molecular Laboratories, Santa Barbara, CA, for expertise in immunohistochemistry. Special thanks are extendedhttps://www.ingentaconnect.com/content/ben/cpps/2001/00000002/00000004/art00010QualTek
2020Nuovo, G;A broad-based approach to differentiate CIN from its mimics: The utility of in situ hybridization and immunohistochemistryAnn Diagn Pathol1515154632330660The hematoxylin and eosin slides of 100 consecutive cases diagnosed as CIN 1-2 were combined with 25 CIN 1 and 25 negative for CIN as documented by in situ HPV testing. The 150 cases were then reviewed blinded and scored as "CIN" or "negative for CIN". Each of the 50 controls was correctly scored. Of the 100 cases, 62 were diagnosed as CIN and the other 38 were scored as negative for CIN on re-review. Each of the CIN cases was positive for HPV as proven by the in situ detection of either HPV DNA or the L1 capsid protein. The 38 cases diagnosed as negative for CIN and 38 of the CIN cases were tested for HPV DNA by in situ hybridization and for a panel of biomarkers that included p16, Ki67, importin-β, exportin-5, and Mcl1 plus the L1 HPV capsid protein. Each of the 38 CIN cases was positive for HPV as well as each biomarker that localized towards the basal aspect of the lesion. Two of the 38 negative for CIN cases were positive for HPV DNA/L1 capsid protein and each of the biomarkers. The other 36 cases were negative for HPV DNA/L1 protein and each of the biomarkers showed baseline expression. Thus, 36% of the diagnoses of CIN 1-2 were incorrect and this could have been prevented with either in situ detection of the viral DNA/capsid protein or the immunohistochemistry detection of a panel of biomarkers that included p16, Ki67, importin-β, exportin-5, and Mcl1. Copyright © 2020 Elsevier Inc. All rights reserved.16,0study was the review of 100 consecutive cases of formalin fixed, paraffin embedded tissues cervical biopsies done for either an abnormal Pap smear and/or a high risk HPV DNA positive result where the diagnosis was listed as CIN 1 or CIN 2. Folio Biosciences provided most ofhttps://www.sciencedirect.com/science/article/pii/S1092913420300563"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2019Nicol, AF;de Andrade, CV;Gomes, SC;Brusadelli, MG;Lodin, HM;Wells, SI;Nuovo, GJ;The distribution of novel biomarkers in carcinoma-in-situ, microinvasive, and squamous cell carcinoma of the uterine cervixAnn Diagn Pathol115-1223830579259Importin-β, exportin-5, p16, Ki-67, Mcl1, PDL1, and cFLIP are each over-expressed in the majority of CIN 1 lesions. These biomarkers, plus HPV E6/E7 RNA, were analyzed in carcinoma-in-situ (CIS), microinvasive, and squamous cell carcinoma (SCC) of the uterine cervix and cervical carcinoma cell lines. Only p16 and Ki-67 continued to be over-expressed in CIS, with a concomitant marked increase in E6/E7 RNA. There was a highly significant increase in PDL1 expression and decrease in Ki-67 (each p < 0.001) in microinvasive cancer compared to CIS whereas p16 and E6/E7 remained stable. As the lesion progressed to SCC, p16 and E6/E7 RNA remained strongly overexpressed with a concomitant over expression of importin-β and Ki67. HPV positive Caski cells showed significant elevations of p16, importin-β, exportin-5 and PDL1 compared to the HPV negative cervical cancer cell line C33A, consistent with viral induction of these biomarkers. The data suggest that PDL1 may be a useful biomarker to differentiate CIS from microinvasive cancer and, thus, anti-PDL1 therapy may inhibit the progression of CIS to the invasive stage. Copyright © 2018 Elsevier Inc. All rights reserved.16,0National Institute of Infectious Diseases Evandro Chagas - INI-Fiocruz, Rio de Janeiro, Brazil.++National Institute of Health of Women, Children, and Adolescents, Fernandes Figueira - IFF-FIOCRUZ, Rio de Janeiro, Brazil.++National Institute of Health of Women, Children, and Adolescents, Fernandes Figueira - IFF-FIOCRUZ, Rio de Janeiro, Brazil.++Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.++Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.++Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.++The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA; Phylogeny Inc, Powell, OH, USA. Electronic address: nuovo.1@osu.eduhttp://dx.doi.org/10.1016/j.anndiagpath.2018.12.001"Phylogeny Inc"
2010Hill, LS;Richardson, TL;Profeta, LT;Shaw, TJ;Hintz, CJ;Twining, BS;Lawrenz, E;Myrick, ML;Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy systemRev Sci Instrum01310381120113077Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 10(6) photons/s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter.16,0The relay box uses a Bud Industries PN series box enclosure (model PN-1341-C) as the base. The circuit consisted of two NTE Electronics solid-state relays (model RS3–1D10–51), two Omron Electronic relays (model G4B-112T-C-U.S.-AC120), one Qualtek Electronics Corp. multifunction module (model 761–18∕003), and two Amphenol rf coaxial BNC connectors (model 31–221-RFX). A schematic of the electronic configuration of the box is seen in Fig. ​Fig.44https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816980QualTek
2018Smyth, E;Thuss-Patience, PC;Immune Checkpoint Inhibition in Gastro-Oesophageal CancerOncol Res Treat272-28041529705787Recently some progress has been made in the palliative treatment of gastric cancer. It was shown that second-line chemotherapy and VEGF-R2-directed treatment can prolong survival. Despite these advances most patients with metastatic gastric cancer live for less than 2 years. Immune-checkpoint blockade with anti-CTLA4, anti-PD-1 and anti-PD-L1 antibodies has revolutionised the treatment of many cancers. Significant benefit was also proven in gastric adenocarcinomas. Nivolumab improves overall survival as third-line treatment in Asian gastric cancer patients and is already registered in Japan. Pembrolizumab shows significant efficacy, especially in PD-L1-positive patients as third-line treatment and is FDA approved for this indication. Trials with avelumab are promising and studies with atezolizumab and durvalumab are also on the way. To extend the subgroup of benefitting patients combinations of PD-1/PD-L1 antibodies with CTLA4, or VEGF-R2 antibodies or combination with chemotherapy are investigated and show promising results. In this article, the existing evidence of PD1 and PD-L1 blockade as monotherapy or in combination with anti-CTLA4, anti-VEGF-R2 and chemotherapy in gastro-oesophageal adenocarcinoma is reviewed and put into perspective. © 2018 S. Karger GmbH, Freiburg.15,0gastro-oesophageal cancer. Unlike the studies with nivolumab, in the KEYNOTE- 12 patients were selected for PD-L1-positive status; however, a different antibody (QualTek 22C3) and scoring system were used. Eligibility washttps://www.karger.com/Article/Abstract/489099QualTek
2003Wang, A;Clapper, J;Guderian, JA;Foy, TM;Fanger, GR;Retter, MW;Skeiky, YA;A novel method for increasing the expression level of recombinant proteinsProtein Expr. Purif.124-13330112821330Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.13,0visualize antigen expression. Slides were counterstained with hematoxylin to visualize cell nuclei. We would like to acknowledge and thank Qualtek for immunohistochemistry results. FACS analysis. HEK293 cells were transientlyhttps://www.sciencedirect.com/science/article/pii/S1046592803000755QualTek
2006Cantwell, C;Waybill, P;Lynch, F;Taubman, K;Inferior vena cava duplication and deep venous thrombosisAnn Vasc Surg295-2962021657549712,0http://dx.doi.org/10.1007/s10016-006-9013-3Frank Lynch[au]
2018Porter, ET;Porter, FS;A Strain Gauge Monitor (SGM) for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pHJ Vis Exp13830124658An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response to diel-cycling hypoxia. A Wheatstone bridge was connected to strain gauges that were attached to the shells of oysters (Crassostrea virginica). The recorded signals allowed for the opening and closing of the bivalves to be recorded continuously over two-day periods of experimentally-induced diel-cycling hypoxia and diel-cycling changes in pH. Here, we describe a protocol for developing an inexpensive strain gauge monitor and describe, in an example laboratory experiment, how we used it to measure the valve gape behavior of Eastern oysters (C. virginica), in response to diel-cycling hypoxia and cyclical changes in pH. Valve gape was measured on oysters subjected to cyclical severe hypoxic (0.6 mg/L) dissolved oxygen conditions with and without cyclical changes in pH, cyclical mild hypoxic (1.7 mg/L) conditions and normoxic (7.3 mg/L) conditions. We demonstrate that when oysters encounter repeated diel cycles, they rapidly close their shells in response to severe hypoxia and close with a time lag to mild hypoxia. When normoxia is restored, they rapidly open again. Oysters did not respond to cyclical pH conditions superimposed on diel cycling severe hypoxia. At reduced oxygen conditions, more than one third of the oysters closed simultaneously. We demonstrate that oysters respond to diel-cycling hypoxia, which must be considered when assessing the behavior of bivalves to dissolved oxygen. The valve SGM can be used to assess responses of bivalve molluscs to changes in dissolved oxygen or contaminants. Sealing techniques to better seal the valve gape strain gauges from sea water need further improvement to increase the longevity of the sensors.11,0Name Company Catalog Number Comments Campbell CR 10x data logger Campbell Scientific, Logan, Utah Or other data logger. At Campbell the CR 10X has been replaced with the CR 1000 Campbell CR 10x multiplexer Campbell Scientific, Logan, Utah Data logger needs to have space for 12 channels Dsub connector male crimp pins TE Connectivity 205089-1 pins for gape sensor leads PCA tape Micro Measurements Corp, NC To seal the strain gauge Duro Quick Gel Ace Hardware Superglue SG13/1000-LY43 or LY41 Omega Engineering Inc., Stanford, CT Strain gauges 32 AWG (7/40) teflon Alpha wires AlphaWire, Elizabeth, NJ 2840/7 Sensor cables, different colors are available 1/16" heat shrink tubing Qualtek B01A3QKKO6 To seal the leads of the sensor cable Weller WES51 Analog Soldering Station Amazon Lots of soldering, need a good soldering iron. https://www.amazon.com/Weller-WES51-Analog-Soldering-Station/dp/B000BRC2XU/ref=sr_1_23?s=hi&ie=UTF8&qid=1505654295 &sr=1-23&keywords=soldering+iron Rosin Soldering Flux Paste Amazon Needed for solderinghttps://www.jove.com/video/57404/a-strain-gauge-monitor-sgm-for-continuous-valve-gape-measurementsQualTek
2019Symeonides, S;Choice of Law in the American Courts in 2018: Thirty-Second Annual SurveyThe American Journal of Comparative Law1-97671This is the Thirty-Second Annual Survey of American Choice-of-Law Cases.1 It is written at the request of the Association of American Law Schools Section on Conflict of Laws,2 and is intended as a service to fellow teachers and to students of conflicts law, both inside and outside of the United States.3 Its purpose remains the same as it has been from the beginning: to inform, rather than to advocate. Occasionally, however, small amounts of criticism or praise escape the author’s self-censoring filters.8,0In re EP Floors Corp., No. 14-18-00610-CV, 2018 WL 4354688 (Tex. App. Sept. 13, 2018); In re Rosewood Private Invs., Inc., No. 05-18-00166-CV, 2018 WL 4403749 (Tex. App. Sept. 17, 2018); Bundy v. Adesa Houston D/B/A Adesa, Inc., No. 01-17-00863-CV, 2018 WL 6053602 (Tex. App. Nov. 20, 2018). For federal court cases, see, e.g., In re McGraw-Hill Global Educ. Holdings LLC, 909 F.3d 48 (3d Cir. 2018); Wall v. Corona Capital, LLC, Nos. 17-2275, 17-2361, 2018 WL 6133390 (3d Cir. Nov. 23, 2018); Al Copeland Invs., LLC v. First Specialty Ins. Corp., 884 F.3d 540 (5th Cir. 2018); Yei A. Sun v. Advanced China Healthcare, Inc., 901 F.3d 1081 (9th Cir. 2018); Hisey v. Qualtek USA, LLC, No. 16-13477, 2018 WL 4896744 (11th Cir. Oct. 9, 2018)https://academic.oup.com/ajcl/article-abstract/67/1/1/5519501QualTek
2011Lundgren, JA;Matsushima, K;Lynch, FC;Frankel, H;Cooney, RN;Angiographic embolization of nonvariceal upper gastrointestinal bleeding: predictors of clinical failureJ Trauma1208-121270521610434Angiographic embolization (AE) has emerged as an important therapy for patients with nonvariceal upper gastrointestinal bleeding (UGIB). We hypothesized that discrete factors predictive of AE failure could be identified. A retrospective review was performed for patients with nonvariceal UGIB who underwent AE from 1999 to 2009 at Penn State Milton S. Hershey Medical Center. AE clinical failure was defined as requirement for another intervention (surgery, endoscopic therapy, or another AE) for nonvariceal UGIB and/or death from bleeding after AE. Statistical analysis was performed using Fisher's exact test and Student's t test to explore the risk of AE failure. Of 48 total AE cases, 17 patients (35.4%) had clinically failed AE. Mortality rate was significantly higher in patients with AE clinical failure than in patients with AE clinical success (64.7% vs. 12.9%, p=0.001). Factors associated with AE clinical failure include anticoagulant use before admission (p=0.001), use of corticosteroids before admission (p=0.045), pre-AE vasopressor use (p=0.038), and embolization using either coils alone (p=0.05) or using coils with or without additional embolic materials (p=0.018). AE clinical failure portends poor prognosis. Caution should be exercised when considering AE, particularly AE using coils, in patients with a history of anticoagulant, corticosteroid, or vasopressor use.0,0http://dx.doi.org/10.1097/TA.0b013e318213faf1Frank Lynch[au]
2008Cherry, RA;Nichols, PA;Snavely, TM;David, MT;Lynch, FC;Prophylactic inferior vena cava filters: do they make a difference in trauma patients?J Trauma544-54865318784566Inferior vena cava filters (IVCF) are used in trauma patients to reduce the incidence of pulmonary embolism (PE). This study investigates the efficacy of prophylactic IVCF (PIVCF) placement from implantation through outpatient follow-up. Data were prospectively collected on PIVCF placed in trauma patients > or =18-years old from 2004 to 2006. Exclusion criteria include therapeutic IVCF, major burns, deviated from a modified EAST protocol, and deaths. Data were collected on age, gender, Injury Severity Score (ISS), filter type, total implant days, PE, deep venous thrombosis (DVT), and filter-related complications. p < 0.05*, chi square test, mean +/- SD. Of 4,936 patients, 280 had an IVCF with 244 meeting inclusion criteria. Study group demographics: 63.5% men; 98.8% blunt; mean age 43.8 +/- 20.3; ISS 26.7 +/- 12.8. There were 176 of 244 (72.1%) patients who met traditional EAST guidelines for PIVCF. PIVCF increased significantly from 29 in 2004 to 127 in 2006 with no difference in the PE rate (0.7% to 0.4%). There were 4 PEs (1.6%) on postprocedure days 7, 14, 18, and 23. Five technical complications occurred: two filter fractures, two caudal migrations, and one filter tilt. A total of 140 retrievable filters had the opportunity for outpatient follow-up for 18 months with 58.6% removed, 15.7% declared permanent, 12.1% lost to follow-up, and 13.6% still considered potential removal candidates. Days to implant: 0 to 32; 3.89 +/- 4.79. Implant days: 15 to 838; mean 231 +/- 162. PIVCF increased significantly without impacting the overall PE rate. There was a 1.6% PE rate among PIVCF, high retrieval rate (59%), low complication rate (0.1%), and satisfactory compliance with traditional EAST guidelines.0,0http://dx.doi.org/10.1097/TA.0b013e31817f980fFrank Lynch[au]
2020de Almeida, DVP;Fong, L;Rettig, MB;Autio, KA;Immune Checkpoint Blockade for Prostate Cancer: Niche Role or Next Breakthrough?Am Soc Clin Oncol Educ Book1-184032343604A number of trials have evaluated the use of single-agent immune checkpoint inhibitors for the treatment of metastatic castration-resistant prostate cancer (mCRPC). The benefit appears to be limited to a small subset of patients, such as those with tumors with microsatellite instability, highlighting the importance of biomarkers to identify which patients may be more likely to respond. Given the lack of efficacy for most patients with mCRPC, our understanding of the mechanisms of primary resistance to checkpoint inhibitors and of the tumor immune microenvironment in prostate cancer is critical. Knowledge gained in these key areas will allow for the identification of novel combination therapies that will circumvent resistance mechanisms and should be tested in clinical trials. Improving our understanding of the effects of androgen deprivation therapy on immune cells and of the most favorable disease setting (e.g., biochemically recurrent vs. castration-resistant prostate cancer) may aid in the optimal use of checkpoint inhibitors in combination with other agents. If successful, this may move immune checkpoint inhibitors into the treatment armamentarium of prostate cancer management.stromal cells). Among the 245 patients with mCRPC who were screened, 35 met criteria for PD-L1 + expression (14.3%) using a prototype assay (QualTek, Goleta, CA), and 23 were enrolled and received treatment. Seven patientshttps://ascopubs.org/doi/abs/10.1200/EDBK_278853QualTek
2020Tancredi, TS;Kissane, JL;Lynch, FC;Li, M;Kong, L;Waybill, PN;The Effect of Immediate Versus Delayed Port Access on 30-Day Infection RateJ Infus Nurs167-17143332287172This study compared the 30-day infection risk of chest ports accessed on the same day as placement and chest ports with delayed initial access. The aim was to evaluate a larger data set that provided evidence for the development of port access guidelines. A retrospective chart review of 3322 chest port placement procedures performed between October 15, 2003, and June 10, 2015, was conducted at the interventional radiology department of a single institution. Procedure notes and health records were reviewed to determine time of initial port access, evidence of infection within a 30-day window of port placement, and causal organism(s) of infection. The results demonstrated that 64 ports (1.93%) met infection criteria within 30 days of placement, including 30 of the 945 ports immediately accessed and 34 of the 2377 ports not immediately accessed (3.17% vs 1.43%; P < .005). Dual lumen devices had a statistically significant higher rate of infection compared with single lumen devices (P = .006). This study concluded that there is a statistically significant higher rate of infection if a port is accessed immediately versus when access is deferred to later than 24 hours after placement.http://dx.doi.org/10.1097/NAN.0000000000000370Frank Lynch[au]
2020Miller, RM;Nworu, C;McKee, L;Balcerzak, D;Pham, L;Pugh, J;Liu, YZ;Gustafson, H;Marwah, E;Lamb, T;Clements, J;Development of an Immunohistochemical Assay to Detect the Ataxia-Telangiectasia Mutated (ATM) Protein in Gastric CarcinomaAppl. Immunohistochem. Mol. Morphol.303-31028431206368Ataxia-telangiectasia mutated (ATM), a key activator of DNA damage response mechanisms, represents a potential biomarker for targeted gastric carcinoma therapies. A phase II study (Study 39; NCT01063517) designed to investigate the combination olaparib plus paclitaxel in patients with recurrent or metastatic gastric cancer did not meet its primary endpoint of progression-free survival; however, an improvement in the secondary endpoint of overall survival was recorded with a greater overall survival benefit noted in patients with ATM-negative tumors. An ATM immunohistochemical (IHC) diagnostic assay was developed to identify patients who may respond favorably to targeted therapies and deployed in the confirmatory phase III GOLD trial (NCT01924533). The VENTANA ATM (Y170) assay was developed for investigational use in formalin-fixed, paraffin-embedded gastric carcinoma samples using an anti-ATM rabbit monoclonal antibody (clone Y170) and was optimized with OptiView DAB IHC Detection Kit on a BenchMark ULTRA instrument. The assay was deployed in studies assessing sensitivity, specificity, robustness, precision, and determining optimal ATM staining cutoff to define ATM-deficiency (ATM-low). The ATM (Y170) assay met all predefined product development acceptance criteria. Multiple parameters were characterized, including repeatability, reproducibility, analytical sensitivity, specificity, robustness, and product stability. The scoring algorithm was defined; gastric carcinoma samples were considered ATM-negative or ATM-positive when <25% or ≥25%, respectively, of tumor cell nuclei expressed ATM at any IHC stain intensity and nuclei of immune and/or endothelial cells expressed ATM at a moderate stain intensity (internal positive control). Results highlight reproducibility of the assay, supporting suitability for investigational use for evaluation of gastric carcinoma samples using tumor cell staining cutoff of <25% to define ATM-deficiency. Using this ATM assay, phase III GOLD trial (NCT01924533) clinical trial did not meet its primary endpoint, only suggesting, but not demonstrating, that assessment of ATM levels by IHC could possibly be useful in assessing the degree of benefit that may be achieved by adding olaparib to paxitaxel when treating gastric carcinoma. The utility of ATM (Y170) assay as a companion diagnostic requires further clinical validation.deficient gastric tumors. Verification studies included ∼400 FFPE gastric carcinoma cases from US Biomax Inc. (Derwood, MD), PrecisionMed (Solana Beach, CA), and Conversant Bio (Huntsville, AL). All FFPE tissue sampleshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147393/"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2019Barta, SK;Zain, J;MacFarlane, AW;Smith, SM;Ruan, J;Fung, HC;Tan, CR;Yang, Y;Alpaugh, RK;Dulaimi, E;Ross, EA;Campbell, KS;Khan, N;Siddharta, R;Fowler, NH;Fisher, RI;Oki, Y;Phase II Study of the PD-1 Inhibitor Pembrolizumab for the Treatment of Relapsed or Refractory Mature T-cell LymphomaClin Lymphoma Myeloma Leuk356-364.e319631029646Programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are frequently expressed in T-cell lymphomas. This provides a rationale for exploration of immune checkpoint inhibitors in the management of T-cell lymphomas. In this phase II single-arm multicenter trial, patients with relapsed or refractory systemic T-cell lymphoma were treated with 200 mg pembrolizumab intravenously every 21 days. The primary endpoint was progression-free survival (PFS). The secondary endpoints were response rate, overall survival, response duration, and safety. We assessed PD-L1, p-AKT expression, and peripheral blood immune cells as potential predictive biomarkers. Of 18 enrolled patients, 13 were evaluable for the primary endpoint. The trial was halted early after a preplanned interim futility analysis. The overall response rate was 33% (95% confidence interval [CI], 9%-55%); 4 patients achieved a complete response (27%; 95% CI, 5%-49%). The median PFS was 3.2 months (95% CI, 1.2-3.7 months), and the median overall survival was 10.6 months (95% CI, 3.2-100 months). The median duration of response was 2.9 months (95% CI, 0-10.1 months). Two of the 4 complete responders remain in remission > 15 months. Rash was the most common adverse event (17%; n = 3). The most common ≥ grade 3 treatment-emergent adverse events were rash and pneumonitis (11%; n = 2 each). Neither PD-L1 nor p-AKT expression were associated with outcomes. However, a higher relative frequency of CD4+ T lymphocytes pre-treatment was associated with improved PFS (hazard ratio, 0.15; 95% CI, 0.03-0.74). Pembrolizumab demonstrated modest single-agent activity in relapsed or refractory T-cell lymphoma. Copyright © 2019 Elsevier Inc. All rights reserved.PD-L1 expression was determined on formalin-fixed, paraffin-embedded archival tumor samples at a central laboratory (QualTek Molecular Laboratories) using the anti-human PD-L1 antibody (clone 22C3) generated at Merck Research Laboratories (Kenilworth, NJ)https://www.sciencedirect.com/science/article/pii/S2152265018317506QualTek
2019Thirukkumaran, CM;Shi, ZQ;Nuovo, GJ;Luider, J;Kopciuk, KA;Dong, Y;Mostafa, AA;Thakur, S;Gratton, K;Yang, A;Chin, AC;Coffey, MC;Jimenez-Zepeda, VH;Stewart, D;Chesi, M;Bergsagel, PL;Morris, D;Oncolytic immunotherapy and bortezomib synergy improves survival of refractory multiple myeloma in a preclinical modelBlood Adv797-8123530850386The oncolytic reovirus (RV) has demonstrated clinical efficacy and minimal toxicity in a variety of cancers, including multiple myeloma (MM). MM is a malignancy of plasma cells that is considered treatable but incurable because of the 90% relapse rate that is primarily from drug resistance. The systemic nature of MM and the antitumor immunosuppression by its tumor microenvironment presents an ongoing therapeutic challenge. In the present study, we demonstrate that RV synergizes with the standard-of-care MM drug bortezomib (BTZ) and, importantly, enhances its therapeutic potential in therapy-resistant human MM cell lines in vitro. Using the syngeneic Vk*MYC BTZ-resistant immunocompetent transplantable MM murine model, we also demonstrate that mice harboring BTZ-insensitive MM tumors respond to the RV/BTZ combination treatment in terms of decreased tumor burden and improved overall survival (P < .00001). We demonstrate that BTZ augments RV replication in tumor-associated endothelial cells and myeloma cells, leading to enhanced viral delivery and thereby stimulating cytokine release, immune activity, apoptosis, and reduction of the MM-associated immune suppression. We conclude that combined RV/BTZ is an attractive therapeutic strategy with no safety signals for the treatment of MM. © 2019 by The American Society of Hematology.Center, Ohio State University, Columbus, OH; 3. Phylogeny Inc., Powell, OH; http://orcid.org/0000-0002-5351-4721. Search for other works by this author on: This Site. PubMed. Google Scholar. , Joanne Luider Joanne Luider. 4https://ashpublications.org/bloodadvances/article-abstract/3/5/797/246735"Phylogeny Inc"
2017Adams, S;Dramatic response of metaplastic breast cancer to chemo-immunotherapyNPJ Breast Cancer8328649648Frequent overexpression of programmed death-ligand 1 has recently been demonstrated in metaplastic breast cancer, which is a rare breast cancer subtype with limited treatment options. This report describes the clinical course of a patient with metastatic metaplastic breast cancer who had a remarkable response to anti-programmed death-1 therapy with pembrolizumab in combination with nab-paclitaxel. Tissue correlates are presented including tumor-infiltrating lymphocytes and high-programmed death-ligand 1 expression in the tumor.Full size image. PD-L1 staining was performed by Qualtek (Newtown, PA) on formalin-fixed paraffin-embedded tumor sections obtained at study baseline utilizing immunohistochemistry (IHC, 22C3 antibody, Merck). Results ;in the right chest wall mass consistent with a partial treatment response, as well as interval decrease of multiple FDG avid nodal, pulmonary, and pleural metastases PD-L1 staining was performed by Qualtek (Newtown, PA) on formalin-fixed paraffin-embedded tumor sections obtained at study baseline utilizing immunohistochemistry (IHC, 22C3 antibody, Merck). Results showed 100% of tumor cells stainhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445614QualTek
2011Cherry, RA;Goodspeed, DC;Lynch, FC;Delgado, J;Reid, SJ;Intraoperative angioembolization in the management of pelvic-fracture related hemodynamic instabilityJ Trauma Manag Outcomes6521569480This case series report discusses patients presenting with hemorrhage and hemodymanic compromise due to severe pelvic fractures and undergoing intraoperative angioembolization (IAE) with other resuscitative procedures. We used portable digital subtraction fluoroscopy units for IAE in patients with severe pelvic hemorrhage and hemodynamic instability (5/03-4/09). Data was collected on demographics, injury severity, resource utilization, and outcomes at our Level 1 trauma center. There were 6,538 adult admissions with 912 having pelvic fractures and 65 of these undergoing pelvic angioembolization. Twelve hemodynamically compromised patients (10 males, 2 females) had intraoperative pelvic angiography (age: 22-79 years; mean 51.3 ± 17.4). Injury severity score (ISS) was 37.5 ± 8.4 (22-50). Mean emergency department (ED) length of stay (LOS) was 57.4 min ± 47.9 with 10 patients transported directly to the OR and 2 to the SICU prior to OR. Ten of 12 patients underwent exploratory laparotomy followed by angioembolization. Mortality was 50%. Among the 6 survivors (ISS 22 - 50), all had a pre-op CT scan, five had an initial base deficit <13, and four were transfused ≤ 6 units pre-incision/pre-procedure. Four of the 6 survivors had unilateral embolization. In contrast, all 6 non-survivors (ISS 29-41) required massive transfusion prior to OR (>6 units PRBCs) with 4 having a based deficit >13. Three of these patients bypassed CT and five underwent bilateral internal iliac embolization (BIIE). IAE for severe pelvic hemorrhage can be successfully performed concurrently with exploratory laparotomy, pelvic packing or other resuscitative procedures. Patients most likely to benefit have a base deficit <13, and do not require massive transfusion prior to IAE or suffer from a vertically unstable pelvis fracture.http://dx.doi.org/10.1186/1752-2897-5-6Frank Lynch[au]
2011Shatalova, EG;Klein-Szanto, AJ;Devarajan, K;Cukierman, E;Clapper, ML;Estrogen and cytochrome P450 1B1 contribute to both early- and late-stage head and neck carcinogenesisCancer Prev Res (Phila)107-1154121205741Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer in the United States. The goal of this study was to evaluate the contribution of estrogens to the development of HNSCCs. Various cell lines derived from early- and late-stage head and neck lesions were used to characterize the expression of estrogen synthesis and metabolism genes, including cytochrome P450 (CYP) 1B1, examine the effect of estrogen on gene expression, and evaluate the role of CYP1B1 and/or estrogen in cell motility, proliferation, and apoptosis. Estrogen metabolism genes (CYP1B1, CYP1A1, catechol-o-methyltransferase, UDP-glucuronosyltransferase 1A1, and glutathione-S-transferase P1) and estrogen receptor (ER) β were expressed in cell lines derived from both premalignant (MSK-Leuk1) and malignant (HNSCC) lesions. Exposure to estrogen induced CYP1B1 2.3- to 3.6-fold relative to vehicle-treated controls (P = 0.0004) in MSK-Leuk1 cells but not in HNSCC cells. CYP1B1 knockdown by shRNA reduced the migration and proliferation of MSK-Leuk1 cells by 57% and 45%, respectively. Exposure of MSK-Leuk1 cells to estrogen inhibited apoptosis by 26%, whereas supplementation with the antiestrogen fulvestrant restored estrogen-dependent apoptosis. Representation of the estrogen pathway in human head and neck tissues from 128 patients was examined using tissue microarrays. The majority of the samples exhibited immunohistochemical staining for ERβ (91.9%), CYP1B1 (99.4%), and 17β-estradiol (88.4%). CYP1B1 and ERβ were elevated in HNSCCs relative to normal epithelium (P = 0.024 and 0.008, respectively). These data provide novel insight into the mechanisms underlying head and neck carcinogenesis and facilitate the identification of new targets for chemopreventive intervention. ©2011 AACR.Immunohistochemical analyses were performed on histological sections of formalin-fixed, paraffin-embedded human head and neck TMAs and cytospins of cultured human head and neck cells. Sections were stained with antibodies against human CYP1B1 (raised in rabbit, Alpha Diagnostics International Inc., San Antonio, TX), estrogen receptor (ER)α (raised in mouse, Lab Vision Products, Fremont, CA), ERβ (raised in mouse, Serotec, Raleigh, NC) and E2 (raised in rabbit, BioGenex, San Ramon, CA) using standard immunohistochemical procedures (QualTek Molecular Laboratories, Newtown, PA). Sodium citrate (pH 6) was used for antigen retrieval. Human breast carcinoma was used as a positive control for each antibody. TMA sections were scanned and images were captured using an Automated Cellular Imaging System (ACIS, ChromaVision, San Juan Capistrano, CA). Pathologically confirmed regions of HNSCC, dysplasia and normal epithelium were scanned using a 40X objective. Following normalization to a threshold (background staining), the staining intensity of each selected area was quantified and expressed in arbitrary unitshttps://cancerpreventionresearch.aacrjournals.org/content/4/1/107.shortQualTek
2004Scott, BB;Zaratin, PF;Clarke, GD;Barnes, MR;Murdock, PR;Lynch, FJ;Duckworth, M;C20orf9-003 (ACI-1), a gene localized on chromosome 20q13.12 encoding for a 49 kD cytoplasmic protein with a putative nucleotide binding siteDNA Seq.1-815115354348Murine NGD5 is a gene identified from NG108-15 cells which is postulated to be involved in opioid receptor function. Here we report the cloning and characterization of a cDNA C20orf9-003 (ACI-1) encoding the human orthologue of the mouse NGD5. Analysis of the genomic structure revealed that C20orf9-003 (ACI-1) contains 13 exons and 12 introns, spanning 52.5kb of genomic DNA and is a variant of C20orf9. Chromosomal localization of human C20orf9-003 (ACI-1) assigned this gene to chromosome 20q13.12. Genes at this locus have been associated with the progression and possibly the development of various cancers. In addition several linkage studies support the possibility that one or more genes affecting obesity are located in 20q13. No function can be clearly assigned to C20orf9-003 (ACI-1), however, the protein has a cytoplasmic subcellular location and the secondary structure contains a Rossman fold like feature which is found in many nucleotide binding proteins.http://dx.doi.org/10.1080/1042517032000160200Frank Lynch[au]
2020Vos, H;Lambein, K;Floris, G;Mariën, B;Berben, L;Neven, P;Wildiers, H;Nevelsteen, I;Smeets, A;Abstract P5-04-23: Characterization of the tumor microenvironment in a large series of ER/PR negative breast cancerPoster Session AbstractsIntroduction: Recent data show that a subgroup of patients with breast cancer might benefit from immune checkpoint blockade (ICB). However, adequate biomarkers to select patients for ICB are lacking. A better understanding of the immune microenvironment of breast cancer is paramount in the quest for appropriate biomarkers. We investigated several immune related parameters in hormone receptor negative breast cancer, attempting to characterize the tumor microenvironment. We observed the immune micro-environment from three angles. Firstly, we counted and further characterised stromal tumour infiltrating lymphocytes (sTILs). Secondly, we measured the expression of PD1 and PD-L1. Lastly, the expression of CD73 on tumor cells, a potential regulatory molecule of immune escape, was analysed. Methods: Resection specimens of 198 patients, diagnosed with ER/PR negative invasive adenocarcinoma of > 2 cm between 2005 and 2010, were analysed. TILs percentages were analysed according to the international guidelines. Immunohistochemical (IHC) analyses of CD3, CD4, CD8, CD68, CD73, FoxP3 and PD1 were assessed. PD-L1 IHC assay and interpretation was performed by QualTek and a modified proportion score (MPS) was used. The immune parameters were correlated with clinicopathological parameters. Results: Clinicopathological characteristics are summarized in Table 1. The median expression of the immune parameters ranges from 0 to 10 and is presented in Table 2. Further, sTIL percentages were categorised into three groups, with 10% and 30% as cut-off, which matches with respectively 31%, 50% and 19% of the patients. Applying a cut-off of 1%, only 17% of the samples showed PD-L1 expression while 97% of the samples showed PD-1 expression on TILs. As could be expected a significant positive association was observed between sTILs and CD3, CD4 and CD8, but also with CD68, FoxP3 and PD-L1. No association could be noted between PD-1 expression and any of the other parameters. When taking patient age into account, older patients have significant less sTILs and expression of CD3, CD4, CD8 and FoxP3. An increase in the fraction of sTILs with 10% was significantly associated with a lower risk of metastasis ([HR] = 0.529, p < 0.01). This also reflects in the observation that an increase in CD3, CD8 and FoxP3 lowers the risk of metastasis ([HR] of respectively 0.940 (p < 0.01), 0.876 (p = 0.02) and 0.658 (p = 0.01)). No significant association between CD73 and the risk of metastasis was observed (p = 0.1098).Methods: Resection specimens of 198 patients, diagnosed with ER/PR negative invasive adenocarcinoma of 2 cm between 2005 and 2010, were analysed. TILs percentages were analysed according to the international guidelines. Immunohistochemical (IHC) analyses of CD3, CD4, CD8, CD68, CD73, FoxP3 and PD1 were assessed. PD-L1 IHC assay and interpretation was performed by QualTek and a modified proportion score (MPS) was used. The immune parameters were correlated with clinicopathological parametershttps://cancerres.aacrjournals.org/content/80/4_Supplement/P5-04-23.shortQualTek
2004Chen, X;Shen, F;Wang, A;Wang, Z;Zhang, Y;Novel Fabry-Perot fiber optic sensor with multiple applicationsSensors for Harsh Environments1-12A novel Intrinsic Fabry-Perot fiber-optic sensor is presented in this paper. The sensors were made through two simple steps: wet chemical etch and fusion splice. Micro air-gaps were generated inside the fibers and functioned as reflective mirrors. This procedure not only provides a simple and cost effective technology for fabricating intrinsic Fabry-Perot Interferometric (IFPI) fiber sensors, but also provides two possible IFPI structures. Both of the fiber cavity between the air-gaps or the air-gap and cleaved fiber end can be used as sensing elements. With these two structures, this sensor can be used to measure the temperature, strain, pressure, refractive index of chemicals and the thin film thickness by itself. Multi-point measurements can also be achieved by multiplexing. Furthermore, it also can be multiplexed with other sensors such as Long Period Gratings (LPG) to provide compensations for other perturbation sensing. Theoretical and experimental studies of two sensor structures are described. Experimental results show that high resolution and high sensitivity can be obtained with appropriate signal processing.In Fig. 15, we show the results generated by the statistic software (Qualtek-4). Y-axis in Fig. 15 represents the percentage of deviation from mean (in percentage) and x-axis represents the number of experiments. Lead In/Out Fiber Sensor 2 Air-gap Air-gap Sensor 1 Thin filmhttps://www.spiedigitallibrary.org/conference-proceedings-of-spie/5590/0000/Novel-Fabry-Perot-fiber-optic-sensor-with-multiple-applications/10.1117/12.581014.shortQualTek
2019Schruf, E;Schroeder, V;Le, HQ;Schonberger, T;Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in an iPSC-derived air-liquid interface modelBioRxivAn abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is a process often observed in patients suffering from idiopathic pulmonary fibrosis (IPF), a fatal disease characterized by progressive fibrotic lung remodeling. However, the origin of this dysfunctional epithelium remains unknown. In this study, we aimed to investigate the effects of a pro-fibrotic milieu, similar to that found in an IPF lung, on human alveolar epithelial progenitor cell differentiation. We developed an induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar type II (ATII)-like cell differentiation and stimulated it with an IPF-relevant cocktail (IPF-RC), composed of cytokines previously reported to be elevated in IPF lungs. iPSC-derived cultures express ATII markers and contain lamellar body-like structures. Stimulation with IPF-RC during the last two weeks of differentiation increases secretion of IPF biomarkers. Transcriptome analysis of IPF-RC treated cultures reveals significant overlap with human IPF data and enrichment of transcripts associated with extracellular matrix organization. IPF-RC stimulation further impairs ATII differentiation by driving a shift towards an airway epithelial-like expression signature. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization in vitro. Our findings reveal how aberrant alveolar epithelial progenitor cell differentiation in a pro-fibrotic environment could contribute to alveolar bronchiolization in the distal IPF lung.Formalin-fixed and paraffin embedded lung samples from human IPF patients were purchased from Folio Biosciences (Powell, OH, US) under the regulatory conditions of the Boehringer Ingelheim corporate policy regarding the acquisition and use of human biospecimenhttps://www.biorxiv.org/content/10.1101/830109v1.abstract"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2019Lee, TW;David, HS;Engstrom, AK;Carpenter, BS;H3K9me2 protects lifespan against the transgenerational burden of germline transcription in C. elegansbioRxivDuring active transcription, the COMPASS complex methylates histone H3 at lysine 4 (H3K4me). In Caenorhabditis elegans, mutations in COMPASS subunits, including WDR-5, extend lifespan and enable the inheritance of increased lifespan in wild-type descendants. Here we show that the increased lifespan of wdr-5 mutants is itself a transgenerational trait that manifests after eighteen generations and correlates with changes in the heterochromatin factor H3K9me2. Additionally, we find that wdr-5 mutant longevity and its inheritance requires the H3K9me2 methyltransferase MET-2 and can be recapitulated by a mutation in the putative H3K9me2 demethylase JHDM-1. These data suggest that lifespan is constrained by reduced H3K9me2 due to transcription-coupled H3K4me. wdr-5 mutants alleviate this burden, extending lifespan and enabling the inheritance of increased lifespan. Thus, H3K9me2 functions in the epigenetic establishment and inheritance of a complex trait. Based on this model, we propose that lifespan is limited by the germline in part because germline transcription reduces heterochromatin.Samples were sent for library preparation and sequencing 367 at either the HudsonAlpha Genomic Services (Huntsville, AL, USA) or the Georgia Genomics and 368 Bioinformatics Core (Athens, GA, USA). For wdr-5 mutant replicates 1 and 2 and for jhdm-1 mutant 369 replicate 1, single-end 50-bp sequencing was performed on the IIlumina HiSeq v4 platform. For wdr-5 370 replicate 3 and jhdm-1 replicate 2, single-end 75-bp sequencing was performed on the Illumina NextSeq 371 platform. Reads were filtered and aligned to genome WS220/ce10 using Bowtie2 (Langmead & Salzberg 372 2012) using default settings. Peaks were called with MACS v2.1.1 (Feng, Liu, Qin, Zhang, & Liu 2012) using 373 the following parameters: --bw 150https://www.biorxiv.org/content/10.1101/782136v1.abstract"HudsonAlpha Genomic Services"
2020Engstrom, AK;Walker, AC;Moudgal, RA;Myrick, DA;The inhibition of LSD1 via sequestration contributes to tau-mediated neurodegenerationbioRxivTauopathies are a class of neurodegenerative diseases associated with pathological tau. However, the mechanism through which tau contributes to neurodegeneration remains unknown. Previously, our lab implicated the histone demethylase LSD1 in tau-induced neurodegeneration by showing that LSD1 localizes to pathological tau aggregates in Alzheimer’s disease cases, and that it is continuously required for the survival of hippocampal and cortical neurons in mice. Here, we utilize the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. In addition, we show that reducing LSD1 in these mice is sufficient to highly exacerbate tau-mediated neurodegeneration. Finally, we find that overexpressing LSD1 in the hippocampus of tauopathy mice, even after pathology has formed, is sufficient to significantly delay neurodegeneration. These results suggest that inhibiting LSD1 via sequestration contributes to tau-mediated neurodegeneration. Thus, LSD1 is a promising therapeutic target for tauopathies such as Alzheimer’s disease.As much of the aqueous layer was recovered as possible, then RNA was 462 precipitated with isoproponal. Pellets were then washed with 75% ethanol and resuspended in 463 50μL of dionized water. RNA library preparation and sequencing were performed by 464 HudsonAlpha Genomic Services Lab. RNA was Poly(A) selected and 300bp size selected. 465 Libraries were sequenced for 25 million 50bp paired end readshttps://www.biorxiv.org/content/10.1101/745133v2.full-text"HudsonAlpha Genomic Services"
2019Engstrom, AK;Walker, AC;Moudgal, RA;Myrick, DA;Pathological Tau Induces Neurodegeneration by Sequestering and Inhibiting LSD1bioRxivTauopathies are a class of neurodegenerative diseases associated with pathological tau. However, the mechanism through which tau contributes to neurodegeneration remains unknown. Previously, our lab implicated the histone demethylase LSD1 in tau-induced neurodegeneration by showing that LSD1 localizes to pathological tau aggregates in Alzheimer’s Disease cases, and that it is continuously required for the survival of hippocampal and cortical neurons in mice. Here, we utilize the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. In addition, we show that reducing LSD1 in these mice is sufficient to highly exacerbate tau-mediated neurodegeneration. Finally, we find that overexpressing LSD1 in the hippocampus of tauopathy mice, even after pathology has formed, is sufficient to significantly delay neurodegeneration. These results suggest that inhibition of LSD1 is a downstream mediator of pathological tau. Thus, LSD1 is a promising therapeutic target for tauopathies such as Alzheimer’s Disease.Pellets were then washed with 75% ethanol and resuspended in 50μL of dionized water. RNA library preparation and sequencing were performed by HudsonAlpha Genomic Services Lab. RNA was Poly(A) selected and 300bp size selected. Libraries were sequenced for 25 million 50bp paired end readshttps://www.biorxiv.org/content/10.1101/745133v1.abstract"HudsonAlpha Genomic Services"
2018Jaime, MDLA;Karott, S;Salem, GH;Krynitsky, J;The High-throughput WAFFL System for Treating and Monitoring Individual Drosophila melanogaster AdultsBioRxivNon-mammalian model organisms have been essential for our understanding of the mechanisms and control of development, disease, and physiology, but are underutilized in pharmacological phenotypic screening assays due to low throughput compared to cell-based systems. To increase the utility of using Drosophila melanogaster in screening, we have designed the whole animal feeding flat (WAFFL), a novel, flexible, and complete system for feeding, monitoring, and assaying flies in a high throughput format. Our system was conceived keeping in mind the use of off-the-shelf, commercial, 96-well consumables and equipment in order to be amenable to experimental needs. Here we provide an overview of the design and 3-D printing manufacture specifications.In addition to the monitoring arena, the MUFFIN unit housed the electronics needed for the back-lighting subsystem, as well as the video acquisition and transmission: a 12V power transformer (Qualtek Electronics Corporation, London, ON) to supply the illumination PCB, 24 Raspberry Pi (RPI) modules which acquired video from the camera boards, USB power ports (Anker, Seattle, WA) to supply the RPIs, a 24-port Ethernet switch (TRENDnet, Torrance, CA) and a five-port Ethernet switch (Netgear, San Jose,https://www.biorxiv.org/content/10.1101/428037v2.abstractQualTek
2020Arumugam, A;Wong, SS;The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction StepBioRxivQuantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection.1–4 It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. With many PCR-based molecular assays, an extraction step is routinely used as part of the protocol. This step can take up a significant amount of time and labor, especially if the extraction is performed manually. Long assay time, partly caused by slow sample preparation steps, has created a large backlog when testing patient samples suspected of COVID-19. Using flu and RSV clinical specimens, we have collected evidence that the RT-qPCR assay can be performed directly on patient sample material from a nasal swab immersed in virus transport medium (VTM) without an RNA extraction step. We have also used this approach to test for the direct detection of SARS-CoV-2 reference materials spiked in VTM. Our data, while preliminary, suggest that using a few microliters of these untreated samples still can lead to sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests and the backlog we currently experience can be reduced drastically. Next, we will confirm our findings using patient samples.The clinical specimens of Influenza A (DLS16-85584), Influenza B (DLS15-33890 and RSV (KH19-00715) were obtained from Discovery Life Sciences Inc. The TaqPath 1-step multiplex master mix (Cat. No. A28525) was purchased from Thermo Fisher Scientific (Waltham, MA)https://www.biorxiv.org/content/10.1101/2020.04.06.028811v1.abstract"Discovery Life Sciences Inc"
2020Barsh, G;Graff, E;Cochran, N;Kaelin, C;Day, K;Prokop, J;PEA15 loss of function and defective cerebral development in the domestic catbioRxivCerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNFα-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species.Genomic DNA was isolated from liver samples using a Qiagen DNeasy Blood & Tissue kit 344 according to the manufacturer’s instructions. DNA sequencing libraries were prepared for 345 sequencing on the Illumina HiSeq X by the HudsonAlpha Genomic Services Lab by Covaris 346 shearing, end repair, adapter ligation, and PCR using standard protocols. Library concentrationshttps://www.biorxiv.org/content/10.1101/2020.02.14.949214v1.abstract"HudsonAlpha Genomic Services"
2020Sandoval-Sierra, JV;Garcia, FIS;Brooks, JH;Effect of short-term prescription opioids on DNA methylation of the OPRM1 promoterbioRxivBackground Long-term opioid use has been associated with hypermethylation of the opioid receptor mu 1 (OPRM1) promoter. Very little is currently known about the early epigenetic response to therapeutic opioids. Here we examine whether we can detect a similar increase in DNA methylation in the days following the use of prescribed opioids. Longitudinal changes in DNA methylation was assayed using the Illumina Infinium Human MethylationEPIC array in a cohort of 33 opioid-naïve participants who underwent standard dental surgery followed by opioid medication self-administration. Saliva samples were collected before surgery (visit 1), and at two postsurgery visits at 2.7 ± 1.5 days (visit 2), and 39 ± 10 days (visit 3) after the discontinuation of opioid analgesics. Results The perioperative methylome underwent significant changes over the three visits. This was unrelated to opioids, and primarily due to postoperative inflammatory response and alteration in cellular composition. Deconvolution of cell heterogeneity indicated an increase in granulocytes at visit 2 and compensatory decline by visit 3. To specifically examine the effect of opioids, we applied a candidate gene approach and evaluated 10 CpGs located in the OPRM1 promoter. There was significant cross-sectional variability in opioid use, and for participants who self-administered the prescribed drugs, the total dosage ranged from 5–210 morphine milligram equivalent (MME). Participants were categorized by cumulative dosage into three groups:The 370 second saliva sample was collected a few days after opioid discontinuation, and the third was 371 collected on a follow-up visit (Fig. 1; Table 1). DNA was purified using the DNA Genotek PrepIT 372 L2P kit according to manufacturer’s instruction. Genome-wide DNA methylation was assayed 373 on the Illumina Infinium Human MethylationEPIC BeadChips following the manufacturer's 374 standard protocol at the HudsonAlpha Genomic Services Lab (https://gsl.hudsonalpha.org)https://www.biorxiv.org/content/10.1101/2020.01.24.919084v1.abstract"HudsonAlpha Genomic Services"
2020Rowe, S;Samson, C;Clough, D;A Framework to Guide the Instruction of Industrial Programmable Logic Controllers in Undergraduate Engineering EducationEducation for Chemical Engineers1-9Programmable logic controllers are ubiquitous in automation practice and 74% of process controls job advertisements seek applicant familiarity with this equipment. However, undergraduate training on these devices is lacking, likely due to expense and pedagogical uncertainty. Without a clear view of how to teach logic controllers little justifies the purchase of these devices in teaching laboratories and engineering programs. During the modernization of process control exercises at the University of Colorado Boulder a finite state machine framework, common to engineering practice, was used to teach programmable logic controllers. Herein we describe this approach, which reliably guided an undergraduate activity where actual commercial controllers drove engineering automation. Ultimately, the cost of a single apparatus was $2,294US, which appeared warranted given feedback from 84 alumni and closer curricular alignment with industrial needs.12" 1/2 nipple, McMaster Carr, 4882K913, 1, $ 5.26, $ 5.26. AC-DC power converter, Phoenix Contact, 2868651, 1, $ 103.38, $ 103.38. IEC connector, Qualtek, 703W-00/08, 1, $ 0.88, $ 0.88. $ 2,293.68. 3. Finite State Machines for Programmable Logic Controllershttps://www.sciencedirect.com/science/article/pii/S1749772820300178QualTek
2020Grigorieva, J;Asmellash, S;Oliveira, C;Roder, H;Net, L;Roder, J;Application of protein set enrichment analysis to correlation of protein functional sets with mass spectral features and multivariate proteomic testsClinical Mass Spectrometry44-5315Mass spectral data from multiple samples are suitable for a hypothesis-free development of clinically useful multivariate tests using modern machine learning techniques. However, the transition from discovery to adoption of proteomic tests has proved challenging. Slow adoption of these tests in clinical practice is, in part, related to insufficient understanding of the biological mechanisms underlying multivariate tests developed based on correlative studies. While identification of individual proteins may provide important insights, elucidation of concerted relationships of sets of proteins with biological pathways can better reflect complex phenomena, such as cancerogenesis and response to treatment. Protein set enrichment analysis (PSEA) allows identification of associations of mass spectral features or test classifications with biological function by looking for consistent correlations across a group of proteins. We evaluated the utility of PSEA for exploring the biological information content of mass spectra, using a sample set with mass spectral data and matched protein expression information. This made it possible to detect significant biological associations with mass spectral peaks without identifying their protein constituents. We demonstrated that the method produces reproducible associations and can be used for elucidation of the mechanisms of action associated with two previously developed multivariate mass spectrometry-based tests. Significant correlations with several host immune response-related processes were found on the level of individual mass spectral features and with test classifications. The results illustrate the utility of the PSEA approach applied to mass spectral data as a method for elucidating biological mechanisms underlying phenotypes related to different physiological states of the organism.Samples were obtained from commercial biobanks Conversant Bio (Huntsville, AL) and Oncology Metrics (Fort Worth, TX). Samples were collected under ethics-approved protocols, according to the requirements of Conversant Bio and Oncology Metrics, and stored at −80 °Chttps://www.sciencedirect.com/science/article/pii/S2376999819300406"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2020Shrader, S;Australian Labradoodle Dystrophinopathy: A Novel Canine Model for the Study of Duchenne Muscular Dystrophy CardiomyopathyThesisObjective: To evaluate the cardiac transcriptome in affected dogs in order to elucidate potential regulators of cardiomyopathy. Methods: Left ventricular myocardial samples from seven dystrophin-deficient and five control (normal) male littermates were included in the analyses, which were performed at the HudsonAlpha Genomic Services Laboratory. Results: Sequencing found 29,740 gene transcripts expressed in the ventricular myocardium samples; of these, there was a statistically significant difference (p 0.05) and a log2-fold change higher than 1.5 between affected and control dogs in 1267 transcripthttps://etd.auburn.edu/handle/10415/7150"HudsonAlpha Genomic Services"
2008List, LCP;Getting Started with the PICO LogoChipThesis1 "AA" battery holder (3 cells) Keystone 2465K-ND 3 "AA" batteries Panasonic P107-ND 1 USB/serial interface board PICO 1 USB cable Qualtek Q361-ND Tools suggested • additional #22 solid hookup wire on a spool (such as Digi-Key part number C2117W-100-ND)http://academics.wellesley.edu/Physics/Rberg/logochip/v3/alpha-h/GettingStartedWithLogoChips3v-0.4.pdfQualTek
2008Dorfman, MD;Identifying roles for non-essential genes in essential processesThesis2002 VI Page 7. Vll Laboratory Technician, Qualtek Molecular Laboratories, Goleta, California, 1998 -2001 GRANTS, AWARDS AND HONORS: NIH Developmental Biology Training Grant, University of Oregon, 2004-2007 Cohttp://scholarsbank.uoregon.edu/xmlui/handle/1794/9169QualTek
2016Kroening, A;REDUCING NOISE EXPOSURES PRODUCED BY VIBRATORY FINISHING MACHINESThesisNoise monitoring for a tumbling operator occurred in May of 2010 at the request of the company, Qualtek Manufacturing, Inc. The company, Qualtek, wanted to see if engineering controls, noise absorbing baffles hung from the ceiling, had been effective at reducing the noise exposure levels for their operators after initial noise monitoring performed in early March 2010 indicated that the noise exposure limits exceeded the OSHA PEL of 90 dBA TWAhttps://digitalcommons.mtech.edu/grad_rsch/85/QualTek
2010Vaaksio, J;Ultraäänipesurin suunnitteluThesislasiputkisulake. Kuva 4: Kaiuttimien virtalahteen kytkenta 6.5 Kayttojannitteen suodatus Laitteen kayttojannitteen suodatus hoidetaan Qualtek Electronicsin valmistamalla EMI- suodatinmoduulilla. Kyseinen moduuli hoitaa seka elektroniikan hakkuriteholahteen ettahttps://www.theseus.fi/bitstream/handle/10024/14433/lopputyo.pdf?sequence=1QualTek
2017Retana, OM;Desarrollo e implementacion de un sistema automatizado para la recarga de baterias en el proceso de manufacturaThesisJumper Weidmuller Bloque de Terminal Weidmuller Bloque de Terminal tierra Weidmuller Entrada de poder Qualtek Connectores Molex Pines Molexhttp://13.65.82.242:8080/xmlui/handle/cenit/1074QualTek
2001ANDSURFACETEXTUREMEASUREMENT, OFAP;DEVELOPMENT OF A LASER BASED TEXTURE MEASURING SYSTEMThesis1 Power Supply (Triple Output): 5V@3A HTTA-16W-A+ (CONDOR) 2 Regulated Power Supply: 24V@2.4A SLS-24-024-TB (SOLA) 1 Brushless DS Fan 031158 (COMAIR.) 1 Fan Guard 09250G (QUALTEK) 1 Circular Power Connector : 10 Connections 97-3102A-18S-1S (AMPHENOL) 1 Power Switch (On-Off Panel Mounted Toggle No part number Switch) 1 Start Switch (Push Button) AB15AP (NKK SWITCHES)http://library.ctr.utexas.edu/digitized/texasarchive/phase1/2981-1.pdfQualTek
2012Nam, A;Development of a coherent optical imaging system for clinical dermatologyThesisBNC SMA USB NKK Switches NKK Switches NKK Switches. NKK Switches Chicago Miiature, Chicago Miniature Chicago Miniature Lascar Electronics Amphenol Amphenol L-com Connectivity S301T M2018SS1G03-RO M2012SS1W01 M2011SS1W01 607-1050C1 1091M1-24V 1091 M5-24V EMT1900 31 -1 0-RFX 901-9889-RFX ECF504-SC5E Description /coniments neon indicator Thermometer; 28 VDC; LCD; -20 degC to 220 degC (External Sensor); 1 degC; (LASCAR electronics# EMT1900) Power connection 80mm Fan Power Cable Panel mount Power Terminal 1Op Power Terminal 6p Fan Power Supply Orion Fans OD8025-24HB Qualtek Electronics (701W-X2/04https://dspace.mit.edu/handle/1721.1/74928QualTek
2001Lautman, K;Codex OuroborusThesis1 C10 47uf SOLID_TANT Digikey P2042-ND 2.85 2.85 Panasonic ECS-F1CE476 47 uf 1 J1 TERMINAL12 12HDR-200 Digikey 277-1246-ND 4.24 4.24 Phoenix Contact 1729115 Terminal block 1 Power inlet (IEC) w/ fuseholder Digikey Q202-ND 2.50 2.50 Qualtek/Fan-S Div. 719W-00/03 1 Fuse, 12VAC, 4A, 5x20 Digikey WK4716-ND 0.19 0.19 Wikmann USA 1911400000 10 pack 45 G1-G45 ZVN4424A T0-92 Digikey ZVN4424A-ND 1.26 56.70 Zetex ZVN4424A T0-92 pkg. 1 PCB ECD 140.00 140.00 ECD 1 U1 4005XL-PC84 84QUAD Insight Electronics XC4005XL-3PC84C 27.00 27.00 Xilinx XC4005XL-PC84-3 FPGA 40 Solenoid Magnetic Sensor Systems S-17-85-37Q 9.15 366.00 Magnetic Sensor Systems S-17-85-37Q Stand-up solenoihttp://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.215.1159&rep=rep1&type=pdfQualTek
2012Park, SH;Evaluating the Feasibility of Producing Shelf-Stable Low-Acid Vegetables Through Pressure-Ohmic-Thermal Sterilization: Studies on Product Quality and Microbiological SafetyThesisAWG #14 copper wires were soldered on each electrode and passed through the bored outer sample holder plug (Ultem®, SABIC Plastics, Pittsfield, MA, USA). The end of the copper wires were soldered to the high pressure electrical feed through and electrically insulated using polyolefin heat shrink tubing (Q2-Z-1/8, Qualtek Electronics, Mentor, OH, USA). A type K thermocouple (0.51 mm diameter, TFAL/CY-020, Omega engineering, Stamford, CT, USA) was positioned between the outer and inner sample holders to measure the temperature under pressurehttp://rave.ohiolink.edu/etdc/view?acc_num=osu1330954417QualTek
2016Giorgetti, M;Studies on the interaction between inhaled drug molecules and mucinThesisPorcine jejunum was sourced from the local abattoir, complete mini Tablets protease inhibitor cocktail were purchased from Roche, CsCl was purchased from Sigma Aldrich (Gillingham, UK), quick seal centrifuge tube Beckman Coulter (High Wycombe, UK), electric mix-paddle purchased from Qualtek Electronics Corporation (Fort Worth, UK), N-ethyl maleimide and Guanidine purchased from Thermo Fisher Scientific (UK), EDTA was purchased from Sigma Aldrich (Gillingham, UK), Cellulose Ester Dialysis membrane (Spectra/Por Biotech)https://ueaeprints.uea.ac.uk/id/eprint/63610/1/Melania_Giorgetti_Studies_on_the_interaction_between_inhaled_drug_molecules_and_mucin.pdfQualTek
2006Livingston, FJ;Development of an Internet Addressable Pneumatically Controlled Instrument for Applying Strain to Cells In-VitroThesisLCD/TouchScreen Screen Apollo Displays www.apollodisplays.com KI-1A-103B Y 1 $603.22 $603.22 Pressure Transducer Omega www.omega.com PX139-030A4V Y 1 $85 $85.00 Power Switch Qualtek Electronics www.qualtekusa.com 764-00/002 1 Power Outlet Qualtek Electronics www.qualtekusa.com 738W-X2/03 2 CAT5 Panel Plug (RJ45 F/F) Diamond Systems www.diamonsystems.com 698002 Y 1 $10.00 $10.00 Dual-port USB cable Diamond Systems www.diamonsystems.com 698012 Y 1 $8.00 $8.00 Power Cable Qualtek Electronics www.qualtekusa.com 212004-01 1 Motherboard Diamond Systems www.diamonsystems.com HRC750-5A256 Y 1 $995.00 $995https://repository.lib.ncsu.edu/handle/1840.16/538QualTek
2009Wensing, P;Real-time computer control of a prototype bipedal systemThesisVendor Vendor Part Num. Quantity P1 AC Plug NEMA 15-30 Male Hubbell HBL8431C StayOnline 6663 1 P2 3-Conductor Wire (12 AWG) - - Graybar - 40 ft P3 AC Plug NEMA 6-20 Male Hubbell HBL5466C StayOnline 6247 1 P4 AC Connector NEMA 6-20 Female Hubbell HBL5469C StayOnline 6188 1 P5 48VDC Power Supply Galil PSR-6-48 Galil PSR-6-48 1 P6 Power Cord Qualtek 211012-06 Digi-Key Q122-ND 1 P7 2-Conductor Wire (18 AWG) RadioShack 278-567 RadioShack 278-567 20 ft P8 48VDC Power Supply Lambda SWS600L-48 Digi-Key 285-1791-ND 4 P9 Barrier Block Cinch 4-142 Digi-Key CBB304-ND 4 P10 Terminal Jumper Cinch 95B Digi-Key CBB318-ND 12 P11 Ring Spade #8 Stud Tyco Electronics 2-320568-3 Digi-Key A27357-ND 4 P12 Tongue Spade #8 Stud Molex 19144-0039 Digi-Key WM18360-ND 30 P13 Tongue Spade #10 Stud (10-12 AWG) Molex 19144-0042 Digi-Key WM18361-ND 10 P14 Capacitor 10000µF Panasonic ECE-T2AP103EA Digi-Key P10038-ND 1 P15 Schottky Blocking Diode Microsemi SBR8050 Digi-Key SBR8050-ND 1 P16 10-Gauge Wire RadioShack 278-569 RadioShack 278-569 35 ft P17 Tongue Space #8 Stud (18-22 AWG) Molex 19144-0003 Digi-Key WM18353-ND 20 P18 Butt Splice Molex 19154-0004 Digi-Key WM18383-ND 20 P19 Hookup Wire (3 Color, 22 AWG) RadioShack 278-1224 RadioShack 278-1224 10 ft P20 Crimps Molex 08-50-0113 Digi-Key WM1114CT-ND 100 P21 Crimp Housing (5 Position) Molex 22-01-3057 Digi-Key WM2003-ND 4 Table Dhttps://kb.osu.edu/handle/1811/36974QualTek
2015Kauffman, A;Ekert, J;Gyurdieva, A;Rycyzyn, M;Hornby, P;Directed differentiation protocols for successful human intestinal organoids derived from multiple induced pluripotent stem cell linesStem Cell Biol Res121Sections (4 μm) of HIO or hu- man tissue bank (MTB330, QualTek Molecular Labs; Goleta, CA) were dried on glass slides (60°C). Steam heat induced epitope recovery (SHIER) system [24] was followed by were incubated in mouse anti-human E-Cadherin (M3612, Dakohttps://pdfs.semanticscholar.org/83ba/c1b5a58ef54051118f5e3dc80e81f9002063.pdfQualTek
2019Gurevich, V;Resilience of Digital Protection Relay's Power Supplies to Powerful Nanosecond PulsesInternational Journal of Research Studies in Electrical and Electronics Engineering51These filters (single- and two-stage) are manufactured by dozens of companies all over the world, eg Shaffner, Epcos, TDK Lambda, API Technologies, Astrodyne, Qualtek Electronics and many others. The most efficient among them are two-stage filters (fighttp://45.113.122.54/pdfs/ijrseee/v5-i1/5.pdfQualTek
2019Dávila Delgado, L;Propuesta de mejora en la gestión de abastecimiento y comercialización de la empresa Leaders in Import S.A.C.Universidad Peruana de Ciencias AplicadasLa empresa Leaders In Import posee una gama de proveedores de diferentes países, entre los principales se encuentran en China y Alemania. Actualmente, la empresa en estudio es la única representante de la marca Qualtek en el Perú; correspondiendo la mayor parte de los productos que se comercializan para la empresa Telefónicahttps://repositorioacademico.upc.edu.pe/handle/10757/625501QualTek
2013Nicholson, A;Janicke, H;Watson, T;An Initial Investigation into Attribution in SCADA SystemsConferenceSupervisory control and Data Acquisition (SCADA) systems play a core role in a nation’s critical infrastructure, overseeing the monitoring and control of systems in electricity, gas supply, logistics services, banks and hospitals. SCADA systems were once separated from other networks and used proprietary communications protocols, hardware and software. Nowadays modern SCADA systems are increasingly directly or indirectly connected to the Internet, use standardised protocols and commercial-off-the-shelf hardware and software. Attacks on these systems have the potential for devastating consequences and attribution of attacks against SCADA systems presents new challenges. This paper investigates the use of techniques to attribute cyber attacks against SCADA systems.We investigate the use of five known technical attribution techniques in SCADA systems.It allows for users to extend the tool to simulate more network protocols using simple scripts. Pothamsetty and Franz (2004) created a number of scripts to emulate the SCADA functionality of a Qualtek device with HTTP, FTP, Telnet and Modbus serviceshttps://www.scienceopen.com/hosted-document?doi=10.14236/ewic/ICSCSR2013.7QualTek
2020Music, M;Iafolla, M;Soosaipillai, A;Batruch, I;Prassas, I;Pintilie, M;Hansen, A;Bedard, P;Lheureux, S;Spreafico, A;Razak, A;Siu, L;Diamandis, E;Predicting response and toxicity to PD-1 inhibition using serum autoantibodies identified from immuno-mass spectrometryF1000Res3379Background: Validated biomarkers are needed to identify patients at increased risk of immune-related adverse events (irAEs) to immune checkpoint blockade (ICB). Antibodies directed against endogenous antigens can change after exposure to ICB. Methods: Patients with different solid tumors stratified into cohorts received pembrolizumab every 3 weeks in a Phase II trial (INSPIRE study). Blood samples were collected prior to first pembrolizumab exposure (baseline) and approximately 7 weeks (pre-cycle 3) into treatment. In a discovery analysis, autoantibody target immuno-mass spectrometry was performed in baseline and pre-cycle 3 pooled sera of 24 INSPIRE patients based on clinical benefit (CBR) and irAEs. Results: Thyroglobulin (Tg) and thyroid peroxidase (TPO) were identified as the candidate autoantibody targets. In the overall cohort of 78 patients, the frequency of CBR and irAEs from pembrolizumab was 31% and 24%, respectively. Patients with an anti-Tg titer increase ≥1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without this increase in unadjusted, cohort adjusted, and multivariable models (OR=17.4, 95% CI 1.8–173.8, p=0.015). Similarly, patients with an anti-TPO titer ≥ 1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without the increase in unadjusted and cohort adjusted (OR=6.1, 95% CI 1.1–32.7, p=0.035) models. Further, the cohort adjusted analysis showed patients with anti-Tg titer greater than median (10.0 IU/mL) at pre-cycle 3 were more likely to have irAEs (OR=4.7, 95% CI 1.2–17.8, p=0.024). Patients with pre-cycle 3 anti-TPO titers greater than median (10.0 IU/mL) had a significant difference in overall survival (23.8 vs 11.5 months; HR=1.8, 95% CI 1.0–3.2, p=0.05). Conclusions: Patient increase ≥1.5x of anti-Tg and anti-TPO titers from baseline to pre-cycle 3 were associated with irAEs from pembrolizumab, and patients with elevated pre-cycle 3 anti-TPO titers had an improvement in overall survival.The PD-L1 immunohistochemistry clone 22C3 was applied to 4-5 μm sections mounted on positively charged ProbeOn slides (QualTek, Goleta, CA). QualTek produced a modified proportion score (MPS) denoting the proportion Cachedhttps://f1000research.com/articles/9-337QualTek
2018Loi, S;Giobbe-Hurder, A;Gombos, A;Bachelot, T;Hui, R;Curigliano, G;Campone, M;Biganzoli, L;Bonnefoi, H;Jerusalem, G;Bartsch, R;Rabaglio-Poretti, M;Kammler, R;Maibach, R;Smyth, M;Di Leo, A;Colleoni, M;Viale, G;Regan, M;Andre, F;Abstract GS2-06: Phase Ib/II study evaluating safety and efficacy of pembrolizumab and trastuzumab in patients with trastuzumab-resistant HER2-positive metastatic breast cancer: Results from the PANACEA (IBCSG 45-13/BIG 4-13/KEYNOTE-014) studyGeneral Session Abstracts6 pts enrolled in phase Ib between April and July 2015; no DLTs were observed. The PD-L1pos cohort enrolled 40 pts between August 2015 and September 2016. The PD-L1neg cohort enrolled May 2016 to April 2017, stopping after 12 pts due to low rate of PD-L1 negativity, maintaining 90% power to detect the target difference in ORR. PD-L1 testing labs changed in April 2016. Prior to this time, QualTek PD-L1 positive was defined as ≥1% on tumor or TILs. Using the Dako 22C3 antibody, positive was defined as tumor PD-L1 combined positive score (CPS)≥1%https://cancerres.aacrjournals.org/content/78/4_Supplement/GS2-06.shortQualTek
2018Yanakova, E;Kolobanova, M;Tiurin, A;Glebov, A;Goryunov, A;The effective space wire behavior model for a virtual prototype of embedded systems2018 IEEE Conference of Russian Young Researchers in Electrical and Electronic Engineering (EIConRus)In Proc. of ED&TC, 1995. [16] EXPRESSION Homepage. http://www.cecs.uci.edu/~aces/index. html. [17] N. Mouratidis. Development of SpaceWire and CAN SystemC Transaction Level Models for ESA IP cores. // Qualtek Sprl., Brussels, Belgium, 2010https://ieeexplore.ieee.org/abstract/document/8317368/QualTek
2017van Brummelen, EMJ;Mergui, M;Preliminary effi cacy and safety of pembrolizumab in nine different solid tumor typesBookto be PD-L1 positive (≥ 1% of tumor or stroma cells or the presence of a distinctive interface pattern in both neoplastic cells and contiguous mononuclear inflammatory cells) as determined by a prototype immunohistochemistry (IHC) assay (QualTek Molecular Laboratorieshttps://dspace.library.uu.nl/bitstream/handle/1874/356786/vBrummelen.pdf?sequence=1#page=141QualTek
2019Don, ML;Pugh, KE;Kline, BJ;Hallameyer, JM;The Design of a Multichannel Time Interval Measurement Interface BoxAwardName Description Quantity P937 USB cable Panel Mount USB Cable - B Female to Micro-B Male 1 VSK-S20-3R3U-T AC/DC CONVERTER 3.3 V 20 W 1 Qualtek 764-00/002 AC Power Entry Module Screw 1 Cmod A7 Digilent Artix-7 FPGA Module 1 Page 14. 7https://apps.dtic.mil/sti/citations/AD1086071QualTek
2005Sylvia Jr, CJ;The therapeutic value of RDP58 in a mouse model of radiation-induced lung damageThesisbuffered formalin (NBF) and shipped to QualTek Molecular Laboratories (Santa Barbara, CA) where they were imbedded in paraffin and sectioned at 3-5 pm. They were then stained with hematoxylin and eosin (H&E). The slides were evaluated for inflammation, edema, congestion, collagen deposition and fibrosis by a board-certified veterinary pathologist at Idexx Laboratories (West Sacramento, CA)http://search.proquest.com/openview/c750f84200a9ceadb02dc147f6cbbf06/1?pq-origsite=gscholar&cbl=18750&diss=yQualTek
2011Hill, LS;System design, construction, implementation, and validation for rapid single-cell classification using imaging multivariate optical computingThesisThe relay box uses a Bud Industries PN series box enclosure (model PN-1341-C) as the base. The circuit consisted of two NTE Electronics solid-state relays (model RS3- 1D10-51), two Omron Electronic relays (model G4B-112T-C-US-AC120), one Qualtekhttp://search.proquest.com/openview/10e0e6fb73d21ae7978a1fbd21d8e42f/1?pq-origsite=gscholar&cbl=18750&diss=yQualTek
2018Mayes, SA;Improving Hyperspectral Imaging Technology through Systems EngineeringThesisToggle Switches 451-1168-ND Electroswitch Digikey 2 BNC Coaxial Connectors ACX1055-ND Amphenol Digikey 2 3 Prong Outlet Connector 703W-00/08 Qualtek Digikey 1 DB 25 Connector A32074-ND Digikey 2 DB 25 Cables 367-1128-ND 2http://search.proquest.com/openview/45fb543cdce9c0c9cc09a19cd5d2d9aa/1?pq-origsite=gscholar&cbl=18750&diss=yQualTek
2014Li, S;Tighe, SW;Nicolet, CM;Grove, D;Levy, S;Farmerie, W;Viale, A;Wright, C;Schweitzer, PA;Gao, Y;Kim, D;Boland, J;Hicks, B;Kim, R;Chhangawala, S;Jafari, N;Raghavachari, N;Gandara, J;Garcia-Reyero, N;Hendrickson, C;Roberson, D;Rosenfeld, J;Smith, T;Underwood, JG;Wang, M;Zumbo, P;Baldwin, DA;Grills, GS;Mason, CE;Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing studyNat. Biotechnol.915-92532925150835High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.417,0Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.++Vermont Cancer Center, University of Vermont, Burlington, Vermont, USA.++Keck School of Medicine, University of Southern California, Los Angeles, California, USA.++The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida, USA.++Memorial Sloan-Kettering Cancer Institute, New York, New York, USA.++Roy J. Carver Biotechnology Center, University of Illinois, Urbana, Illinois, USA.++Biotechnology Resource Center, Institute of Biotechnology, Cornell University, Ithaca, New York, USA.++Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.++Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.++NIH/NCI/SAIC-Frederick, Gaithersburg, Maryland, USA.++NIH/NCI/SAIC-Frederick, Gaithersburg, Maryland, USA.++Genome Center, University of California, Davis, Davis, California, USA.++Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.++Center for Genetic Medicine, Northwestern University, Chicago, Illinois, USA.++NIH/NHLBI, Bethesda, Maryland, USA.++Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.++Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Starkville, Mississippi, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++NIH/NCI/SAIC-Frederick, Gaithersburg, Maryland, USA.++Division of High Performance and Research Computing, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA.++PerkinElmer Inc., Seattle, Washington, USA.++University of Washington, Department of Genome Sciences. Seattle, Washington, USA.++Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.++Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.++Pathonomics LLC, Philadelphia, Pennsylvania, USA.++Biotechnology Resource Center, Institute of Biotechnology, Cornell University, Ithaca, New York, USA.++Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.http://dx.doi.org/10.1038/nbt.2972HudsonAlpha
2017Lim, JS;Ibaseta, A;Fischer, MM;Cancilla, B;O'Young, G;Cristea, S;Luca, VC;Yang, D;Jahchan, NS;Hamard, C;Antoine, M;Wislez, M;Kong, C;Cain, J;Liu, YW;Kapoun, AM;Garcia, KC;Hoey, T;Murriel, CL;Sage, J;Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancerNature360-364545765428489825The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.401,0... Luminex assay for midkine protein in human plasma Human plasma samples from cancer-free normal donors were purchased from BioreclamationIVT. SCLC donor plasma was sourced from Conversant Biologics (Conversant Bio). The samples were collected, processed and distributed in accordance with IRB approval following informed patient consent. Plasma samples were assayed by following the Luminex assay prhttps://www.nature.com/articles/nature22323"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2013Singh, R;Low, ET;Ooi, LC;Ong-Abdullah, M;Ting, NC;Nagappan, J;Nookiah, R;Amiruddin, MD;Rosli, R;Manaf, MA;Chan, KL;Halim, MA;Azizi, N;Lakey, N;Smith, SW;Budiman, MA;Hogan, M;Bacher, B;Van Brunt, A;Wang, C;Ordway, JM;Sambanthamurthi, R;Martienssen, RA;The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICKNature340-344500746223883930A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation.401,0We thank Johannes Jansen (PRI, Wageningen, Netherlands) for assistance with JoinMap, and Beijing Genome Institute, The Genome Institute at Washington University, Tufts University Core Facility and the Arizona Genome Institute for mapping and genome sequencing. We thank Kulim, Sime Darby, FELDA Agricultural Services and Applied Agricultural Resources for providing materials and phenotype information of individual palms. Phylogeny, Inc.https://www.nature.com/articles/nature12356?draft=collection"Phylogeny Inc"
2016Snyder, MW;Kircher, M;Hill, AJ;Daza, RM;Shendure, J;Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-OriginCell57-681641-226771485Nucleosome positioning varies between cell types. By deep sequencing cell-free DNA (cfDNA), isolated from circulating blood plasma, we generated maps of genome-wide in vivo nucleosome occupancy and found that short cfDNA fragments harbor footprints of transcription factors. The cfDNA nucleosome occupancies correlate well with the nuclear architecture, gene structure, and expression observed in cells, suggesting that they could inform the cell type of origin. Nucleosome spacing inferred from cfDNA in healthy individuals correlates most strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how nucleosome footprints can be used to infer cell types contributing to cfDNA in pathological states such as cancer. Since this strategy does not rely on genetic differences to distinguish between contributing tissues, it may enable the noninvasive monitoring of a much broader set of clinical conditions than currently possible. Copyright © 2016 Elsevier Inc. All rights reserved.304,0... man peripheral blood plasma from healthy donors, donors with clinical diagnosis of Stage IV cancer, and donors with clinical diagnosis with autoimmune disease (Tables S1, S4 and S5) was obtained from Conversant Bio (Huntsville, Alabama, USA) or PlasmaLab International (Everett, Washington, USA). Plasma was stored at −80°C and thawed on the bench-top immediately before use. Cell-free DNA was purhttps://www.sciencedirect.com/science/article/pii/S009286741501569X"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2013Ong, CT;Van Bortle, K;Ramos, E;Corces, VG;Poly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in DrosophilaCell148-159155124055367Insulators mediate inter- and intrachromosomal contacts to regulate enhancer-promoter interactions and establish chromosome domains. The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive. Here, we demonstrate that Drosophila insulator proteins are poly(ADP-ribosyl)ated and that mutation of the poly(ADP-ribose) polymerase (Parp) gene impairs their function. This modification is not essential for DNA occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). However, poly(ADP-ribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo. Consistent with these findings, the nuclear clustering of CP190 complexes is disrupted in Parp mutant cells. Importantly, poly(ADP-ribosyl)ation facilitates intrachromosomal interactions between insulator sites measured by 4C. These data suggest that the role of insulators in organizing the three-dimensional architecture of the genome may be modulated by poly(ADP-ribosyl)ation. Copyright © 2013 Elsevier Inc. All rights reserved.304,0We would like to thank the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology for their help in performing Illumina sequencing of ChIP-Seq samples. This work was supported by U.S. Public Health Service Award R01 GM035463 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.http://dx.doi.org/10.1016/j.cell.2013.08.052HudsonAlpha
2013Mukhopadhyay, S;Wen, X;Ratti, N;Loktev, A;Rangell, L;Scales, SJ;Jackson, PK;The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signalingCell210-2231521-223332756The primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia. Here, we describe a conserved vertebrate G-protein-coupled receptor, Gpr161, which localizes to primary cilia in a Tulp3/IFT-A-dependent manner. Complete loss of Gpr161 in mouse causes midgestation lethality and increased Shh signaling in the neural tube, phenocopying Tulp3/IFT-A mutants. Constitutive Gpr161 activity increases cAMP levels and represses Shh signaling by determining the processing of Gli3 to its repressor form. Conversely, Shh signaling directs Gpr161 to be internalized from cilia, preventing its activity. Thus, Gpr161 defines a morphogenetic pathway coupling protein kinase A activation to Shh signaling during neural tube development. Copyright © 2013 Elsevier Inc. All rights reserved.304,0For the ISH experiments using radiolabeled probes, custom service from Phylogeny Inc. (Columbus, OH) was used. Both antisense and sense radiolabeled riboprobes were synthesized in vitro according to the manufacturer’s specifications (Ambion) and labeled with 35S-UTP (1,000 Ci/mmol; NEG039H, PerkinElmer LAS Canada, Inc.).https://www.sciencedirect.com/science/article/pii/S0092867412015486"Phylogeny Inc"
2018Smith, JJ;Timoshevskaya, N;Ye, C;Holt, C;Keinath, MC;Parker, HJ;Cook, ME;Hess, JE;Narum, SR;Lamanna, F;Kaessmann, H;Timoshevskiy, VA;Waterbury, CKM;Saraceno, C;Wiedemann, LM;Robb, SMC;Baker, C;Eichler, EE;Hockman, D;Sauka-Spengler, T;Yandell, M;Krumlauf, R;Elgar, G;Amemiya, CT;The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolutionNat. Genet.270-27750229358652The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys' unique biology and evolutionary/comparative perspective.280,0Sequencing. Fragment libraries were prepared by Covaris shearing of sperm genomic DNA isolated from a single individual and size selected to achieve average insert sizes of ~205 and 231bp. These libraries were sequenced on the Illumina HiSeq2000 platform. Two separate 4-kb mate pair libraries were generated. One 4-kb library was prepared and sequenced by the Genomic Services Laboratory at HudsonAlpha (Huntsville, AL) and another was prepared and sequenced using the standard Illumina mate-pair kit. Two 4-kb libraries were prepared and sequenced by Lucigen (Middleton, WI). Long reads were prepared by the University of Florida Interdisciplinary Center for Biotechnology Research (Gainesville, FL) and sequenced using Pacific Biosciences (Menlo Park, CA) XL/C2 chemistry on a Single Molecule, Real-Time (SMRT) Sequencing platform.http://dx.doi.org/10.1038/s41588-017-0036-1HudsonAlpha
2016Imsland, F;McGowan, K;Rubin, CJ;Henegar, C;Sundström, E;Berglund, J;Schwochow, D;Gustafson, U;Imsland, P;Lindblad-Toh, K;Lindgren, G;Mikko, S;Millon, L;Wade, C;Schubert, M;Orlando, L;Penedo, MC;Barsh, GS;Andersson, L;Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horsesNat. Genet.152-15848226691985Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.280,0Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.++Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.++Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.++Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.++Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.++Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.++Menntaskólinn við Hamrahlíð, Reykjavík, Iceland.++Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.++Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.++Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.++Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.++Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.++Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.++Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.++Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.https://www.nature.com/ng/journal/v48/n2/abs/ng.3475.html"HudsonAlpha Genomic Services"
2012Xu, B;Ionita-Laza, I;Roos, JL;Boone, B;Woodrick, S;Sun, Y;Levy, S;Gogos, JA;Karayiorgou, M;De novo gene mutations highlight patterns of genetic and neural complexity in schizophreniaNat. Genet.1365-1369441223042115To evaluate evidence for de novo etiologies in schizophrenia, we sequenced at high coverage the exomes of families recruited from two populations with distinct demographic structures and history. We sequenced a total of 795 exomes from 231 parent-proband trios enriched for sporadic schizophrenia cases, as well as 34 unaffected trios. We observed in cases an excess of de novo nonsynonymous single-nucleotide variants as well as a higher prevalence of gene-disruptive de novo mutations relative to controls. We found four genes (LAMA2, DPYD, TRRAP and VPS39) affected by recurrent de novo events within or across the two populations, which is unlikely to have occurred by chance. We show that de novo mutations affect genes with diverse functions and developmental profiles, but we also find a substantial contribution of mutations in genes with higher expression in early fetal life. Our results help define the genomic and neural architecture of schizophrenia.280,0We are enormously grateful to all the families who participated in this research. We thank H. Pretorius and nursing sisters R. van Wyk, C. Botha and H. van den Berg for their assistance with subject recruitment, family history assessments and diagnostic evaluations. We thank S. Laura Lundy for valuable assistance with clinical database maintenance and Laura Rodriguez-Murillo for help with Supplementary Fig. 9. We also wish to thank Brooks Plummer and Melanie Robinson and the HudsonAlpha Genomics Services Laboratory for experimental support. Finally, we thank Prof. Mienie for the thymine loading test. This work was partially supported by National Institute of Mental Health (NIMH) grants MH061399 (to M.K.) and MH077235 (to J.A.G.) and the Lieber Center for Schizophrenia Research at Columbia University. BX was partially supported by a NARSAD Young Investigator Award.http://dx.doi.org/10.1038/ng.2446HudsonAlpha
2011Xu, B;Roos, JL;Dexheimer, P;Boone, B;Plummer, B;Levy, S;Gogos, JA;Karayiorgou, M;Exome sequencing supports a de novo mutational paradigm for schizophreniaNat. Genet.864-86843921822266Despite its high heritability, a large fraction of individuals with schizophrenia do not have a family history of the disease (sporadic cases). Here we examined the possibility that rare de novo protein-altering mutations contribute to the genetic component of schizophrenia by sequencing the exomes of 53 sporadic cases, 22 unaffected controls and their parents. We identified 40 de novo mutations in 27 cases affecting 40 genes, including a potentially disruptive mutation in DGCR2, a gene located in the schizophrenia-predisposing 22q11.2 microdeletion region. A comparison to rare inherited variants indicated that the identified de novo mutations show a large excess of non-synonymous changes in schizophrenia cases, as well as a greater potential to affect protein structure and function. Our analyses suggest a major role for de novo mutations in schizophrenia as well as a large mutational target, which together provide a plausible explanation for the high global incidence and persistence of the disease.280,0BX, JAG and MK designed the study, interpreted the data and prepared the manuscript; BX developed the analysis pipeline and had the primary role in analysis and validation of sequence data; JLR collected the samples and was the primary clinician on the project; SL and BP performed exome library construction, capture and sequencing; PD contributed to the analysis of the data; BB contributed to the primary sequence data analysis; SL supervised the sequencing project at HudsonAlpha Institute and contributed to the manuscript.http://dx.doi.org/10.1038/ng.902HudsonAlpha
2019Fedoriw, A;Rajapurkar, SR;O'Brien, S;Gerhart, SV;Mitchell, LH;Adams, ND;Rioux, N;Lingaraj, T;Ribich, SA;Pappalardi, MB;Shah, N;Laraio, J;Liu, Y;Butticello, M;Carpenter, CL;Creasy, C;Korenchuk, S;McCabe, MT;McHugh, CF;Nagarajan, R;Wagner, C;Zappacosta, F;Annan, R;Concha, NO;Thomas, RA;Hart, TK;Smith, JJ;Copeland, RA;Moyer, MP;Campbell, J;Stickland, K;Mills, J;Jacques-O'Hagan, S;Allain, C;Johnston, D;Raimondi, A;Porter Scott, M;Waters, N;Swinger, K;Boriack-Sjodin, A;Riera, T;Shapiro, G;Chesworth, R;Prinjha, RK;Kruger, RG;Barbash, O;Mohammad, HP;Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP LossCancer Cell100-114.e2536131257072Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection. Copyright © 2019 Elsevier Inc. All rights reserved.274,0GSK3368715 was evaluated at 20, 5, 1.25, 0.3125 and 0.078 mM in a total of 10 patient samples. DLBCL patient cells from 10 unique donors were received as frozen samples from Conversant Bio. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. The use of human tissue samples was reviewed and approved by GSK Research & Development Compliance (RDC) Human Biological Sample Use Committee. Samples were thawed rapidly and diluted in 10 ml of IMDM + 10% FBS and washed. The supernatant was discarded and the cell pellets were resuspended in a known volume of IMDM + 10% FBS. To assess the effect of the test compound on DLBCL CFC, a methylcellulose media formulation containing 10% ALCM was used. The cultures were incubated in a humidified incubator at 37 C, 5% CO2 for 14 days and the colonies then manually enumerated.https://www.sciencedirect.com/science/article/pii/S1535610819302569"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Li, J;Stagg, NJ;Johnston, J;Harris, MJ;Menzies, SA;DiCara, D;Clark, V;Hristopoulos, M;Cook, R;Slaga, D;Nakamura, R;McCarty, L;Sukumaran, S;Luis, E;Ye, Z;Wu, TD;Sumiyoshi, T;Danilenko, D;Lee, GY;Totpal, K;Ellerman, D;Hötzel, I;James, JR;Junttila, TT;Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell KillingCancer Cell383-39531328262555The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies. Copyright © 2017 Genentech. Published by Elsevier Inc. All rights reserved.274,0... ec). CD4+ or CD8+ cells were used as effectors in a 3:1 effector:target ratio. In Vitro Cytotoxicity Assay: Human Plasma Cells and Primary MM Samples Human BMMCs from MM patients were procured from Conversant Bio. Human bone marrow aspirates of healthy donors were procured from AllCells. All human biospecimens were collected, processed, and distributed in full ethical and regulatory compliance whttps://www.sciencedirect.com/science/article/pii/S1535610817300156"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2014Graves, JD;Kordich, JJ;Huang, TH;Piasecki, J;Bush, TL;Sullivan, T;Foltz, IN;Chang, W;Douangpanya, H;Dang, T;O'Neill, JW;Mallari, R;Zhao, X;Branstetter, DG;Rossi, JM;Long, AM;Huang, X;Holland, PM;Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activityCancer Cell177-18926225043603Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into why Apo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadministration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combination of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistant cells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higher order clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy. Copyright © 2014 Elsevier Inc. All rights reserved.274,0Non-small-cell lung tumor samples were collected under Institutional Review Board approval for each institution, with appropriate informed consent, from surgical resections or after biopsy. Freshly resected human tissue samples were obtained from the following institutions: Asterand USA, Bio-Options, MT Group, and Conversant Bio.https://www.sciencedirect.com/science/article/pii/S1535610814001913"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Toy, W;Weir, H;Razavi, P;Lawson, M;Goeppert, AU;Mazzola, AM;Smith, A;Wilson, J;Morrow, C;Wong, WL;De Stanchina, E;Carlson, KE;Martin, TS;Uddin, S;Li, Z;Fanning, S;Katzenellenbogen, JA;Greene, G;Baselga, J;Chandarlapaty, S;Activating ESR1 Mutations Differentially Affect the Efficacy of ER AntagonistsCancer Discov277-2877327986707Recent studies have identified somatic ESR1 mutations in patients with metastatic breast cancer and found some of them to promote estrogen-independent activation of the receptor. The degree to which all recurrent mutants can drive estrogen-independent activities and reduced sensitivity to ER antagonists like fulvestrant is not established. In this report, we characterize the spectrum of ESR1 mutations from more than 900 patients. ESR1 mutations were detected in 10%, with D538G being the most frequent (36%), followed by Y537S (14%). Several novel, activating mutations were also detected (e.g., L469V, V422del, and Y537D). Although many mutations lead to constitutive activity and reduced sensitivity to ER antagonists, only select mutants such as Y537S caused a magnitude of change associated with fulvestrant resistance in vivo Correspondingly, tumors driven by Y537S, but not D5358G, E380Q, or S463P, were less effectively inhibited by fulvestrant than more potent and bioavailable antagonists, including AZD9496. These data point to a need for antagonists with optimal pharmacokinetic properties to realize clinical efficacy against certain ESR1 mutants.Significance: A diversity of activating ESR1 mutations exist, only some of which confer resistance to existing ER antagonists that might be overcome by next-generation inhibitors such as AZD9496. Cancer Discov; 7(3); 277-87. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 235. ©2016 American Association for Cancer Research.200,0... n). PATIENT-DERIVED XENOGRAFTS (PDX) CTC-714 PDX model was derived from patient circulating tumor cells (CTC) cultures, consented according to the Human Biological Samples Policy and purchased from Conversant Biologics. The CTCs were obtained from a 63-year-old patient with stage IV ER+ breast cancer after 42 days of fulvestrant therapy and 26 days of eribulin therapy. To generate the PDX, EpCAM ... n). PATIENT-DERIVED XENOGRAFTS (PDX) CTC-714 PDX model was derived from patient circulating tumor cells (CTC) cultures, consented according to the Human Biological Samples Policy and purchased from Conversant Biologics. The CTCs were obtained from a 63-year-old patient with stage IV ER+ breast cancer after 42 days of fulvestrant therapy and 26 days of eribulin therapy. To generate the PDX, EpCAMhttp://cancerdiscovery.aacrjournals.org/content/7/3/277.abstract"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2016Wang, G;Lu, X;Dey, P;Deng, P;Wu, CC;Jiang, S;Fang, Z;Zhao, K;Konaparthi, R;Hua, S;Zhang, J;Li-Ning-Tapia, EM;Kapoor, A;Wu, CJ;Patel, NB;Guo, Z;Ramamoorthy, V;Tieu, TN;Heffernan, T;Zhao, D;Shang, X;Khadka, S;Hou, P;Hu, B;Jin, EJ;Yao, W;Pan, X;Ding, Z;Shi, Y;Li, L;Chang, Q;Troncoso, P;Logothetis, CJ;McArthur, MJ;Chin, L;Wang, YA;DePinho, RA;Targeting YAP-Dependent MDSC Infiltration Impairs Tumor ProgressionCancer Discov80-956126701088The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival. ©2015 American Association for Cancer Research.200,0... d R&D Biosystems (Minneaplois, MN). Cxcr2 antibody was obtained from Bioss and R & D Biosystems. CD45 and Ly-6G antibodies were obtained from Biolegend. Prostate tissue micrcroarray was obtained from Folio Bioscience. CHROMATIN IMMUNOPRECIPITATION Chromatin immunoprecipitation (ChIP) was performed as described (26) using YAP1 antibody from Novus. Briefly, 5ug of Rabbit IgG (Santa Cruz) or YAP1 a ... d R&D Biosystems (Minneaplois, MN). Cxcr2 antibody was obtained from Bioss and R & D Biosystems. CD45 and Ly-6G antibodies were obtained from Biolegend. Prostate tissue micrcroarray was obtained from Folio Bioscience. CHROMATIN IMMUNOPRECIPITATION Chromatin immunoprecipitation (ChIP) was performed as described (26) using YAP1 antibody from Novus. Briefly, 5ug of Rabbit IgG (Santa Cruz) or YAP1 ahttp://cancerdiscovery.aacrjournals.org/content/6/1/80.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2014Brissova, M;Aamodt, K;Brahmachary, P;Prasad, N;Hong, JY;Dai, C;Mellati, M;Shostak, A;Poffenberger, G;Aramandla, R;Levy, SE;Powers, AC;Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes β cell regenerationCell Metab.498-51119324561261Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature, islet microenvironment, and β cell mass, we transiently increased VEGF-A production by β cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to β cell loss. After withdrawal of the VEGF-A stimulus, β cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing β cells. Bone marrow-derived macrophages (MΦs) recruited to the site of β cell injury were crucial for the β cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated β cells will improve strategies aimed at β cell regeneration and expansion. Copyright © 2014 Elsevier Inc. All rights reserved.182,0Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Electronic address: marcela.brissova@vanderbilt.edu.++Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA; Department of Biology, University of Alabama, Huntsville, Huntsville, AL 35899, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA. Electronic address: al.powers@vanderbilt.edu.http://dx.doi.org/10.1016/j.cmet.2014.02.001HudsonAlpha
2019Melamed, Z;López-Erauskin, J;Baughn, MW;Zhang, O;Drenner, K;Sun, Y;Freyermuth, F;McMahon, MA;Beccari, MS;Artates, JW;Ohkubo, T;Rodriguez, M;Lin, N;Wu, D;Bennett, CF;Rigo, F;Da Cruz, S;Ravits, J;Lagier-Tourenne, C;Cleveland, DW;Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegenerationNat. Neurosci.180-19022230643298Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from patients with sporadic ALS and familial ALS with GGGGCC repeat expansion in the C9orf72 gene, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS-FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.178,0... titute for the kind contribution of the SIN18 vector. We thank B. Ren for providing the Illumina sequencing platform. We thank A. Goginashvili for valued input on the manuscript. We thank J. Nuovo at Phylogeny Inc. (Columbus, OH) for performing the CISH experiment. This work was supported by grants from NINDS/NIH R01-NS27036 to D.W.C, Target ALS (S20A00) and NINDS/NIH (R01-NS087227) to C.L.-T. andhttps://www.nature.com/articles/s41593-018-0293-z"Phylogeny Inc"
2017Le Pichon, CE;Meilandt, WJ;Dominguez, S;Solanoy, H;Lin, H;Ngu, H;Gogineni, A;Sengupta Ghosh, A;Jiang, Z;Lee, SH;Maloney, J;Gandham, VD;Pozniak, CD;Wang, B;Lee, S;Siu, M;Patel, S;Modrusan, Z;Liu, X;Rudhard, Y;Baca, M;Gustafson, A;Kaminker, J;Carano, RAD;Huang, EJ;Foreman, O;Weimer, R;Scearce-Levie, K;Lewcock, JW;Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative diseaseSci Transl Med940328814543Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.168,0AD (early-stage AD) or confirmed AD along with age-matched controls from Folio Biosciences or Banner Sun Health Research Institute. All human tissues were collected with informed consents and institutional review board approvals.https://stm.sciencemag.org/content/9/403/eaag0394?intcmp=trendmd-stm"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2015Sun, LL;Ellerman, D;Mathieu, M;Hristopoulos, M;Chen, X;Li, Y;Yan, X;Clark, R;Reyes, A;Stefanich, E;Mai, E;Young, J;Johnson, C;Huseni, M;Wang, X;Chen, Y;Wang, P;Wang, H;Dybdal, N;Chu, YW;Chiorazzi, N;Scheer, JM;Junttila, T;Totpal, K;Dennis, MS;Ebens, AJ;Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignanciesSci Transl Med287ra70728725972002Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies. Copyright © 2015, American Association for the Advancement of Science.168,0We have obtained 40 ml of whole blood collected in Streck tubes, separated into plasma and extracted cfRNA samples from two Stage IV Prostate cancer patients and three healthy controls from Conversant Bio and generated cfRNA sequencing data with UMIs added. For the two cancer patients, it has been determined that TMPRSS2-ETV4 and TMPRSS2-ERG fusion were the most likely real fusion events, because TMPRSS2 fusion are detected in 50% of prostate cancer (Tomlins et al., 2008). The three healthy individuals had a primary diagnosis of normal, and therefore, should contain no real fusion events and serve as negative controlshttps://academic.oup.com/bioinformatics/article-abstract/35/14/i225/5529228"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Pearlman, R;Frankel, WL;Swanson, B;Zhao, W;Yilmaz, A;Miller, K;Bacher, J;Bigley, C;Nelsen, L;Goodfellow, PJ;Goldberg, RM;Paskett, E;Shields, PG;Freudenheim, JL;Stanich, PP;Lattimer, I;Arnold, M;Liyanarachchi, S;Kalady, M;Heald, B;Greenwood, C;Paquette, I;Prues, M;Draper, DJ;Lindeman, C;Kuebler, JP;Reynolds, K;Brell, JM;Shaper, AA;Mahesh, S;Buie, N;Weeman, K;Shine, K;Haut, M;Edwards, J;Bastola, S;Wickham, K;Khanduja, KS;Zacks, R;Pritchard, CC;Shirts, BH;Jacobson, A;Allen, B;de la Chapelle, A;Hampel, H;, ;Prevalence and Spectrum of Germline Cancer Susceptibility Gene Mutations Among Patients With Early-Onset Colorectal CancerJAMA Oncol464-4713427978560Hereditary cancer syndromes infer high cancer risks and require intensive cancer surveillance, yet the prevalence and spectrum of these conditions among unselected patients with early-onset colorectal cancer (CRC) is largely undetermined. To determine the frequency and spectrum of cancer susceptibility gene mutations among patients with early-onset CRC. Overall, 450 patients diagnosed with colorectal cancer younger than 50 years were prospectively accrued from 51 hospitals into the Ohio Colorectal Cancer Prevention Initiative from January 1, 2013, to June 20, 2016. Mismatch repair (MMR) deficiency was determined by microsatellite instability and/or immunohistochemistry. Germline DNA was tested for mutations in 25 cancer susceptibility genes using next-generation sequencing. Mutation prevalence and spectrum in patients with early-onset CRC was determined. Clinical characteristics were assessed by mutation status. In total 450 patients younger than 50 years were included in the study, and 75 gene mutations were found in 72 patients (16%). Forty-eight patients (10.7%) had MMR-deficient tumors, and 40 patients (83.3%) had at least 1 gene mutation: 37 had Lynch syndrome (13, MLH1 [including one with constitutional MLH1 methylation]; 16, MSH2; 1, MSH2/monoallelic MUTYH; 2, MSH6; 5, PMS2); 1 patient had the APC c.3920T>A, p.I1307K mutation and a PMS2 variant; 9 patients (18.8%) had double somatic MMR mutations (including 2 with germline biallelic MUTYH mutations); and 1 patient had somatic MLH1 methylation. Four hundred two patients (89.3%) had MMR-proficient tumors, and 32 patients (8%) had at least 1 gene mutation: 9 had mutations in high-penetrance CRC genes (5, APC; 1, APC/PMS2; 2, biallelic MUTYH; 1, SMAD4); 13 patients had mutations in high- or moderate-penetrance genes not traditionally associated with CRC (3, ATM; 1, ATM/CHEK2; 2, BRCA1; 4, BRCA2; 1, CDKN2A; 2, PALB2); 10 patients had mutations in low-penetrance CRC genes (3, APC c.3920T>A, p.I1307K; 7, monoallelic MUTYH). Importantly, 24 of 72 patients (33.3%) who were mutation positive did not meet established genetic testing criteria for the gene(s) in which they had a mutation. Of 450 patients with early-onset CRC, 72 (16%) had gene mutations. Given the high frequency and wide spectrum of mutations, genetic counseling and testing with a multigene panel could be considered for all patients with early-onset CRC.166,0Myriad Genetics Inc donated the next-generation sequencing testing for mismatch repair–proficient patients with CRC diagnosed younger than 50 years for this study. Dr Kalady discloses honorarium from Helomics and Transenterix and is a member of the speakers’ bureau for Helomics. Ms Heald discloses a consulting or advising role with Invitae and Myriad Genetics Inc and is a member of the speakers’ bureau for Myriad Genetics Inc. Dr Paquette has received honoraria from Medtronic and has served on the speakers’ bureau. Dr Kuebler discloses employment with Columbus Oncology Associates, LLC. Dr Mahesh discloses stock in Merck, Pfizer, Kite pharmaceuticals, Oncothyreon, and Exelixis. Ms Buie discloses research funding from Merck, Galera, Amgen, and Celgene. Dr Haut discloses research funding from Amgen, Celgene, Incyte, Conversant Bio, Evidera, EMD Serono, Vector Oncology, Bristol Myers, GlaxoSmithKline, Eli Lilly, Luitpold pharmaceuticals, and Sanofi pharmaceuticals. Mr Allen discloses employment and stock with Myriad Genetics Inc. Dr de la Chapelle discloses a patent or intellectual property interest with Genzyme and Ipsogenhttps://jamanetwork.com/journals/jamaoncology/article-abstract/2593042"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2015Li, L;Lyu, X;Hou, C;Takenaka, N;Nguyen, HQ;Ong, CT;Cubeñas-Potts, C;Hu, M;Lei, EP;Bosco, G;Qin, ZS;Corces, VG;Widespread rearrangement of 3D chromatin organization underlies polycomb-mediated stress-induced silencingMol. Cell216-23158225818644Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are formed in regions containing active genes and clusters of architectural protein binding sites. The transcription of most genes is repressed after temperature stress in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. Heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies in the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be reconfigured quickly. Copyright © 2015 Elsevier Inc. All rights reserved.147,0We thank Drs. John T. Lis for providing antibodies against RNAPII, and Vincent Pirrotta and Richard Jones for providing antibodies against Pc. We would like to thank the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology, and specially Braden Boone, Angela Jones and Terri Pointer, for their help in performing Illumina sequencing of ChIP-Seq samples. This work was supported by U.S. Public Health Service Awards R01 GM035463 to V.G.C., R01 HG005119 to Z.S.Q., R01 GM069462 to G.B, and funds from the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases DK015602 to E.P.L.http://dx.doi.org/10.1016/j.molcel.2015.02.023HudsonAlpha
2011Wood, AM;Van Bortle, K;Ramos, E;Takenaka, N;Rohrbaugh, M;Jones, BC;Jones, KC;Corces, VG;Regulation of chromatin organization and inducible gene expression by a Drosophila insulatorMol. Cell29-3844121981916Insulators are multiprotein-DNA complexes thought to affect gene expression by mediating inter- and intrachromosomal interactions. Drosophila insulators contain specific DNA-binding proteins plus common components, such as CP190, that facilitate these interactions. Here, we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA-binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to DNA to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli. Copyright © 2011 Elsevier Inc. All rights reserved.147,0Chromatin immunoprecipitation was performed as described (Bushey et al., 2009). To generate sequencing libraries ChIP DNA was prepared for adaptor ligation by end repair (‘End-It DNA End Repair Kit’ - Epicentre Cat# ER0720) and addition of ‘A’ base to 3′ ends (Klenow 3′-5′ exo- NEB Cat# M0212S). Illumina adaptors (Illumina Cat# PE-102-1001) were titrated according to prepared DNA ChIP sample concentration, and ligated with T4 ligase (NEB Cat# M0202S). Ligated ChIP samples were PCR amplified using Illumina primers and Phusion DNA polymerase (NEB Cat# F-530L) and size selected for 200–300bp by gel extraction. ChIP libraries were sequenced at the HudsonAlpha Institute for Biotechnology using an Illumina GAII sequencer.http://dx.doi.org/10.1016/j.molcel.2011.07.035HudsonAlpha
2018Dejanovic, B;Huntley, MA;De Mazière, A;Meilandt, WJ;Wu, T;Srinivasan, K;Jiang, Z;Gandham, V;Friedman, BA;Ngu, H;Foreman, O;Carano, RAD;Chih, B;Klumperman, J;Bakalarski, C;Hanson, JE;Sheng, M;Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q AntibodiesNeuron1322-1336.e7100630392797Synapse loss and Tau pathology are hallmarks of Alzheimer's disease (AD) and other tauopathies, but how Tau pathology causes synapse loss is unclear. We used unbiased proteomic analysis of postsynaptic densities (PSDs) in Tau-P301S transgenic mice to identify Tau-dependent alterations in synapses prior to overt neurodegeneration. Multiple proteins and pathways were altered in Tau-P301S PSDs, including depletion of a set of GTPase-regulatory proteins that leads to actin cytoskeletal defects and loss of dendritic spines. Furthermore, we found striking accumulation of complement C1q in the PSDs of Tau-P301S mice and AD patients. At synapses, C1q decorated perisynaptic membranes, accumulated in correlation with phospho-Tau, and was associated with augmented microglial engulfment of synapses and decline of synapse density. A C1q-blocking antibody inhibited microglial synapse removal in cultured neurons and in Tau-P301S mice, rescuing synapse density. Thus, inhibiting complement-mediated synapse removal by microglia could be a potential therapeutic target for Tau-associated neurodegeneration. Copyright © 2018 Elsevier Inc. All rights reserved.140,0Biological Samples Human brain tissue Folio Biosciences N/Ahttps://www.sciencedirect.com/science/article/pii/S0896627318309024"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2014Fecteau, JF;Corral, LG;Ghia, EM;Gaidarova, S;Futalan, D;Bharati, IS;Cathers, B;Schwaederlé, M;Cui, B;Lopez-Girona, A;Messmer, D;Kipps, TJ;Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21(WAF1/Cip1)-dependent mechanism independent of functional p53Blood1637-16441241024990888Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL. © 2014 by The American Society of Hematology.132,0Blood samples were collected from CLL patients at the University of California San Diego Moores Cancer Center who satisfied diagnostic and immunophenotypic criteria for common B-cell CLL, and who provided written, informed consent, in compliance with the Declaration of Helsinki18 and the Institutional Review Board of the University of California San Diego. Peripheral blood mononuclear cells were isolated by density centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), resuspended in 90% fetal calf serum (FCS) (Omega Scientific, Tarzana, CA) and 10% DMSO for viable storage in liquid nitrogen. Alternatively, viably frozen CLL cells were purchased from AllCells (Emeryville, CA) or Conversant Biologics (Huntsville, AL). Samples with .95% CD191CD51 CLL cells were used without further purification throughout this study.http://www.bloodjournal.org/content/124/10/1637.short"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2018Schafer, PH;Ye, Y;Wu, L;Kosek, J;Ringheim, G;Yang, Z;Liu, L;Thomas, M;Palmisano, M;Chopra, R;Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosusAnn. Rheum. Dis.1516-1523771029945920IKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacology of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos, was studied in SLE patient cells and in a phase 1 healthy volunteer study. CRBN, IKZF1 and IKZF3 gene expression was measured in peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy volunteers. Ikaros and Aiolos protein levels were measured by Western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured in SLE PBMC cultures treated for 7 days with iberdomide. Fifty-six healthy volunteers were randomised to a single dose of iberdomide (0.03-6 mg, n=6 across seven cohorts) or placebo (n=2/cohort). CD19+ B cells, CD3+ T cells and intracellular Aiolos were measured by flow cytometry. Interleukin (IL)-2 and IL-1β production was stimulated with anti-CD3 and lipopolysaccharide, respectively, in an ex vivo whole blood assay. SLE patient PBMCs expressed significantly higher CRBN (1.5-fold), IKZF1 (2.1-fold) and IKZF3 (4.1-fold) mRNA levels compared with healthy volunteers. Iberdomide significantly reduced Ikaros and Aiolos protein levels in B cells, T cells and monocytes. In SLE PBMC cultures, iberdomide inhibited anti-dsDNA and anti-phospholipid autoantibody production (IC50 ≈10 nM). Single doses of iberdomide (0.3-6 mg) in healthy volunteers decreased intracellular Aiolos (minimum mean per cent of baseline: ≈12%-28% (B cells); ≈0%-33% (T cells)), decreased absolute CD19+ B cells, increased IL-2 and decreased IL-1β production ex vivo. These findings demonstrate pharmacodynamic activity of iberdomide and support its further clinical development for the treatment of SLE. NCT01733875; Results. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.128,0study in healthy volunteers. Methods. Gene expression studies. Viably frozen PBMCs from 11 patients with SLE and 10 healthy volunteers were obtained from Conversant Bio (Huntsville, Alabama, USA). Informed consent washttps://ard.bmj.com/content/77/10/1516.abstract"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2019Furie, R;Werth, VP;Merola, JF;Stevenson, L;Reynolds, TL;Naik, H;Wang, W;Christmann, R;Gardet, A;Pellerin, A;Hamann, S;Auluck, P;Barbey, C;Gulati, P;Rabah, D;Franchimont, N;Monoclonal antibody targeting BDCA2 ameliorates skin lesions in systemic lupus erythematosusJ. Clin. Invest.1359-1371129330645203Plasmacytoid DCs (pDC) produce large amounts of type I IFN (IFN-I), cytokines convincingly linked to systemic lupus erythematosus (SLE) pathogenesis. BIIB059 is a humanized mAb that binds blood DC antigen 2 (BDCA2), a pDC-specific receptor that inhibits the production of IFN-I and other inflammatory mediators when ligated. A first-in-human study was conducted to assess safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) effects of single BIIB059 doses in healthy volunteers (HV) and patients with SLE with active cutaneous disease as well as proof of biological activity and preliminary clinical response in the SLE cohort. A randomized, double-blind, placebo-controlled clinical trial was conducted in HV (n = 54) and patients with SLE (n = 12). All subjects were monitored for adverse events. Serum BIIB059 concentrations, BDCA2 levels on pDCs, and IFN-responsive biomarkers in whole blood and skin biopsies were measured. Skin disease activity was determined using the Cutaneous Lupus Erythematosus Disease Area and Severity Index Activity (CLASI-A). Single doses of BIIB059 were associated with favorable safety and PK profiles. BIIB059 administration led to BDCA2 internalization on pDCs, which correlated with circulating BIIB059 levels. BIIB059 administration in patients with SLE decreased expression of IFN response genes in blood, normalized MxA expression, reduced immune infiltrates in skin lesions, and decreased CLASI-A score. Single doses of BIIB059 were associated with favorable safety and PK/PD profiles and robust target engagement and biological activity, supporting further development of BIIB059 in SLE. The data suggest that targeting pDCs may be beneficial for patients with SLE, especially those with cutaneous manifestations. ClinicalTrials.gov NCT02106897. Biogen Inc.128,0... un-exposed skin of HVs matched approximately by age and ethnicity to the BIIB059-treated cohort (mean age = 34; range, 28–37; White, 6/8; Asian, 1/8; Black or African American, 1/8; female, 8/8) by Folio Biosciences, then processed into paraffin blocks for transfer to Biogen. IHC for MxA, IFITM3, CD45, and dual IHC for CD123/CD31 were performed on 1 section from each specimen on Ventana Discover ... un-exposed skin of HVs matched approximately by age and ethnicity to the BIIB059-treated cohort (mean age = 34; range, 28–37; White, 6/8; Asian, 1/8; Black or African American, 1/8; female, 8/8) by Folio Biosciences, then processed into paraffin blocks for transfer to Biogen. IHC for MxA, IFITM3, CD45, and dual IHC for CD123/CD31 were performed on 1 section from each specimen on Ventana Discover ... un-exposed skin of HVs matched approximately by age and ethnicity to the BIIB059-treated cohort (mean age = 34; range, 28–37; White, 6/8; Asian, 1/8; Black or African American, 1/8; female, 8/8) by Folio Biosciences, then processed into paraffin blocks for transfer to Biogen. IHC for MxA, IFITM3, CD45, and dual IHC for CD123/CD31 were performed on 1 section from each specimen on Ventana Discoverhttps://www.jci.org/articles/view/124466"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2018Liles, JT;Corkey, BK;Notte, GT;Budas, GR;Lansdon, EB;Hinojosa-Kirschenbaum, F;Badal, SS;Lee, M;Schultz, BE;Wise, S;Pendem, S;Graupe, M;Castonguay, L;Koch, KA;Wong, MH;Papalia, GA;French, DM;Sullivan, T;Huntzicker, EG;Ma, FY;Nikolic-Paterson, DJ;Altuhaifi, T;Yang, H;Fogo, AB;Breckenridge, DG;ASK1 contributes to fibrosis and dysfunction in models of kidney diseaseJ. Clin. Invest.4485-45001281030024858Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.128,0... NAL BIOPSIES AND DB/DB ENOS–/– MOUSE KIDNEYS Formalin-fixed paraffin-embedded renal biopsies from 10 patients with DKD and from 7 donors without kidney disease (“healthy”) were obtained from Folio Biosciences. Kidney sections were prepared from db/db eNOS–/– mice that were treated with 0.3% GS-444217 in chow for 8 weeks (_n_ = 8–10). Sections were immunostained for p-p38 using a rab ... NAL BIOPSIES AND DB/DB ENOS–/– MOUSE KIDNEYS Formalin-fixed paraffin-embedded renal biopsies from 10 patients with DKD and from 7 donors without kidney disease (“healthy”) were obtained from Folio Biosciences. Kidney sections were prepared from db/db eNOS–/– mice that were treated with 0.3% GS-444217 in chow for 8 weeks (_n_ = 8–10). Sections were immunostained for p-p38 using a rab ... NAL BIOPSIES AND DB/DB ENOS–/– MOUSE KIDNEYS Formalin-fixed paraffin-embedded renal biopsies from 10 patients with DKD and from 7 donors without kidney disease (“healthy”) were obtained from Folio Biosciences. Kidney sections were prepared from db/db eNOS–/– mice that were treated with 0.3% GS-444217 in chow for 8 weeks (_n_ = 8–10). Sections were immunostained for p-p38 using a rabhttps://www.jci.org/articles/view/99768/files/pdf"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2016Vilgelm, AE;Johnson, CA;Prasad, N;Yang, J;Chen, SC;Ayers, GD;Pawlikowski, JS;Raman, D;Sosman, JA;Kelley, M;Ecsedy, JA;Shyr, Y;Levy, SE;Richmond, A;Connecting the Dots: Therapy-Induced Senescence and a Tumor-Suppressive Immune MicroenvironmentJ. Natl. Cancer Inst.djv406108626719346Tumor cell senescence is a common outcome of anticancer therapy. Here we investigated how therapy-induced senescence (TIS) affects tumor-infiltrating leukocytes (TILs) and the efficacy of immunotherapy in melanoma. Tumor senescence was induced by AURKA or CDK4/6 inhibitors (AURKAi, CDK4/6i). Transcriptomes of six mouse tumors with differential response to AURKAi were analyzed by RNA sequencing, and TILs were characterized by flow cytometry. Chemokine RNA and protein expression were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Therapeutic response was queried in immunodeficient mice, in mice with CCL5-deficient tumors, and in mice cotreated with CD137 agonist to activate TILs. CCL5 expression in reference to TIS and markers of TILs was studied in human melanoma tumors using patient-derived xenografts (n = 3 patients, n = 3 mice each), in AURKAi clinical trial samples (n = 3 patients, before/after therapy), and in The Cancer Genome Atlas (n = 278). All statistical tests were two-sided. AURKAi response was associated with induction of the immune transcriptome (P = 3.5 x 10-29) while resistance inversely correlated with TIL numbers (Spearman r = -0.87, P < .001). AURKAi and CDK4/6i promoted the recruitment of TILs by inducing CCL5 secretion in melanoma cells (P ≤ .005) in an NF-κB-dependent manner. Therapeutic response to AURKAi was impaired in immunodeficient compared with immunocompetent mice (0% vs 67% tumors regressed, P = .01) and in mice bearing CCL5-deficient vs control tumors (P = .61 vs P = .02); however, AURKAi response was greatly enhanced in mice also receiving T-cell-activating immunotherapy (P < .001). In human tumors, CCL5 expression was also induced by AURKAi (P ≤ .02) and CDK4/6i (P = .01) and was associated with increased immune marker expression (P = 1.40 x 10-93). Senescent melanoma cells secret CCL5, which promotes recruitment of TILs. Combining TIS with immunotherapy that enhances tumor cell killing by TILs is a promising novel approach to improve melanoma outcomes. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.126,0Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE). anna.e.vilgelm@vanderbilt.edu.++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).++Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN (AEV, CAJ, JY, JSP, DR, AR); Department of Cancer Biology (AEV, AJ, JY, SCC, JSP, DR, AR), Center for Quantitative Sciences (SCC), Division of Cancer Biostatistics, Department of Biostatistics (GDA, YS), Division of Hematology/Oncology, Department of Medicine (JAS), Vanderbilt University Medical Center, Nashville, TN; HudsonAlpha Institute for Biotechnology, Huntsville, Alabama (NP, SEL); Division of Surgical Oncology, Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN (MK); Translational Medicine, Takeda Pharmaceuticals International Co, Cambridge, MA (JAE).http://dx.doi.org/10.1093/jnci/djv406HudsonAlpha
2019Hudson, WH;Prokhnevska, N;Gensheimer, J;Akondy, R;McGuire, DJ;Ahmed, R;Kissick, HT;Expression of novel long noncoding RNAs defines virus-specific effector and memory CD8+ T cellsNat Commun19610130643116In response to viral infection, CD8+ T cells undergo expansion and differentiate into distinct classes of effector cells. After clearance of the virus, a small population of long-lived memory cells persists. Comprehensive studies have defined the protein-coding transcriptional changes associated with this process. Here we expand on this prior work by performing RNA-sequencing to identify changes in long noncoding RNA (lncRNA) expression in human and mouse CD8+ T cells responding to viral infection. We identify hundreds of unannotated lncRNAs and show that expression profiles of both known and novel lncRNAs are sufficient to define naive, effector, and memory CD8+ T cell subsets, implying that they may be involved in fate decisions during antigen-driven differentiation. Additionally, in comparing mouse and human lncRNA expression, we find that lncRNAs with conserved sequence undergo similar changes in expression in the two species, suggesting an evolutionarily conserved role for lncRNAs during CD8+ T cell differentiation.121,0... of Emory University. RNA Isolation and sequencing RNA was isolated from sorted cells of LCMV-infected mice with the Qiagen AllPrep Micro Kit. Library preparation and sequencing were performed by the HudsonAlpha Genomic Services Laboratory via their low-input RNA-sequencing protocol: poly(A)-selected RNA was amplified using the Ovation RNA-Seq System V2 kit (Nugen) and libraries were sequenced onhttps://www.nature.com/articles/s41467-018-07956-7"HudsonAlpha Genomic Services"
2017Potts, AH;Vakulskas, CA;Pannuri, A;Yakhnin, H;Babitzke, P;Romeo, T;Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomicsNat Commun15968129150605CsrA is a post-transcriptional regulatory protein that is widely distributed among bacteria. This protein influences bacterial lifestyle decisions by binding to the 5' untranslated and/or early coding regions of mRNA targets, causing changes in translation initiation, RNA stability, and/or transcription elongation. Here, we assess the contribution of CsrA to gene expression in Escherichia coli on a global scale. UV crosslinking immunoprecipitation and sequencing (CLIP-seq) identify RNAs that interact directly with CsrA in vivo, while ribosome profiling and RNA-seq uncover the impact of CsrA on translation, RNA abundance, and RNA stability. This combination of approaches reveals unprecedented detail about the regulatory role of CsrA, including novel binding targets and physiological roles, such as in envelope function and iron homeostasis. Our findings highlight the integration of CsrA throughout the E. coli regulatory network, where it orchestrates vast effects on gene expression.121,0;... nufacturer’s instructions with one modification. Adaptors and reverse transcription primers were diluted 1 in 5 in water before use. Libraries were sequenced using HiSeq 2500 50SE (Illumina) by the HudsonAlpha Genomic Services Laboratory. Sequencing reads were demultiplexed and trimmed with cutadapt64 to remove adaptor sequences. Reads longer than 12 bases were then mapped to E. coli rRNA sequehttps://www.nature.com/articles/s41467-017-01613-1HudsonAlpha
2017Christopher, MA;Myrick, DA;Barwick, BG;Engstrom, AK;Porter-Stransky, KA;Boss, JM;Weinshenker, D;Levey, AI;Katz, DJ;LSD1 protects against hippocampal and cortical neurodegenerationNat Commun8058128993646To investigate the mechanisms that maintain differentiated cells, here we inducibly delete the histone demethylase LSD1/KDM1A in adult mice. Loss of LSD1 leads to paralysis, along with widespread hippocampus and cortex neurodegeneration, and learning and memory defects. We focus on the hippocampus neuronal cell death, as well as the potential link between LSD1 and human neurodegenerative disease and find that loss of LSD1 induces transcription changes in common neurodegeneration pathways, along with the re-activation of stem cell genes, in the degenerating hippocampus. These data implicate LSD1 in the prevention of neurodegeneration via the inhibition of inappropriate transcription. Surprisingly, we also find that transcriptional changes in the hippocampus are similar to Alzheimer's disease (AD) and frontotemporal dementia (FTD) cases, and LSD1 is specifically mislocalized to pathological protein aggregates in these cases. These data raise the possibility that pathological aggregation could compromise the function of LSD1 in AD and FTD."LSD1 is a histone demethylase that plays many roles during development. Here, the authors provide evidence that loss of LSD1 in adult mice leads to paralysis and neurodegeneration in the hippocampus and cortex and suggest a potential link between LSD1 and human neurodegenerative disease.121,0... s possible, then RNA was precipitated with isopropanol. Pellets were then washed with 75% ethanol and resuspended in 50 µL deionized water. RNA library preparation and sequencing were performed by HudsonAlpha Genomic Services Lab. RNA was Poly(A) selected and 300 bp size selected. Libraries were sequenced for 25 million 50 bp paired-end reads. RNA-seq analysis Short read FASTQ files were qhttps://www.nature.com/articles/s41467-017-00922-9"HudsonAlpha Genomic Services"
2016Wakeman, CA;Moore, JL;Noto, MJ;Zhang, Y;Singleton, MD;Prentice, BM;Gilston, BA;Doster, RS;Gaddy, JA;Chazin, WJ;Caprioli, RM;Skaar, EP;The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interactionNat Commun11951727301800Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections.121,0Total RNA was harvested using a combination of LETS buffer (0.1 M LiCl; 10 mM EDTA; 10 mM Tris HCl pH 7.4; 1% SDS) and TRI Reagent as previously described61. qRT–PCR was performed using SYBR green supermix (Bio-Rad) following manufacturer’s instructions using the primers listed in Supplementary Table 1. RNA samples for RNA-seq were submitted to HudsonAlpha (Huntsville, AL) for ribosomal reduction, 50 bp paired-end (PE) sequencing with 12.5 million reads per sample and subsequent data analysis. The statistical test used for this data set was a moderated t-test with a corrected P value cutoff of 0.05, asymptotic P value computation and Benjamini–Hochberg multiple testing correctionhttp://dx.doi.org/10.1038/ncomms11951HudsonAlpha
2015Bhate, A;Parker, DJ;Bebee, TW;Ahn, J;Arif, W;Rashan, EH;Chorghade, S;Chau, A;Lee, JH;Anakk, S;Carstens, RP;Xiao, X;Kalsotra, A;ESRP2 controls an adult splicing programme in hepatocytes to support postnatal liver maturationNat Commun8768626531099Although major genetic networks controlling early liver specification and morphogenesis are known, the mechanisms responsible for postnatal hepatic maturation are poorly understood. Here we employ global analyses of the mouse liver transcriptome to demonstrate that postnatal remodelling of the liver is accompanied by large-scale transcriptional and post-transcriptional transitions that are cell-type-specific and temporally coordinated. Combining detailed expression analyses with gain- and loss-of-function studies, we identify epithelial splicing regulatory protein 2 (ESRP2) as a conserved regulatory factor that controls the neonatal-to-adult switch of ∼20% of splice isoforms in mouse and human hepatocytes. The normal shift in splicing coincides tightly with dramatic postnatal induction of ESRP2 in hepatocytes. We further demonstrate that forced expression of ESRP2 in immature mouse and human hepatocytes is sufficient to drive a reciprocal shift in splicing and causes various physiological abnormalities. These findings define a direct role for ESRP2 in the generation of conserved repertoires of adult splice isoforms that facilitate terminal differentiation and maturation of hepatocytes.121,0... RI digested amplified PCR products for Esrp2 cDNA in pDP19 vector (Ambion). Sense and anti-sense RNA strands were generated as previously described43. In situ hybridization for Esrp2 was performed by Phylogeny Inc. (Columbus, Ohio.) The C57BL/6 mouse samples were prepared for in situ hybridization as previously described. Histology and immunohistochemistry Liver tissues from wild-type (n=3) and Ehttps://www.nature.com/articles/ncomms9768"Phylogeny Inc"
2014Sugihara, K;Kobayashi, Y;Suzuki, A;Tamura, N;Motamedchaboki, K;Huang, CT;Akama, TO;Pecotte, J;Frost, P;Bauer, C;Jimenez, JB;Nakayama, J;Aoki, D;Fukuda, MN;Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosisNat Commun4478525047118Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel β3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans.121,0... wa (positive) and A431 (negative) cells. A hybridoma clone was subcloned by a limited dilution, establishing the 3B2 clone. Paraffin tissue sections endometriosis patients (_n_=35) were obtained from Folio Biosciences (Columbus, OH). A normal uterine endometrium tissue microarray (60 cores) and a normal tissue microarray (60 cores) were obtained from Imgenex (San Diego, CA). Antigen retrieval was ... wa (positive) and A431 (negative) cells. A hybridoma clone was subcloned by a limited dilution, establishing the 3B2 clone. Paraffin tissue sections endometriosis patients (_n_=35) were obtained from Folio Biosciences (Columbus, OH). A normal uterine endometrium tissue microarray (60 cores) and a normal tissue microarray (60 cores) were obtained from Imgenex (San Diego, CA). Antigen retrieval was ... wa (positive) and A431 (negative) cells. A hybridoma clone was subcloned by a limited dilution, establishing the 3B2 clone. Paraffin tissue sections endometriosis patients (_n_=35) were obtained from Folio Biosciences (Columbus, OH). A normal uterine endometrium tissue microarray (60 cores) and a normal tissue microarray (60 cores) were obtained from Imgenex (San Diego, CA). Antigen retrieval washttps://www.nature.com/articles/ncomms5478?origin=ppub"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2013Yang, J;Sung, E;Donlin-Asp, PG;Corces, VG;A subset of Drosophila Myc sites remain associated with mitotic chromosomes colocalized with insulator proteinsNat Commun1464423403565Myc has been characterized as a transcription factor that activates expression of genes involved in pluripotency and cancer, and as a component of the replication complex. Here we find that Myc is present at promoters and enhancers of Drosophila melanogaster genes during interphase. Myc colocalizes with Orc2, which is part of the prereplication complex, during G1. As is the case in mammals, Myc associates preferentially with paused genes, suggesting that it may also be involved in the release of RNA polymerase II from the promoter-proximal pausing in Drosophila. Interestingly, about 40% of Myc sites present in interphase persists during mitosis. None of the Myc mitotic sites correspond to enhancers, and only some correspond to promoters. The rest of the mitotic Myc sites overlap with binding sites for multiple insulator proteins that are also maintained in mitosis. These results suggest alternative mechanisms to explain the role of Myc in pluripotency and cancer.121,0ChIP was performed with ~4 × 107 cells. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Nuclear lysates were sonicated to generate 200– 1000 bp DNA fragments. ChIP was then performed with 6 μL of Drosophila α-Myc antibody (Santa Cruz Biotechnology, sc-28208), α-CP190, α-CTCF or α-BEAF-32 antibodies 50, 51. Libraries were prepared with the Illumina TruSeq DNA Sample Preparation Kit. Fragments in the 200–300 bp ranged were selected and sequenced in an Illumina HiSeq sequencer at the HudsonAlpha Institute for Biotechnology.http://dx.doi.org/10.1038/ncomms2469HudsonAlpha
2016Yang, Z;Shah, K;Khodadadi-Jamayran, A;Jiang, H;Dpy30 is critical for maintaining the identity and function of adult hematopoietic stem cellsJ. Exp. Med.2349-23642131127647347As the major histone H3K4 methyltransferases in mammals, the Set1/Mll complexes play important roles in animal development and are associated with many diseases, including hematological malignancies. However, the role of the H3K4 methylation activity of these complexes in fate determination of hematopoietic stem and progenitor cells (HSCs and HPCs) remains elusive. Here, we address this question by generating a conditional knockout mouse for Dpy30, which is a common core subunit of all Set1/Mll complexes and facilitates genome-wide H3K4 methylation in cells. Dpy30 loss in the adult hematopoietic system results in severe pancytopenia but striking accumulation of HSCs and early HPCs that are defective in multilineage reconstitution, suggesting a differentiation block. In mixed bone marrow chimeras, Dpy30-deficient HSCs cannot differentiate or efficiently up-regulate lineage-regulatory genes, and eventually fail to sustain for long term with significant loss of HSC signature gene expression. Our molecular analyses reveal that Dpy30 directly and preferentially controls H3K4 methylation and expression of many hematopoietic development-associated genes including several key transcriptional and chromatin regulators involved in HSC function. Collectively, our results establish a critical and selective role of Dpy30 and the H3K4 methylation activity of the Set1/Mll complexes for maintaining the identity and function of adult HSCs. © 2016 Yang et al.120,0Total RNAs and ChIP (and their corresponding input) DNAs were sent to the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology (Huntsville, AL) for sequencing, including library construction. In brief, the quality of the total RNA and DNA was assessed using the Agilent 2100 Bioanalyzer. Two rounds of polyA+ selection was performed for RNA samples, followed by conversion to cDNAs.http://dx.doi.org/10.1084/jem.20160185HudsonAlpha
2014Ramesh, R;Kozhaya, L;McKevitt, K;Djuretic, IM;Carlson, TJ;Quintero, MA;McCauley, JL;Abreu, MT;Unutmaz, D;Sundrud, MS;Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoidsJ. Exp. Med.89-104211124395888IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease.120,0... sue samples. All experiments using human blood and tissue samples were conducted in accordance with IRB protocols approved by committees at Research Blood Components, New York Blood Center, OneBlood, Conversant Bio., and the University of Miami. Peripheral blood from healthy adult volunteers was purchased from Research Blood Components (Boston, MA), New York Blood Center (New York, NY), and OneBlohttp://jem.rupress.org/content/211/1/89.short"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Ramaker, RC;Savic, D;Hardigan, AA;Newberry, K;Cooper, GM;Myers, RM;Cooper, SJ;A genome-wide interactome of DNA-associated proteins in the human liverGenome Res.1950-1960271129021291Large-scale efforts like the ENCODE Project have made tremendous progress in cataloging the genomic binding patterns of DNA-associated proteins (DAPs), such as transcription factors (TFs). However, most chromatin immunoprecipitation-sequencing (ChIP-seq) analyses have focused on a few immortalized cell lines whose activities and physiology differ in important ways from endogenous cells and tissues. Consequently, binding data from primary human tissue are essential to improving our understanding of in vivo gene regulation. Here, we identify and analyze more than 440,000 binding sites using ChIP-seq data for 20 DAPs in two human liver tissue samples. We integrated binding data with transcriptome and phased WGS data to investigate allelic DAP interactions and the impact of heterozygous sequence variation on the expression of neighboring genes. Our tissue-based data set exhibits binding patterns more consistent with liver biology than cell lines, and we describe uses of these data to better prioritize impactful noncoding variation. Collectively, our rich data set offers novel insights into genome function in human liver tissue and provides a valuable resource for assessing disease-related disruptions. © 2017 Ramaker et al.; Published by Cold Spring Harbor Laboratory Press.119,0HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA.http://genome.cshlp.org/content/27/11/1950.short"HudsonAlpha Genomic Services"
2015Weckselblatt, B;Hermetz, KE;Rudd, MK;Unbalanced translocations arise from diverse mutational mechanisms including chromothripsisGenome Res.937-94725726070663Unbalanced translocations are a relatively common type of copy number variation and a major contributor to neurodevelopmental disorders. We analyzed the breakpoints of 57 unique unbalanced translocations to investigate the mechanisms of how they form. Fifty-one are simple unbalanced translocations between two different chromosome ends, and six rearrangements have more than three breakpoints involving two to five chromosomes. Sequencing 37 breakpoint junctions revealed that simple translocations have between 0 and 4 base pairs (bp) of microhomology (n = 26), short inserted sequences (n = 8), or paralogous repeats (n = 3) at the junctions, indicating that translocations do not arise primarily from nonallelic homologous recombination but instead form most often via nonhomologous end joining or microhomology-mediated break-induced replication. Three simple translocations fuse genes that are predicted to produce in-frame transcripts of SIRPG-WWOX, SMOC2-PROX1, and PIEZO2-MTA1, which may lead to gain of function. Three complex translocations have inversions, insertions, and multiple breakpoint junctions between only two chromosomes. Whole-genome sequencing and fluorescence in situ hybridization analysis of two de novo translocations revealed at least 18 and 33 breakpoints involving five different chromosomes. Breakpoint sequencing of one maternally inherited translocation involving four chromosomes uncovered multiple breakpoints with inversions and insertions. All of these breakpoint junctions had 0-4 bp of microhomology consistent with chromothripsis, and both de novo events occurred on paternal alleles. Together with other studies, these data suggest that germline chromothripsis arises in the paternal genome and may be transmitted maternally. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of the molecular mechanisms behind translocation formation. © 2015 Weckselblatt et al.; Published by Cold Spring Harbor Laboratory Press.119,0We used Agilent SureSelect Target Enrichment to pull down 40-kb regions around breakpoints fine-mapped by custom array CGH. SureSelect followed by Illumina HiSeq sequencing was performed at HudsonAlpha Genomic Services Laboratory, and sequence analysis was performed as described previously (Hermetz et al. 2014). Custom SureSelect library numbers (ELID) are listed in Supplemental Tables S1 and S2. Arrays and SureSelect libraries were designed using the GRCh37/hg19 genome build, and we kept genomic coordinates in this version so that the design IDs correspond to the coordinates in our tables. Providing genomic coordinates in this genome build does not affect our conclusions.http://genome.cshlp.org/content/25/7/937.short"HudsonAlpha Genomic Services"
2012Yang, J;Ramos, E;Corces, VG;The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila speciesGenome Res.2199-2207221122895281Understanding the relationship between genome organization and expression is central to understanding genome function. Closely apposed genes in a head-to-head orientation share the same upstream region and are likely to be coregulated. Here we identify the Drosophila BEAF-32 insulator as a cis regulatory element separating close head-to-head genes with different transcription regulation modes. We then compare the binding landscapes of the BEAF-32 insulator protein in four different Drosophila genomes and highlight the evolutionarily conserved presence of this protein between close adjacent genes. We find that changes in binding of BEAF-32 to sites in the genome of different Drosophila species correlate with alterations in genome organization caused by DNA rearrangements or genome size expansion. The cross-talk between BEAF-32 genomic distribution and genome organization contributes to new gene-expression profiles, which in turn translate into specific and distinct phenotypes. The results suggest a mechanism for the establishment of differences in transcription patterns during evolution.119,0on a Branson Sonifier 250 with output control set at 1.5. Libraries were prepared with the Illumina TruSeq DNA Sample Preparation Kit and sequenced at the HudsonAlpha Institute for Biotechnology.http://dx.doi.org/10.1101/gr.142125.112HudsonAlpha
2012Van Bortle, K;Ramos, E;Takenaka, N;Yang, J;Wahi, JE;Corces, VG;Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domainsGenome Res.2176-2187221122722341Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.119,0Ligated ChIP samples were PCR-amplified using Illumina primers and Phusion DNA polymerase (NEB Cat# F-530L) and size selected for 200–300 bp by gel extraction. ChIP libraries were sequenced at the HudsonAlpha Institute for Biotechnology, using an Illumina HiSeq 2000. Sequences were mapped to the dm3 genome with Bowtie 0.12.3 (Langmead 2010) using default settings. Peaks were then called with MACS 1.4.0alpha2 (Zhang et al. 2008) using equal numbers of unique reads for input and ChIP samples and a P-value cutoff of 1 × 10−10.http://dx.doi.org/10.1101/gr.136788.111HudsonAlpha
2012Kellner, WA;Ramos, E;Van Bortle, K;Takenaka, N;Corces, VG;Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promotersGenome Res.1081-108822622508764Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. Here, we show that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism.119,0We thank Mattias Mannervik for the gift of CBP antibody and members of the Corces Lab for invaluable discussions. We also thank The Genomic Services Lab at the HudsonAlpha Institute for Biotechnology for their help in performing Illumina sequencing of ChIP-seq samples. This work was supported by U.S. Public Health Service Award GM35463 from the National Institutes of Health.http://dx.doi.org/10.1101/gr.136929.111HudsonAlpha
2019Ma, X;Shao, Y;Tian, L;Flasch, DA;Mulder, HL;Edmonson, MN;Liu, Y;Chen, X;Newman, S;Nakitandwe, J;Li, Y;Li, B;Shen, S;Wang, Z;Shurtleff, S;Robison, LL;Levy, S;Easton, J;Zhang, J;Analysis of error profiles in deep next-generation sequencing dataGenome Biol.5020130867008Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions. By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10-5 to 10-4, which is 10- to 100-fold lower than generally considered achievable (10-3) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10-5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10-4 for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing.119,0Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA. Xiaotu.Ma@stjude.org.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Key Laboratory of Pediatric Hematology and Oncology Ministry of Health, Department of Hematology and Oncology, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China.++Key Laboratory of Pediatric Hematology and Oncology Ministry of Health, Department of Hematology and Oncology, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Epidemiology and Cancer Control, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL, 35806, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.++Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA. Jinghui.Zhang@stjude.org.http://dx.doi.org/10.1186/s13059-019-1659-6HudsonAlpha
2014Van Bortle, K;Nichols, MH;Li, L;Ong, CT;Takenaka, N;Qin, ZS;Corces, VG;Insulator function and topological domain border strength scale with architectural protein occupancyGenome Biol.R8215624981874Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains.119,0We are especially thankful to Drs Dale Dorsett, Hugo Bellen, Giovanni Bosco, and Jørgen Johansen for graciously sharing antibodies against Rad21, Barren, CAP-H2, and Chromator. We thank the Drosophila Genomic Resource Center (supported by NIH grant OD010949-10) for reagents, and The Genomic Services Lab at the HudsonAlpha Institute for Biotechnology for their help in performing Illumina sequencing of ChIP-Seq samples. This work was supported by US Public Health Service Award R01GM035463 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.http://dx.doi.org/10.1186/gb-2014-15-5-r82HudsonAlpha
2013Day, K;Waite, LL;Thalacker-Mercer, A;West, A;Bamman, MM;Brooks, JD;Myers, RM;Absher, D;Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscapeGenome Biol.R10214924034465DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects. We found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific age CGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle age CGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains. Our data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.119,0;... samples All tissue samples were collected according to protocols and guidelines at their respective institutions. Iron deficient anemia buffy coat samples were commercially available and collected by Conversant Bio (Huntsville, AL, USA). Blood DNAs were isolated using the Agencourt Genfind v2 kit (Beckman Coulter, Indianapolis, IN, USA) at HudsonAlpha Institute for Biotechnology (HAIB). Frozen poshttps://genomebiology.biomedcentral.com/articles/10.1186/gb-2013-14-9-r102HudsonAlpha
2015Jagannathan, S;Abdel-Malek, MA;Malek, E;Vad, N;Latif, T;Anderson, KC;Driscoll, JJ;Pharmacologic screens reveal metformin that suppresses GRP78-dependent autophagy to enhance the anti-myeloma effect of bortezomibLeukemia2184-2191291126108695Although the therapeutic benefit of proteasome inhibition in multiple myeloma remains unchallenged, drug resistance inevitably emerges through mechanisms that remain elusive. Bortezomib provokes unwanted protein accumulation and the endoplasmic reticulum stress to activate the unfolded protein response (UPR) and autophagy as compensatory mechanisms that restore protein homeostasis. High-throughput screens to detect pharmacologics that modulated autophagy to enhance the anti-myeloma effect of bortezomib revealed metformin, a widely used antidiabetic agent with proven efficacy and limited adverse effects. Metformin co-treatment with bortezomib suppressed induction of the critical UPR effector glucose-regulated protein 78 (GRP78) to impair autophagosome formation and enhance apoptosis. Gene expression profiling of newly diagnosed myeloma patient tumors further correlated the hyperexpression of GRP78-encoding HSPA5 with reduced clinical response to bortezomib. The effect of bortezomib was enhanced with metformin co-treatment using myeloma patient tumor cells and the chemoresistant, stem cell-like side population that may contribute to disease recurrence. The relevance of the findings was confirmed in vivo as shown by metformin co-treatment with bortezomib that delayed the growth of myeloma xenotransplants. Taken together, our results suggest that metformin suppresses GRP78, a key driver of bortezomib-induced autophagy, and support the pharmacologic repositioning of metformin to enhance the anti-myeloma benefit of bortezomib.117,0Eagle's medium. Bone marrow aspirates were obtained after approval institutional review board or from ConversantBio (Huntsville, AL, USA) and CD138 + cells then isolated (Miltenyi Biotec, San Diego, CA, USA). Side populationhttps://www.nature.com/articles/leu2015157"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2015Jagannathan, S;Vad, N;Vallabhapurapu, S;Vallabhapurapu, S;Anderson, KC;Driscoll, JJ;MiR-29b replacement inhibits proteasomes and disrupts aggresome+autophagosome formation to enhance the antimyeloma benefit of bortezomibLeukemia727-73829325234165Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Although proteasome inhibitors (PIs) have transformed management of multiple myeloma (MM), drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism. Genome-wide profiling identified microRNAs (miRs) differentially expressed in bortezomib-resistant myeloma cells compared with drug-naive cells. The effect of individual miRs on proteasomal degradation of short-lived fluorescent reporter proteins was then determined in live cells. MiR-29b was significantly reduced in bortezomib-resistant cells as well as in cells resistant to second-generation PIs carfilzomib and ixazomib. Luciferase reporter assays demonstrated that miR-29b targeted PSME4 that encodes the proteasome activator PA200. Synthetically engineered miR-29b replacements impaired the growth of myeloma cells, patient tumor cells and xenotransplants. MiR-29b replacements also decreased PA200 association with proteasomes, reduced the proteasome's peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through ubiquitin-independent mechanisms. Immunofluorescence studies revealed that miR-29b replacements enhanced the bortezomib-induced accumulation of ubiquitinated proteins but did not reveal aggresome or autophagosome formation. Taken together, our study identifies miR-29b replacements as the first-in-class miR-based PIs that also disrupt the autophagy pathway and highlight their potential to synergistically enhance the antimyeloma effect of bortezomib.117,0(c) Effect of control or miR-29b on myeloma patient tumor cells. CD138 + PCs from bone marrow (BM; ConversantBio, Huntsville, AL, USA) were isolated using microbead-positive selection (Miltenyi Biotec, Auburn, CA, USA)https://www.nature.com/articles/leu2014279"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Cubeñas-Potts, C;Rowley, MJ;Lyu, X;Li, G;Lei, EP;Corces, VG;Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architectureNucleic Acids Res.1714-173045427899590Eukaryotic gene expression is regulated by enhancer-promoter interactions but the molecular mechanisms that govern specificity have remained elusive. Genome-wide studies utilizing STARR-seq identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). We hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. By evaluating ChIP-seq occupancy of architectural proteins, typical enhancer-associated proteins, and histone modifications, we determine that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, we map the interactions of each enhancer class utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.102,0New ChIP experiments were performed in Kc167 cells for GAF, H3, H3K27me3, Ibf1, Ibf2, Pita, Nup98 and ZIPIC as described previously with minor modifications (28). More precisely, antibody-bound protein complexes were isolated with Protein A or Protein G Dynabeads (ThermoFisher Scientific) and library generation was completed utilizing the KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) and size selected with Agencourt AMPure XP beads (Beckman Coulter Inc). ChIP-seq libraries were sequenced on an Illumina HiSeq 2500 at the HudsonAlpha Genomic Services Laboratory.https://academic.oup.com/nar/article-abstract/45/4/1714/2605738"HudsonAlpha Genomic Services"
2013Kellner, WA;Van Bortle, K;Li, L;Ramos, E;Takenaka, N;Corces, VG;Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sitesNucleic Acids Res.9274-9283412023945939Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.102,0Illumina adaptors (Illumina Cat# PE-102–1001) were titrated according to prepared DNA ChIP sample concentration and ligated with T4 ligase (NEB Cat# M0202S). Ligated ChIP samples were PCR-amplified using Illumina primers and Phusion DNA polymerase (NEB Cat# F-530L) and size selected for 200–300 bp by gel extraction. ChIP libraries were sequenced at the HudsonAlpha Institute for Biotechnology, using an Illumina HiSeq 2000 sequencer.http://dx.doi.org/10.1093/nar/gkt722HudsonAlpha
2003Winkler, DG;Sutherland, MK;Geoghegan, JC;Yu, C;Hayes, T;Skonier, JE;Shpektor, D;Jonas, M;Kovacevich, BR;Staehling-Hampton, K;Appleby, M;Brunkow, ME;Latham, JA;Osteocyte control of bone formation via sclerostin, a novel BMP antagonistEMBO J.6267-6276222314633986There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss.98,0Mouse embryos (15.5 days old) were fixed in 4% phosphate-buffered formaldehyde pH 7.2 overnight, dehydrated and embedded in paraffin. Sections (5 µm thick) were prepared and in situ hybridization performed by Phylogeny, Inc. (Columbus, OH) using [35S]UTP-(1000 Ci/mmol, Amersham Bioscience) labeled sense and antisense SOST RNA probes (Lyons et al., 1990).https://www.embopress.org/doi/full/10.1093/emboj/cdg599"Phylogeny Inc"
2017Edwardson, CF;Hollibaugh, JT;Metatranscriptomic analysis of prokaryotic communities active in sulfur and arsenic cycling in Mono Lake, California, USAISME J2195-2208111028548659This study evaluates the transcriptionally active, dissimilatory sulfur- and arsenic-cycling components of the microbial community in alkaline, hypersaline Mono Lake, CA, USA. We sampled five depths spanning the redox gradient (10, 15, 18, 25 and 31 m) during maximum thermal stratification. We used custom databases to identify transcripts of genes encoding complex iron-sulfur molybdoenzyme (CISM) proteins, with a focus on arsenic (arrA, aioA and arxA) and sulfur cycling (dsrA, aprA and soxB), and assigned them to taxonomic bins. We also report on the distribution of transcripts related to the ars arsenic detoxification pathway. Transcripts from detoxification pathways were not abundant in oxic surface waters (10 m). Arsenic cycling in the suboxic and microaerophilic zones of the water column (15 and 18 m) was dominated by arsenite-oxidizing members of the Gammaproteobacteria most closely affiliated with Thioalkalivibrio and Halomonas, transcribing arxA. We observed a transition to arsenate-reducing bacteria belonging to the Deltaproteobacteria and Firmicutes transcribing arsenate reductase (arrA) in anoxic bottom waters of the lake (25 and 31 m). Sulfur cycling at 15 and 18 m was dominated by Gammaproteobacteria (Thioalkalivibrio and Thioalkalimicrobium) oxidizing reduced S species, with a transition to sulfate-reducing Deltaproteobacteria at 25 and 31 m. Genes related to arsenic and sulfur oxidation from Thioalkalivibrio were more highly transcribed at 15 m relative to other depths. Our data highlight the importance of Thioalkalivibrio to arsenic and sulfur biogeochemistry in Mono Lake and identify new taxa that appear capable of transforming arsenic.97,0synthesis kits. Libraries (~225 bp insert) were prepared using Illumina TruSeq technology. Samples were pooled and run on one lane of 150 × 2 Illumina HiSeq 2500 Rapid Run at HudsonAlpha Genomic Services. Reads havehttps://www.nature.com/articles/ismej201780"HudsonAlpha Genomic Services"
2014Steffen, MM;Dearth, SP;Dill, BD;Li, Z;Larsen, KM;Campagna, SR;Wilhelm, SW;Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in MicrocystisISME J2080-209281024858783The cyanobacterium Microcystis aeruginosa is a globally distributed bloom-forming organism that degrades freshwater systems around the world. Factors that drive its dispersion, diversification and success remain, however, poorly understood. To develop insight into cellular-level responses to nutrient drivers of eutrophication, RNA sequencing was coupled to a comprehensive metabolomics survey of M. aeruginosa sp. NIES 843 grown in various nutrient-reduced conditions. Transcriptomes were generated for cultures grown in nutrient-replete (with nitrate as the nitrogen (N) source), nitrogen-reduced (with nitrate, urea or ammonium acting as the N sources) and phosphate-reduced conditions. Extensive expression differences (up to 696 genes for urea-grown cells) relative to the control treatment were observed, demonstrating that the chemical variant of nitrogen available to cells affected transcriptional activity. Of particular note, a high number of transposase genes (up to 81) were significantly and reproducibly up-regulated relative to the control when grown on urea. Conversely, phosphorus (P) reduction resulted in a significant cessation in transcription of transposase genes, indicating that variation in nutrient chemistry may influence transcription of transposases and may impact the highly mosaic genomic architecture of M. aeruginosa. Corresponding metabolomes showed comparably few differences between treatments, suggesting broad changes to gene transcription are required to maintain metabolic homeostasis under nutrient reduction. The combined observations provide novel and extensive insight into the complex cellular interactions that take place in this important bloom-forming organism during variable nutrient conditions and highlight a potential unknown molecular mechanism that may drive Microcystis blooms and evolution.97,0; Scientific, Waltham, MA, USA). Subsequent sample processing, including rRNA subtraction, library prep and sequencing, were performed by the HudsonAlpha Genomic Services Laboratory (Huntsville, AL, USA). RNA quality washttps://www.nature.com/articles/ismej201478HudsonAlpha
2014Picard, M;Zhang, J;Hancock, S;Derbeneva, O;Golhar, R;Golik, P;O'Hearn, S;Levy, S;Potluri, P;Lvova, M;Davila, A;Lin, CS;Perin, JC;Rappaport, EF;Hakonarson, H;Trounce, IA;Procaccio, V;Wallace, DC;Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogrammingProc. Natl. Acad. Sci. U.S.A.E4033-E40421113825192935Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.97,0Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;++School of Biological Sciences, The University of Hong Kong, Hong Kong, People's Republic of China;++Trovagene, San Diego, CA 92130;++Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;++Center for Applied Genomics, Division of Genetics, Department of Pediatrics, and.++Institute of Genetics and Biotechnology, Warsaw University, 00-927, Warsaw, Poland;++Morton Mower Central Research Laboratory, Sinai Hospital of Baltimore, Baltimore, MD 21215;++Genomics Sevices Laboratory, HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806;++Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;++Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;++Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;++Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;++Nucleic Acid/Protein Research Core Facility, Children's Hospital of Philadelphia, Philadelphia, PA 19104;++Nucleic Acid/Protein Research Core Facility, Children's Hospital of Philadelphia, Philadelphia, PA 19104;++Trovagene, San Diego, CA 92130;++Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia; and.++Department of Biochemistry and Genetics, National Center for Neurodegenerative and Mitochondrial Diseases, Centre Hospitalier Universitaire d'Angers, 49933 Angers, France.++Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia and the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; wallaced1@email.chop.edu.http://dx.doi.org/10.1073/pnas.1414028111HudsonAlpha
2012Higgins, G;Roper, KM;Watson, IJ;Blackhall, FH;Rom, WN;Pass, HI;Ainscough, JF;Coverley, D;Variant Ciz1 is a circulating biomarker for early-stage lung cancerProc. Natl. Acad. Sci. U.S.A.E3128-E31351094523074256There is an unmet need for circulating biomarkers that can detect early-stage lung cancer. Here we show that a variant form of the nuclear matrix-associated DNA replication factor Ciz1 is present in 34/35 lung tumors but not in adjacent tissue, giving rise to stable protein quantifiable by Western blot in less than a microliter of plasma from lung cancer patients. In two independent sets, with 170 and 160 samples, respectively, variant Ciz1 correctly identified patients who had stage 1 lung cancer with clinically useful accuracy. For set 1, mean variant Ciz1 level in individuals without diagnosed tumors established a threshold that correctly classified 98% of small cell lung cancers (SCLC) and non-SCLC patients [receiver operator characteristic area under the curve (AUC) 0.958]. Within set 2, comparison of patients with stage 1 non-SCLC with asymptomatic age-matched smokers or individuals with benign lung nodules correctly classified 95% of patients (AUCs 0.913 and 0.905), with overall specificity of 76% and 71%, respectively. Moreover, using the mean of controls in set 1, we achieved 95% sensitivity among patients with stage 1 non-SCLC patients in set 2 with 74% specificity, demonstrating the robustness of the classification. RNAi-mediated selective depletion of variant Ciz1 is sufficient to restrain the growth of tumor cells that express it, identifying variant Ciz1 as a functionally relevant driver of cell proliferation in vitro and in vivo. The data show that variant Ciz1 is a strong candidate for a cancer-specific single marker capable of identifying early-stage lung cancer within at-risk groups without resort to invasive procedures.97,0... anti-rabbit Alexa Fluor 488 (Invitrogen, Folio Bioscience). Subcellular fractionation was carried out as described (21). For immunohistochemistry, 2B was applied to SCLC tissue microarray ARY-HH0117 (Folio Biosciences) and visualized using Ultraview Universal DAB (Ventana) with hematoxylin counterstain. Duplicate cores representing 35 SCLC (grade 4) and tissue from five normal or cancer-adjacent l ... nal antibody 2B (Fig. S2 [http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1210107109/-/DCSupplemental/pnas.201210107SI.pdf?targetid=nameddest=SF2]) and then anti-rabbit Alexa Fluor 488 (Invitrogen, Folio Bioscience). Subcellular fractionation was carried out as described (21). For immunohistochemistry, 2B was applied to SCLC tissue microarray ARY-HH0117 (Folio Biosciences) and visualized using U ... nal antibody 2B (Fig. S2 [http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1210107109/-/DCSupplemental/pnas.201210107SI.pdf?targetid=nameddest=SF2]) and then anti-rabbit Alexa Fluor 488 (Invitrogen, Folio Bioscience). Subcellular fractionation was carried out as described (21). For immunohistochemistry, 2B was applied to SCLC tissue microarray ARY-HH0117 (Folio Biosciences) and visualized using U;. Submit; About: Editorial Board; PNAS Staff; FAQ; Rights and Permissions; Site Map. Contact; Journal Club; Subscribe: Subscription Rates; Subscriptions FAQ; Open Access; Recommend PNAS to Your Librarian. Log in; Log out; My Cart. Main menuhttps://www.pnas.org/content/109/45/E3128.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio";"Phylogeny Inc"
2012Fabbri, M;Paone, A;Calore, F;Galli, R;Gaudio, E;Santhanam, R;Lovat, F;Fadda, P;Mao, C;Nuovo, GJ;Zanesi, N;Crawford, M;Ozer, GH;Wernicke, D;Alder, H;Caligiuri, MA;Nana-Sinkam, P;Perrotti, D;Croce, CM;MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory responseProc. Natl. Acad. Sci. U.S.A.E2110-E21161093122753494MicroRNAs (miRNAs) are small noncoding RNAs, 19-24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor-immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.97,0We thank Phylogeny, Inc. for performing in situ hybridization in its entirety and locked nucleic acid in situ hybridization experiments and for supporting these experiment; Dr. Cecilia FernandezCymering and Dr. Stefano Volinia for supervision of statistical analysis; and Dr. Kay Huebner for critical reading of the manuscript. This work was supported by National Institutes of Health Grants R21 5R21CA150297 (to P.N.-S.) and R01 CA135030, R01 CA124541, and RC2 CA148302 (to C.M.C.). M.F. is supported by a 2009 Kimmel Foundation Fellowship.https://www.pnas.org/content/109/31/E2110.short"Phylogeny Inc"
2018Roberts, BS;Hardigan, AA;Moore, DE;Ramaker, RC;Jones, AL;Fitz-Gerald, MB;Cooper, GM;Wilcox, CM;Kimberly, RP;Myers, RM;Discovery and Validation of Circulating Biomarkers of Colorectal Adenoma by High-Depth Small RNA SequencingClin. Cancer Res.2092-209924929490987Purpose: Colorectal cancer is the third most common cancer worldwide, causing approximately 700,000 deaths each year. The majority of colorectal cancers begin as adenomas. Definitive screening for colorectal adenomas is currently accomplished through colonoscopy but, owing largely to costs and invasiveness, is typically limited to patient groups at higher risk by virtue of age or family history. We sought to determine if blood-based small RNA markers could detect colorectal adenoma.Experimental Design: We applied high-depth small RNA sequencing to plasma from a large (n = 189) cohort of patients, balanced for age, sex, and ancestry. Our analytical methodology allowed for the detection of both microRNAs and other small RNA species. We replicated sequencing results by qPCR on plasma samples from an independent cohort (n = 140).Results: We found several small RNA species with significant associations to colorectal adenoma, including both microRNAs and non-microRNA small RNAs. These associations were robust to correction for patient covariates, including age. Among the adenoma-associated small RNAs, two, a miR-335-5p isoform and an un-annotated small RNA, were validated by qPCR in an independent cohort. A classifier trained on measures of these two RNAs in the discovery cohort yields an AUC of 0.755 (0.775 with age) for adenoma detection in the independent cohort. This classifier accurately detects adenomas in patients under 50 and is robust to sex or ancestry.Conclusions: Circulating small RNAs (including but not limited to miRNAs) discovered by sequencing and validated by qPCR identify patients with colorectal adenomas effectively. Clin Cancer Res; 24(9); 2092-9. ©2018 AACR. ©2018 American Association for Cancer Research.96,0HudsonAlpha Institute for Biotechnology, Huntsville, Alabama.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama.++Center for Clinical and Translational Science, University of Alabama at Birmingham, Birmingham, Alabama.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama.++Department of Medicine, Division of Gastroenterology and Hepatology, University of Alabama at Birmingham, Birmingham, Alabama.++Center for Clinical and Translational Science, University of Alabama at Birmingham, Birmingham, Alabama.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama. rmyers@hudsonalpha.org.http://dx.doi.org/10.1158/1078-0432.CCR-17-1960HudsonAlpha
2018Tucker, DW;Getchell, CR;McCarthy, ET;Ohman, AW;Sasamoto, N;Xu, S;Ko, JY;Gupta, M;Shafrir, A;Medina, JE;Lee, JJ;MacDonald, LA;Malik, A;Hasselblatt, KT;Li, W;Zhang, H;Kaplan, SJ;Murphy, GF;Hirsch, MS;Liu, JF;Matulonis, UA;Terry, KL;Lian, CG;Dinulescu, DM;Epigenetic Reprogramming Strategies to Reverse Global Loss of 5-Hydroxymethylcytosine, a Prognostic Factor for Poor Survival in High-grade Serous Ovarian CancerClin. Cancer Res.1389-140124629263182Purpose: A major challenge in platinum-based cancer therapy is the clinical management of chemoresistant tumors, which have a largely unknown pathogenesis at the level of epigenetic regulation.Experimental Design: We evaluated the potential of using global loss of 5-hydroxymethylcytosine (5-hmC) levels as a novel diagnostic and prognostic epigenetic marker to better assess platinum-based chemotherapy response and clinical outcome in high-grade serous tumors (HGSOC), the most common and deadliest subtype of ovarian cancer. Furthermore, we identified a targetable pathway to reverse these epigenetic changes, both genetically and pharmacologically.Results: This study shows that decreased 5-hmC levels are an epigenetic hallmark for malignancy and tumor progression in HGSOC. In addition, global 5-hmC loss is associated with a decreased response to platinum-based chemotherapy, shorter time to relapse, and poor overall survival in patients newly diagnosed with HGSOC. Interestingly, the rescue of 5-hmC loss restores sensitivity to platinum chemotherapy in vitro and in vivo, decreases the percentage of tumor cells with cancer stem cell markers, and increases overall survival in an aggressive animal model of platinum-resistant disease.Conclusions: Consequently, a global analysis of patient 5-hmC levels should be included in future clinical trials, which use pretreatment with epigenetic adjuvants to elevate 5-hmC levels and improve the efficacy of current chemotherapies. Identifying prognostic epigenetic markers and altering chemotherapeutic regimens to incorporate DNMTi pretreatment in tumors with low 5-hmC levels could have important clinical implications for newly diagnosed HGSOC disease. Clin Cancer Res; 24(6); 1389-401. ©2017 AACR. ©2017 American Association for Cancer Research.96,0... s of 5-hmC levels in a large biorepository of HGSOC cases by using a patient tumor microarray (TMA) from the Dana-Farber Cancer Institute (DFCI, Boston, MA; ref. 22) and a commercially available TMA (Folio Biosciences). We observed strong 5-hmC staining in normal stroma (Fig. 1A [/pmc/articles/PMC5951622/figure/F1/]), which was used as an internal staining control. Samples that were deemed to not ... s of 5-hmC levels in a large biorepository of HGSOC cases by using a patient tumor microarray (TMA) from the Dana-Farber Cancer Institute (DFCI, Boston, MA; ref. 22) and a commercially available TMA (Folio Biosciences). We observed strong 5-hmC staining in normal stroma (Fig. 1A [/pmc/articles/PMC5951622/figure/F1/]), which was used as an internal staining control. Samples that were deemed to not ... s of 5-hmC levels in a large biorepository of HGSOC cases by using a patient tumor microarray (TMA) from the Dana-Farber Cancer Institute (DFCI, Boston, MA; ref. 22) and a commercially available TMA (Folio Biosciences). We observed strong 5-hmC staining in normal stroma (Fig. 1A [/pmc/articles/PMC5951622/figure/F1/]), which was used as an internal staining control. Samples that were deemed to nothttp://clincancerres.aacrjournals.org/content/24/6/1389.abstract"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2017Yeh, TC;O'Connor, G;Petteruti, P;Dulak, A;Hattersley, M;Barrett, JC;Chen, H;Identification of CCR2 and CD180 as Robust Pharmacodynamic Tumor and Blood Biomarkers for Clinical Use with BRD4/BET InhibitorsClin. Cancer Res.1025-103523428073847Purpose: AZD5153 is a novel BRD4/BET inhibitor with a distinctive bivalent bromodomain binding mode. To support its clinical development, we identified pharmacodynamic (PD) biomarkers for use in clinical trials to establish target engagement.Experimental Design: CCR2 and CD180 mRNAs, initially identified from whole transcriptome profiling, were further evaluated by quantitative PCR in hematologic cell lines, xenografts, and whole blood from rat, healthy volunteers, and patients with cancer. MYC and HEXIM1 mRNAs were also evaluated.Results: RNA-sequencing data showed consistent decreases in CCR2/CD180 expression across multiple hematologic cell lines upon AZD5153 treatment. Evaluation of dose dependence in MV4,11 cells confirmed activity at clinically relevant concentrations. In vivo downregulation of CCR2/CD180 mRNAs (>80%) was demonstrated in MV4,11 and KMS-11 xenograft tumors at efficacious AZD5153 doses. Consistent with in vitro rat blood data, an in vivo rat study confirmed greater inhibition of CCR2/CD180 mRNA in whole blood versus MYC at an efficacious dose. Finally, in vitro treatment of whole blood from healthy volunteers and patients with cancer demonstrated, in contrast to MYC, almost complete downregulation of CCR2/CD180 at predicted clinically achievable concentrations.Conclusions: Our data strongly support the use of CCR2 and CD180 mRNAs as whole blood PD biomarkers for BRD4 inhibitors, especially in situations where paired tumor biopsies are unavailable. In addition, they can be used as tumor-based PD biomarkers for hematologic tumors. MYC mRNA is useful as a hematologic tumor-based biomarker but suboptimal as a whole blood biomarker. Utility of HEXIM1 mRNA may be limited to higher concentrations. Clin Cancer Res; 23(4); 1025-35. ©2017 AACR. ©2017 American Association for Cancer Research.96,0Whole blood was collected in 10 mL EDTA vaccutainers (Becton Dickinson) from two control healthy volunteers and 11 patients with cancer with solid tumor or hematologic cancers through Conversant Bio. Compound incubation and sample processing was carried out at Conversant Bio within 2 hours of blood collection. Briefly, 3 mL aliquots of blood were incubated with DMSO or serial dilutions of AZD5153 for 2 hours at 37 C. Afterward, 2.5 mL from each condition was transferred to PAXgene RNA tubes (PreAnalytiX),http://clincancerres.aacrjournals.org/content/23/4/1025.abstract"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2015Blume-Jensen, P;Berman, DM;Rimm, DL;Shipitsin, M;Putzi, M;Nifong, TP;Small, C;Choudhury, S;Capela, T;Coupal, L;Ernst, C;Hurley, A;Kaprelyants, A;Chang, H;Giladi, E;Nardone, J;Dunyak, J;Loda, M;Klein, EA;Magi-Galluzzi, C;Latour, M;Epstein, JI;Kantoff, P;Saad, F;Development and clinical validation of an in situ biopsy-based multimarker assay for risk stratification in prostate cancerClin. Cancer Res.2591-2600211125733599Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish "favorable" versus "nonfavorable" pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on "false-negative" and "false-positive" rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non-GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy. ©2015 American Association for Cancer Research.96,0To develop a robust assay, multiple institutions were recruited representing typical North American patient cohorts (Table 1): Urology Austin, Chesapeake Urology Associates, Cleveland Clinic, Michigan Urology, and Folio Biosciences. Biopsy sample inclusion/exclusion criteria matched those that would be in place during routine clinical use of the assay. Because they are usually not candidates for active surveillance, patients with biopsy Gleason 4þ3 were excluded, except for a limited number of biopsies that had been discordantly graded as both 3þ4 and 4þ3 by two expert pathologists. Annotations, including information on matched biopsy and prostatectomy pathology reports, were obtainedhttp://clincancerres.aacrjournals.org/content/21/11/2591.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2015Garcia-Manero, G;Khoury, HJ;Jabbour, E;Lancet, J;Winski, SL;Cable, L;Rush, S;Maloney, L;Hogeland, G;Ptaszynski, M;Calvo, MC;Bohannan, Z;List, A;Kantarjian, H;Komrokji, R;A phase I study of oral ARRY-614, a p38 MAPK/Tie2 dual inhibitor, in patients with low or intermediate-1 risk myelodysplastic syndromesClin. Cancer Res.985-99421525480830Data suggest that activity of p38 MAPK and Tie2 kinases is dysregulated in myelodysplastic syndromes (MDS) and may be targets for novel therapies. A phase I study of ARRY-614, an oral dual inhibitor of p38 MAPK and Tie2, was conducted in patients with low or intermediate-1 International Prognostic Scoring System risk MDS to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary responses by International Working Group 2006 criteria. Forty-five patients received ARRY-614 either once daily or twice daily in dose escalation (400, 600, 900, or 1,200 mg once daily; 200 or 300 mg twice daily) or expansion cohorts. The 300 mg twice daily schedule was not tolerated, and an MTD was not reached for once daily dosing. Treatment-related adverse events were primarily grade 1-2, with the most common being rash, diarrhea, dry skin, fatigue and anorexia. Interpatient PK variability was high, although exposure was sufficient to achieve reduction in p38 MAPK activation in bone marrow and in the levels of circulating biomarkers. Disease responses were observed in 14 of 44 (32%) evaluable patients, 13 (93%) of whom had previously been treated with a hypomethylating agent. Responses were observed in all lineages, with 5 patients experiencing bilineage responses. Three of 25 red blood cell transfusion-dependent (TD) patients achieved transfusion independence (TI) and 5 of 7 platelet TD patients achieved TI. ARRY-614 was well tolerated and has sufficient activity to warrant further evaluation in this patient population. We recommend 1,200 mg once daily as the optimal dose for further study. ©2014 American Association for Cancer Research.96,0... s (Aurora, CO) proprietary CellMap customized algorithm for p-p38 and CC3, respectively. Age-defined FFPE bone marrow biopsies from apparently healthy subjects (60–74 years old) were obtained from Folio Biosciences (Columbus, OH). Plasma was isolated from blood samples collected at predetermined time points for evidence of p38 MAPK inhibition through changes in circulating cytokine and/or grow ... s (Aurora, CO) proprietary CellMap customized algorithm for p-p38 and CC3, respectively. Age-defined FFPE bone marrow biopsies from apparently healthy subjects (60–74 years old) were obtained from Folio Biosciences (Columbus, OH). Plasma was isolated from blood samples collected at predetermined time points for evidence of p38 MAPK inhibition through changes in circulating cytokine and/or grow ... s (Aurora, CO) proprietary CellMap customized algorithm for p-p38 and CC3, respectively. Age-defined FFPE bone marrow biopsies from apparently healthy subjects (60–74 years old) were obtained from Folio Biosciences (Columbus, OH). Plasma was isolated from blood samples collected at predetermined time points for evidence of p38 MAPK inhibition through changes in circulating cytokine and/or growhttp://clincancerres.aacrjournals.org/content/21/5/985.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2014Smith, DC;Eisenberg, PD;Manikhas, G;Chugh, R;Gubens, MA;Stagg, RJ;Kapoun, AM;Xu, L;Dupont, J;Sikic, B;A phase I dose escalation and expansion study of the anticancer stem cell agent demcizumab (anti-DLL4) in patients with previously treated solid tumorsClin. Cancer Res.6295-6303202425324140This phase I trial evaluated the safety, pharmacokinetics, and pharmacodynamics of demcizumab (OMP-21M18), a humanized IgG2 mAb targeting the Notch ligand DLL4 in adult patients with advanced malignancies. Standard 3+3 design, with demcizumab 0.5, 1, 2.5, or 5 mg/kg weekly or 2.5, 5, or 10 mg/kg every other week, with an expansion cohort at 10 mg/kg every other week. Dose-limiting toxicities (DLT) were assessed during the first 28 days. Fifty-five patients received demcizumab (15 weekly, 18 every other week, 21 expansion cohort, 1 loading dose). No more than one DLT was seen at any dose level. The MTD was not reached for either schedule. Treatment-related adverse events occurring in >10% of patients were hypertension or blood pressure increased (47%), fatigue (31%), anemia (22%), headache (20%), nausea (13%), hypoalbuminemia (11%), dizziness (11%), and dyspnea (11%). One patient dosed at 2.5 mg/kg developed reversible right-sided heart failure after 63 days on treatment and 4 dosed at 10 mg/kg developed congestive heart failure after ≥98 days on treatment. Five patients were hospitalized with bleeding episodes (2 episodes of tumor-associated bleeding). Sixteen of 25 (64%) evaluable patients at 10 mg/kg had evidence of stabilization of disease or response. Demcizumab was generally well tolerated at doses ≤5 mg weekly with disease stabilization and decreases in tumor size demonstrating antitumor activity. Hypertension was the most common adverse event that was clearly related to treatment. Prolonged administration was associated with an increased risk of congestive heart failure. ©2014 American Association for Cancer Research.96,0In addition, control plasma and whole blood samples were sourced and collected (Conversant Bio) using the same methods described above from 8 cancer patients who were not treated with demcizumab. Affymetrix human gene chip U133 Plus 2.0 arrays were used for profiling the gene expression levels in the whole blood and in the hair follicle samples. (Almac CLIA certified laboratory) To obtain the expression levels of each probe set, the raw CEL files in each dataset were processed for background adjustment and signal intensity normalization with GC Robust Multi-array Average (GCRMA) algorithm in the open-source bioconductor software (www.bioconductor.org)http://clincancerres.aacrjournals.org/content/20/24/6295.short"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2013Xing, F;Kobayashi, A;Okuda, H;Watabe, M;Pai, SK;Pandey, PR;Hirota, S;Wilber, A;Mo, YY;Moore, BE;Liu, W;Fukuda, K;Iiizumi, M;Sharma, S;Liu, Y;Wu, K;Peralta, E;Watabe, K;Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brainEMBO Mol Med384-3965323495140Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL-1β which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.92,0... ocyte (UC1) was a kind gift from Dr Russell Piper (University of California-San Francisco). Primary human breast tumour cells which maintained in xenograft tumour of NOD/SCID mouse were obtained from Conversant Biologics, Inc. shRNA-expressing lentiviral plasmids for IL-1β and HES5 were obtained from OpenBiosystems. Recombinant IL-1β, 1-Pyrrolidinecarbodithioic acid ammonium salt (PDTC) and -[N- ... ocyte (UC1) was a kind gift from Dr Russell Piper (University of California-San Francisco). Primary human breast tumour cells which maintained in xenograft tumour of NOD/SCID mouse were obtained from Conversant Biologics, Inc. shRNA-expressing lentiviral plasmids for IL-1β and HES5 were obtained from OpenBiosystems. Recombinant IL-1β, 1-Pyrrolidinecarbodithioic acid ammonium salt (PDTC) and -[N-https://www.embopress.org/doi/abs/10.1002/emmm.201201623"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Kirtane, AR;Sadhukha, T;Kim, H;Khanna, V;Koniar, B;Panyam, J;Fibrinolytic Enzyme Cotherapy Improves Tumor Perfusion and Therapeutic Efficacy of Anticancer NanomedicineCancer Res.1465-147577628108516Elevated interstitial fluid pressure and solid stress within tumors contribute to poor intratumoral distribution of nanomedicine. In this study, we hypothesized that the presence of fibrin in tumor extracellular matrix contributes to hindered intratumoral distribution of nanocarriers and that this can be overcome through the use of a fibrinolytic enzyme such as tissue plasminogen activator (tPA). Analysis of fibrin expression in human tumor biopsies showed significant fibrin staining in nearly all tumor types evaluated. However, staining was heterogeneous across and within tumor types. We determined the effect of fibrin on the diffusion, intratumoral distribution, and therapeutic efficacy of nanocarriers. Diffusivity of nanocarriers in fibrin matrices was limited and could be improved significantly by coincubation with tPA. In vivo, coadministration of tPA improved the anticancer efficacy of nanoparticle-encapsulated paclitaxel in subcutaneous syngeneic mouse melanoma and orthotopic xenograft lung cancer models. Furthermore, treatment with tPA led to decompression of blood vessels and improved tumor perfusion. Cotreatment with tPA resulted in greater intratumoral penetration of a model nanocarrier (Doxil), leading to enhanced availability of the drug in the tumor core. Fibrinolytics such as tPA are already approved for other indications. Fibrinolytic cotherapy is therefore a rapidly translatable strategy for improving therapeutic effectiveness of anticancer nanomedicine. Cancer Res; 77(6); 1465-75. ©2017 AACR. ©2017 American Association for Cancer Research.91,0... anticancer nanomedicine in syngeneic mouse melanoma and xenograft lung cancer models. Go to: MATERIALS AND METHODS MATERIALS Tissue microarrays of various human tumor biopsies were obtained from Folio Biosciences (Ohio, USA). Anti-human fibrin(ogen) antibody was purchased from Dako (California, USA). Poly(lactide-_co_-glycolide) (PLGA) was purchased from Lactel (Alabama, USA). L-lactide, bovi ... anticancer nanomedicine in syngeneic mouse melanoma and xenograft lung cancer models. Go to: MATERIALS AND METHODS MATERIALS Tissue microarrays of various human tumor biopsies were obtained from Folio Biosciences (Ohio, USA). Anti-human fibrin(ogen) antibody was purchased from Dako (California, USA). Poly(lactide-_co_-glycolide) (PLGA) was purchased from Lactel (Alabama, USA). L-lactide, bovi ... anticancer nanomedicine in syngeneic mouse melanoma and xenograft lung cancer models. Go to: MATERIALS AND METHODS MATERIALS Tissue microarrays of various human tumor biopsies were obtained from Folio Biosciences (Ohio, USA). Anti-human fibrin(ogen) antibody was purchased from Dako (California, USA). Poly(lactide-_co_-glycolide) (PLGA) was purchased from Lactel (Alabama, USA). L-lactide, bovihttp://cancerres.aacrjournals.org/content/77/6/1465.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2013Okuda, H;Xing, F;Pandey, PR;Sharma, S;Watabe, M;Pai, SK;Mo, YY;Iiizumi-Gairani, M;Hirota, S;Liu, Y;Wu, K;Pochampally, R;Watabe, K;miR-7 suppresses brain metastasis of breast cancer stem-like cells by modulating KLF4Cancer Res.1434-144473423384942Despite significant improvement in survival rates of patients with breast cancer, prognosis of metastatic disease is still dismal. Cancer stem-like cells (CSC) are considered to play a role in metastatic progression of breast cancer; however, the exact pathologic role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24(-)/CD44(+)/ESA(+)) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ-specific manner. The results of our microRNA (miRNA) profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this miRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain but not bone metastasis-free survival of patients with breast cancer, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or upregulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer.91,0mixture. Breast tumor cells that were directly transplanted to animal only one generation without in vitro culture were obtained from Conversant Biologics. Stem cell populations were isolated as described previously (14). Iso ;... odium pyruvate, 1% of non-essential amino acids and 1% of vitamin mixture. Breast tumor cells that were directly transplanted to animal only one generation without in vitro culture were obtained from conversant biologics, Stem cell populations were isolated as described previously (14). Isolated CSCs were cultured in DMEM/F12 medium with 2%B27, 20ng/ml EGF, 4μg/ml insulin and 0.4% of BSA. MICROR ... odium pyruvate, 1% of non-essential amino acids and 1% of vitamin mixture. Breast tumor cells that were directly transplanted to animal only one generation without in vitro culture were obtained from conversant biologics, Stem cell populations were isolated as described previously (14). Isolated CSCs were cultured in DMEM/F12 medium with 2%B27, 20ng/ml EGF, 4μg/ml insulin and 0.4% of BSA. MICRORhttp://dx.doi.org/10.1158/0008-5472.CAN-12-2037"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2012Ren, H;Tan, ZP;Zhu, X;Crosby, K;Haack, H;Ren, JM;Beausoleil, S;Moritz, A;Innocenti, G;Rush, J;Zhang, Y;Zhou, XM;Gu, TL;Yang, YF;Comb, MJ;Identification of anaplastic lymphoma kinase as a potential therapeutic target in ovarian cancerCancer Res.3312-3323721322570254Ovarian cancer is the leading cause of death from gynecologic cancer. Improvement in the clinical outcome of patients is likely to be achieved by the identification of molecular events that underlie the oncogenesis of ovarian cancer. Here we show that the anaplastic lymphoma kinase (ALK) is aberrantly activated in ovarian cancer. Using an unbiased and global phosphoproteomic approach, we profiled 69 Chinese primary ovarian tumor tissues and found ALK to be aberrantly expressed and phosphorylated in 4 tumors. Genetic characterization of these ALK-positive tumors indicated that full-length ALK expression in two serous carcinoma patients is consistent with ALK gene copy number gain, whereas a stromal sarcoma patient carries a novel transmembrane ALK fusion gene: FN1-ALK. Biochemical and functional analysis showed that both full-length ALK and FN1-ALK are oncogenic, and tumors expressing ALK or FN1-ALK are sensitive to ALK kinase inhibitors. Furthermore, immunohistochemical analysis of ovarian tumor tissue microarray detected aberrant ALK expression in 2% to 4% serous carcinoma patients. Our findings provide new insights into the pathogenesis of ovarian cancer and identify ALK as a potential therapeutic target in a subset of serous ovarian carcinoma and stromal sarcoma patients. ©2012 AACR.91,0Histologic typing and grading were carried out according to International Federation of Gynecology and Obstetrics (FIGO) guidelines. General pathologic information of the 69 ovarian tumor patients is listed in Supplementary Table S1. FFPE ovarian tumor tissue microarray (TMA) slides were purchased from Folio Biosciences and Biochain Institute, Inc.http://cancerres.aacrjournals.org/content/72/13/3312.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2008Jiang, P;Enomoto, A;Jijiwa, M;Kato, T;Hasegawa, T;Ishida, M;Sato, T;Asai, N;Murakumo, Y;Takahashi, M;An actin-binding protein Girdin regulates the motility of breast cancer cellsCancer Res.1310-131868518316593Girdin (girders of actin filaments) is a novel actin-binding Akt substrate that plays an important role in actin organization and Akt-dependent cell motility in fibroblasts. Here, we find that Girdin is expressed in a variety of cancer cell lines, including the breast cancer cell line MDA-MB-231, and is phosphorylated by the stimulation of insulin-like growth factor (IGF-I). In vitro migration and invasion assays showed that Girdin is required for the IGF-I-dependent cell movement of MDA-MB-231 cells. Short hairpin interfering RNA directed against Girdin markedly inhibited the metastasis of s.c. transplanted MDA-MB-231 cells in nude mice. In addition, Girdin is highly expressed in a variety of human malignant tissues, including breast, colon, lung, and uterine cervical carcinomas. These findings highlight the important role of Girdin in tumor progression in which the Akt signaling pathway is aberrantly activated.91,0Tissue arrays of human malignant tumors and matched normal adjacent tissues were purchased from Folio Biosciences, Inc. Immunohistochemisty was performed using standard techniques. Antigen retrieval was performed with microwave treatment in a 0.01 mol/L citrate buffer (pH 6.0) at 95jC for 10 min. For fluorescence staining of the breast carcinoma tissue array, the section was stained with anti-Girdin antibody, followed with Alexa 594–conjugated secondary antibody. Nuclei were stained with 4¶,6-diamidino-2-phenylindole (DAPI; Invitrogen) and observed with a confocal laser-scanning microscopehttp://cancerres.aacrjournals.org/content/68/5/1310.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2007Aziz, MH;Manoharan, HT;Church, DR;Dreckschmidt, NE;Zhong, W;Oberley, TD;Wilding, G;Verma, AK;Protein kinase Cepsilon interacts with signal transducers and activators of transcription 3 (Stat3), phosphorylates Stat3Ser727, and regulates its constitutive activation in prostate cancerCancer Res.8828-8838671817875724Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cepsilon (PKCepsilon), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCepsilon expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCepsilon mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCepsilon. The interaction of PKCepsilon with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCepsilon. Localization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCepsilon with Stat3 was PKCepsilon isoform specific. Inhibition of PKCepsilon protein expression in DU145 cells using specific PKCepsilon small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCepsilon activation is essential for constitutive activation of Stat3 and prostate cancer progression.91,0Prostate cancer specimens were obtained at radical prostatectomy. The University of Wisconsin Human Subjects Committee approved the use of these human prostate specimens. Tissue microarrays were obtained from Folio Biosciences.4 Tissue microarrays were produced by relocating tissue from conventional histologic paraffin blocks (Folio Biosciences). This was done using a needle to biopsy a standard histology section and placing the core into an array on a recipient paraffin block. Optimal sectioning of arrays was obtained with f5-Am sections. Using this technology, each tissue is treated in an identical manner to avoid slideto-slide variations in the conventional sectionshttp://cancerres.aacrjournals.org/content/67/18/8828.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2006Perk, J;Gil-Bazo, I;Chin, Y;de Candia, P;Chen, JJ;Zhao, Y;Chao, S;Cheong, W;Ke, Y;Al-Ahmadie, H;Gerald, WL;Brogi, E;Benezra, R;Reassessment of id1 protein expression in human mammary, prostate, and bladder cancers using a monospecific rabbit monoclonal anti-id1 antibodyCancer Res.10870-10877662217108123Id proteins are a class of dominant-negative antagonists of helix-loop-helix transcription factors and have been shown to control differentiation of a variety of cell types in diverse organisms. Although the importance of Id1 in tumor endothelial cells is well established, the expression and role of the Id1 protein in human cancer cells is controversial. To explore this issue, we developed and characterized a highly specific rabbit monoclonal antibody against Id1 to assess its expression in human breast, prostate, and bladder malignancies. Our results show that in usual types of human mammary carcinomas, the Id1 protein is expressed exclusively in the endothelium. Interestingly, we detected nuclear expression of the Id1 protein in the tumor cells in 10 of 45 cases of poorly differentiated and highly aggressive carcinoma with metaplastic morphology. Similarly, only 1 of 30 prostate cancer samples showed Id1-positive tumor cells, whereas in almost all, endothelial cells showed high Id1 expression. Intriguingly, whereas normal prostate glands do not show any Id1 protein expression, basal layer cells of benign prostate glands in proximity to tumors expressed high levels of the Id1 protein. In contrast to the lack of Id1 expression in the usual types of mammary and prostate cancers, the majority of transitional cell bladder tumors showed Id1 protein expression in both tumor and endothelial cells. These results suggest that further refinement of Id1 expression patterns in a variety of tumor types will be necessary to identify and study the functional roles played by Id1 in human neoplastic processes.91,0Two slides of commercially available (Folio Biosciences, Columbus, OH) TMAs were also analyzed. The two slides included a total of 143 cores, of which 62 samples were common types of breast cancer, and the rest of the samples were benign (slides #ARY-HH0058, ‘‘Breast Carcinoma and Normal TMA’’ and #ARY-HH0088, ‘‘Breast Carcinoma and Matching Normal TMA’’).http://cancerres.aacrjournals.org/content/66/22/10870.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2006Swales, KE;Korbonits, M;Carpenter, R;Walsh, DT;Warner, TD;Bishop-Bailey, D;The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expressionCancer Res.10120-10126662017047076Bile acids are present at high concentrations in breast cysts and in the plasma of postmenopausal women with breast cancer. The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. FXR was detected in normal and tumor breast tissue, with a high level of expression in ductal epithelial cells of normal breast and infiltrating ductal carcinoma cells. FXR was also present in the human breast carcinoma cells, MCF-7 and MDA-MB-468. Activation of FXR by high concentrations of ligands induced MCF-7 and MDA-MB-468 apoptosis. At lower concentrations that had no direct effect on viability, the FXR agonist GW4064 induced expression of mRNA for the FXR target genes, small heterodimer partner (SHP), intestinal bile acid binding protein, and multidrug resistance-associated protein 2 (MRP-2), and repressed the expression of the SHP target gene aromatase. In contrast to MRP-2, mRNA for the breast cancer target genes MDR-3, MRP-1, and solute carrier transporter 7A5 were decreased. Although multidrug resistance transporters were regulated and are known FXR target genes, GW4064 had no effect on the cell death induced by the anticancer drug paclitaxel. Our findings show for the first time that FXR is expressed in breast cancer tissue and has multiple properties that could be used for the treatment of breast cancer.91,0Samples of human infiltrating ductal breast carcinoma and near tumor normal samples were obtained from 10 patients after receiving ethical approval and individual patient consent. Protein was extracted using Cytobuster (Novagen, Merck Biosciences, Nottingham, United Kingdom). Breast carcinoma, matched tumor, and normal adjacent tissue microarrays were purchased from Folio Biosciences, Inc. (Columbus, OH). Immunohistochemistry was done using standard techniques as previously described (19).http://cancerres.aacrjournals.org/content/66/20/10120.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2016Amendola, LM;Jarvik, GP;Leo, MC;McLaughlin, HM;Akkari, Y;Amaral, MD;Berg, JS;Biswas, S;Bowling, KM;Conlin, LK;Cooper, GM;Dorschner, MO;Dulik, MC;Ghazani, AA;Ghosh, R;Green, RC;Hart, R;Horton, C;Johnston, JJ;Lebo, MS;Milosavljevic, A;Ou, J;Pak, CM;Patel, RY;Punj, S;Richards, CS;Salama, J;Strande, NT;Yang, Y;Plon, SE;Biesecker, LG;Rehm, HL;Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research ConsortiumAm. J. Hum. Genet.1067-107698627181684Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.90,0Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address: pair@u.washington.edu.++Center for Health Research, Kaiser Permanente, Portland, OR 97227, USA.++Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, MA 02139, USA.++Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA.++Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Center for Precision Diagnostics, Department of Pathology, University of Washington, Seattle, WA 98195, USA.++Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.++Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.++Baylor College of Medicine, Houston, TX 77030, USA.++Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, MA 02139, USA; Brigham and Women's Hospital and Harvard Medical School, Cambridge, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.++Clinical Diagnostics, Ambry Genetics, Aliso Viejo, CA 92656, USA.++Intramural Research Program, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, MA 02139, USA; Brigham and Women's Hospital and Harvard Medical School, Cambridge, MA 02115, USA.++Baylor College of Medicine, Houston, TX 77030, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.++Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239, USA.++Baylor College of Medicine, Houston, TX 77030, USA.++Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239, USA.++Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.++Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA.++Baylor College of Medicine, Houston, TX 77030, USA.++Baylor College of Medicine, Houston, TX 77030, USA.++Intramural Research Program, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, MA 02139, USA; Brigham and Women's Hospital and Harvard Medical School, Cambridge, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: hrehm@partners.org.http://dx.doi.org/10.1016/j.ajhg.2016.03.024HudsonAlpha
2016Green, RC;Goddard, KAB;Jarvik, GP;Amendola, LM;Appelbaum, PS;Berg, JS;Bernhardt, BA;Biesecker, LG;Biswas, S;Blout, CL;Bowling, KM;Brothers, KB;Burke, W;Caga-Anan, CF;Chinnaiyan, AM;Chung, WK;Clayton, EW;Cooper, GM;East, K;Evans, JP;Fullerton, SM;Garraway, LA;Garrett, JR;Gray, SW;Henderson, GE;Hindorff, LA;Holm, IA;Lewis, MH;Hutter, CM;Janne, PA;Joffe, S;Kaufman, D;Knoppers, BM;Koenig, BA;Krantz, ID;Manolio, TA;McCullough, L;McEwen, J;McGuire, A;Muzny, D;Myers, RM;Nickerson, DA;Ou, J;Parsons, DW;Petersen, GM;Plon, SE;Rehm, HL;Roberts, JS;Robinson, D;Salama, JS;Scollon, S;Sharp, RR;Shirts, B;Spinner, NB;Tabor, HK;Tarczy-Hornoch, P;Veenstra, DL;Wagle, N;Weck, K;Wilfond, BS;Wilhelmsen, K;Wolf, SM;Wynn, J;Yu, JH;, ;Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic MedicineAm. J. Hum. Genet.1051-106698627181682Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine. Copyright © 2016 American Society of Human Genetics. All rights reserved.90,0Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Medical School, Boston, MA 02115, USA; Partners Personalized Medicine, Boston, MA 02139, USA. Electronic address: rcgreen@genetics.med.harvard.edu.++Center for Health Research, Kaiser Permanente Northwest, Portland, OR 97227, USA.++Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; Clinical Sequencing Exploratory Research Coordinating Center, University of Washington, Seattle, WA 98195, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; Clinical Sequencing Exploratory Research Coordinating Center, University of Washington, Seattle, WA 98195, USA.++Department of Psychiatry, Columbia University Medical Center and New York State Psychiatric Institute, New York, NY 10032, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.++Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.++Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.++Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Department of Pediatrics, University of Louisville, Louisville, KY 40202, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; Clinical Sequencing Exploratory Research Coordinating Center, University of Washington, Seattle, WA 98195, USA; Department of Bioethics and Humanities, Department of Medicine, University of Washington, Seattle, WA 98195, USA.++National Cancer Institute, NIH, Bethesda, MD 20892, USA.++Michigan Center for Translational Pathology, Ann Arbor, MI 48109, USA; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA; Departments of Pathology and Urology, University of Michigan, Ann Arbor, MI 48109, USA; Howard Hughes Medical Institute, Ann Arbor, MI 48109, USA.++Department of Pediatrics, Columbia University, New York, NY 10029, USA; Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA.++Center for Biomedical Ethics and Society, Vanderbilt University, Nashville, TN 37203, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.++Department of Bioethics and Humanities, Department of Medicine, University of Washington, Seattle, WA 98195, USA.++Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology and Center for Cancer Precision Medicine, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.++Children's Mercy Bioethics Center, Children's Mercy Hospital, Kansas City, MO 64108, USA; Departments of Pediatrics and Philosophy, University of Missouri - Kansas City, Kansas City, MO 64110, USA.++Harvard Medical School, Boston, MA 02115, USA; Dana-Farber Cancer Institute, Boston, MA 02115, USA.++Department of Social Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.++Division of Genomic Medicine, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Harvard Medical School, Boston, MA 02115, USA; Division of Genetics and Genomics and the Manton Center for Orphan Diseases Research, Boston Children's Hospital, Boston, MA 02115, USA.++Berman Institute of Bioethics, Johns Hopkins, Baltimore, MD 21205, USA.++Division of Genomic Medicine, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Harvard Medical School, Boston, MA 02115, USA; Dana-Farber Cancer Institute, Boston, MA 02115, USA.++Department of Medical Ethics & Health Policy, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.++Division of Genomics and Society, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Centre of Genomics and Policy, Faculty of Medicine, Department of Human Genetics, McGill University, Montreal, QC H3A 1B1, Canada.++Institute for Health and Aging, University of California, San Francisco, San Francisco, CA 94118, USA.++Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.++Division of Genomic Medicine, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Center for Medical Ethics and Health Policy, Baylor College of Medicine, Houston, TX 77030, USA.++Division of Genomics and Society, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.++Center for Medical Ethics and Health Policy, Baylor College of Medicine, Houston, TX 77030, USA.++Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.++Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Clinical Sequencing Exploratory Research Coordinating Center, University of Washington, Seattle, WA 98195, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; Clinical Sequencing Exploratory Research Coordinating Center, University of Washington, Seattle, WA 98195, USA.++Baylor College of Medicine and Texas Children's Cancer Center, Houston, TX 77030, USA.++Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.++Baylor College of Medicine and Texas Children's Cancer Center, Houston, TX 77030, USA.++Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Medical School, Boston, MA 02115, USA; Partners Personalized Medicine, Boston, MA 02139, USA; Laboratory for Molecular Medicine, Partners HealthCare, Cambridge, MA 02139, USA.++Department of Health Behavior & Health Education, University of Michigan School of Public Health, Ann Arbor, MI 48109, USA.++Michigan Center for Translational Pathology, Ann Arbor, MI 48109, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; Clinical Sequencing Exploratory Research Coordinating Center, University of Washington, Seattle, WA 98195, USA.++Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.++Biomedical Ethics Research Program, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.++Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.++Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.++Department of Pediatrics and Seattle Children's Research Institute, University of Washington, Seattle, WA, USA.++Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; University of Washington, Seattle, WA 98105, USA.++Department of Pharmacy, University of Washington, Seattle, WA 98195, USA.++Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology and Center for Cancer Precision Medicine, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.++Department of Pediatrics and Seattle Children's Research Institute, University of Washington, Seattle, WA, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.++Law School, Medical School, and Consortium on Law and Values in Health, Environment, & the Life Sciences, Minneapolis, University of Minnesota, MN 55455, USA.++Department of Pediatrics, Columbia University, New York, NY 10029, USA.++Department of Pediatrics, University of Washington, Seattle, WA 98195, USA.++http://dx.doi.org/10.1016/j.ajhg.2016.04.011HudsonAlpha
2014Gee, HY;Ashraf, S;Wan, X;Vega-Warner, V;Esteve-Rudd, J;Lovric, S;Fang, H;Hurd, TW;Sadowski, CE;Allen, SJ;Otto, EA;Korkmaz, E;Washburn, J;Levy, S;Williams, DS;Bakkaloglu, SA;Zolotnitskaya, A;Ozaltin, F;Zhou, W;Hildebrandt, F;Mutations in EMP2 cause childhood-onset nephrotic syndromeAm. J. Hum. Genet.884-89094624814193Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.90,0Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.++Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.++Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.++Jules Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.++Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.++Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.++Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.++Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.++Nephrogenetics Laboratory, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey.++Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI 48109, USA.++HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA.++Jules Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.++Department of Pediatric Nephrology, Faculty of Medicine, Gazi University, Ankara 06570, Turkey.++New York Medical College, Valhalla, NY 10595, USA.++Nephrogenetics Laboratory, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey; Department of Pediatric Nephrology, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey; Center for Biobanking and Genomics, Hacettepe University, Ankara 06100, Turkey.++Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address: friedhelm.hildebrandt@childrens.harvard.edu.http://dx.doi.org/10.1016/j.ajhg.2014.04.010HudsonAlpha
2014Gee, HY;Otto, EA;Hurd, TW;Ashraf, S;Chaki, M;Cluckey, A;Vega-Warner, V;Saisawat, P;Diaz, KA;Fang, H;Kohl, S;Allen, SJ;Airik, R;Zhou, W;Ramaswami, G;Janssen, S;Fu, C;Innis, JL;Weber, S;Vester, U;Davis, EE;Katsanis, N;Fathy, HM;Jeck, N;Klaus, G;Nayir, A;Rahim, KA;Al Attrach, I;Al Hassoun, I;Ozturk, S;Drozdz, D;Helmchen, U;O'Toole, JF;Attanasio, M;Lewis, RA;Nürnberg, G;Nürnberg, P;Washburn, J;MacDonald, J;Innis, JW;Levy, S;Hildebrandt, F;Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathiesKidney Int.880-88785424257694Rare single-gene disorders cause chronic disease. However, half of the 6000 recessive single gene causes of disease are still unknown. Because recessive disease genes can illuminate, at least in part, disease pathomechanism, their identification offers direct opportunities for improved clinical management and potentially treatment. Rare diseases comprise the majority of chronic kidney disease (CKD) in children but are notoriously difficult to diagnose. Whole-exome resequencing facilitates identification of recessive disease genes. However, its utility is impeded by the large number of genetic variants detected. We here overcome this limitation by combining homozygosity mapping with whole-exome resequencing in 10 sib pairs with a nephronophthisis-related ciliopathy, which represents the most frequent genetic cause of CKD in the first three decades of life. In 7 of 10 sibships with a histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy, we detect the causative gene. In six sibships, we identify mutations of known nephronophthisis-related ciliopathy genes, while in two additional sibships we found mutations in the known CKD-causing genes SLC4A1 and AGXT as phenocopies of nephronophthisis-related ciliopathy. Thus, whole-exome resequencing establishes an efficient, noninvasive approach towards early detection and causation-based diagnosis of rare kidney diseases. This approach can be extended to other rare recessive disorders, thereby providing accurate diagnosis and facilitating the study of disease mechanisms.84,0Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.++Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.++Department of Pediatrics, University Children's Hospital, University Essen, Essen, Germany.++Department of Pediatrics, University Children's Hospital, University Essen, Essen, Germany.++Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina, USA.++Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina, USA.++The Pediatric Nephrology Unit, Alexandria University, Alexandria, Egypt.++Zentrum für Kinder- und Jugendmedizin am UKGM, Marburg, Germany.++Zentrum für Kinder- und Jugendmedizin am UKGM, Marburg, Germany.++Department of Pediatric Nephrology, Faculty of Medicine, University of Istanbul, Istanbul, Turkey.++Department of Pediatric Nephrology, Children's Hospital King Fahad Medical City, Riyadh, Saudi Arabia.++Division of Pediatric Nephrology, Tawam Hospital, UAE University, Al Ain, UAE.++King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia.++Nephrology, Haseki Training and Research Hospital, Bezmialem Vakif University Faculty of Medicine, Istanbul, Turkey.++Dialysis Unit, Polish-American Children's Hospital, Collegium Medicum of Jagiellonian University, Cracow, Poland.++Universitätsklinikum Hamburg-Eppendorf, III. Medizinische Klinik, University of Hamburg, Hamburg, Germany.++Division of Nephrology, Department of Internal Medicine, MetroHealth Medical Center, and Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.++Department of Internal Medicine and Eugene McDermott Center for Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas, USA.++Departments of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas, USA.++Cologne Center for Genomics, Center for Molecular Medicine Cologne, and Cologne Excellence Cluster on Cellular Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.++Cologne Center for Genomics, Center for Molecular Medicine Cologne, and Cologne Excellence Cluster on Cellular Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.++Biomedical Research Core Facilities, University of Michigan, Ann Arbor, Michigan, USA.++Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA.++1] Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA [2] HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.++1] Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.http://dx.doi.org/10.1038/ki.2013.450HudsonAlpha
2019Rowley, MJ;Lyu, X;Rana, V;Ando-Kuri, M;Karns, R;Bosco, G;Corces, VG;Condensin II Counteracts Cohesin and RNA Polymerase II in the Establishment of 3D Chromatin OrganizationCell Rep2890-2903.e3261130865881Interaction domains in Drosophila chromosomes form by segregation of active and inactive chromatin in the absence of CTCF loops, but the role of transcription versus other architectural proteins in chromatin organization is unclear. Here, we find that positioning of RNAPII via transcription elongation is essential in the formation of gene loops, which in turn interact to form compartmental domains. Inhibition of transcription elongation or depletion of cohesin decreases gene looping and formation of active compartmental domains. In contrast, depletion of condensin II, which also localizes to active chromatin, causes increased gene looping, formation of compartmental domains, and stronger intra-chromosomal compartmental interactions. Condensin II has a similar role in maintaining inter-chromosomal interactions responsible for pairing between homologous chromosomes, whereas inhibition of transcription elongation or cohesin depletion has little effect on homolog pairing. The results suggest distinct roles for cohesin and condensin II in the establishment of 3D nuclear organization in Drosophila. Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.83,0We would like to thank the HudsonAlpha Institute for Biotechnology Genomic Services Lab for their help with Illumina sequencing. This work was supported by NIH Pathway to Independence Award K99/R00 GM127671 (M.J.R.) and U.S. Public Health Service Award (R01) GM035463 (V.G.C.) from the NIH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.http://dx.doi.org/10.1016/j.celrep.2019.01.116HudsonAlpha
2017Jung, YH;Sauria, MEG;Lyu, X;Cheema, MS;Ausio, J;Taylor, J;Corces, VG;Chromatin States in Mouse Sperm Correlate with Embryonic and Adult Regulatory LandscapesCell Rep1366-138218628178516The mammalian sperm genome is thought to lack substantial information for the regulation of future expression after fertilization. Here, we show that most promoters in mouse sperm are flanked by well-positioned nucleosomes marked by active histone modifications. Analysis of these modifications suggests that many enhancers and super-enhancers functional in embryonic and adult tissues are already specified in sperm. The sperm genome is bound by CTCF and cohesin at sites that are also present in round spermatids and embryonic stem cells (ESCs). These sites mediate interactions that organize the sperm genome into domains and compartments that overlap extensively with those found in mESCs. These results suggest that sperm carry a rich source of regulatory information, encoded in part by its three-dimensional folding specified by CTCF and cohesin. This information may contribute to future expression during embryonic and adult life, suggesting mechanisms by which environmental effects on the paternal germline are transmitted transgenerationally. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.83,0We would like to thank the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology, and specially Drs. Braden Boone, Angela Jones and Terri Pointer, for their help in performing Illumina sequencing of samples. This work was supported by U.S. Public Health Service Award R01 GM035463 from the National Institutes of Health to VGC and Natural Sciences and Engineering Research Council of Canada (NSERC) 46399-2012 grant to JA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.http://dx.doi.org/10.1016/j.celrep.2017.01.034HudsonAlpha
2018Thompson, ML;Finnila, CR;Bowling, KM;Brothers, KB;Neu, MB;Amaral, MD;Hiatt, SM;East, KM;Gray, DE;Lawlor, JMJ;Kelley, WV;Lose, EJ;Rich, CA;Simmons, S;Levy, SE;Myers, RM;Barsh, GS;Bebin, EM;Cooper, GM;Genomic sequencing identifies secondary findings in a cohort of parent study participantsGenet. Med.1635-1643201229790872Clinically relevant secondary variants were identified in parents enrolled with a child with developmental delay and intellectual disability. Exome/genome sequencing and analysis of 789 "unaffected" parents was performed. Pathogenic/likely pathogenic variants were identified in 21 genes within 25 individuals (3.2%), with 11 (1.4%) participants harboring variation in a gene defined as clinically actionable by the American College of Medical Genetics and Genomics. These 25 individuals self-reported either relevant clinical diagnoses (5); relevant family history or symptoms (13); or no relevant family history, symptoms, or clinical diagnoses (7). A limited carrier screen was performed yielding 15 variants in 48 (6.1%) parents. Parents were also analyzed as mate pairs (n = 365) to identify cases in which both parents were carriers for the same recessive disease, yielding three such cases (0.8%), two of which had children with the relevant recessive disease. Four participants had two findings (one carrier and one noncarrier variant). In total, 71 of the 789 enrolled parents (9.0%) received secondary findings. We provide an overview of the rates and types of clinically relevant secondary findings, which may be useful in the design and implementation of research and clinical sequencing efforts to identify such findings.82,0to change their preferences at the return of results appointment. Whole exome and genome sequencing Blood samples were sent for sequencing at the HudsonAlpha Genomic Services Laboratory (http://gsl.hudsonalpha.org). Genomic DNA was isolated from peripheralhttps://pdfs.semanticscholar.org/5ff0/d42b8dc3aefbc600e155a8db6a77cd410bd3.pdf"HudsonAlpha Genomic Services"
2018Pena, LDM;Jiang, YH;Schoch, K;Spillmann, RC;Walley, N;Stong, N;Rapisardo Horn, S;Sullivan, JA;McConkie-Rosell, A;Kansagra, S;Smith, EC;El-Dairi, M;Bellet, J;Keels, MA;Jasien, J;Kranz, PG;Noel, R;Nagaraj, SK;Lark, RK;Wechsler, DSG;Del Gaudio, D;Leung, ML;Hendon, LG;Parker, CC;Jones, KL;, ;Goldstein, DB;Shashi, V;Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseasesGenet. Med.464-46920428914269PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.82,0Kimberly Splinter Harvard University (CC) David P. Bick HudsonAlpha Camille L. Birch HudsonAlpha Braden E. Boone HudsonAlpha Donna M. Brown HudsonAlpha Daniel C. Dorset HudsonAlpha Lori H. Handley HudsonAlpha Howard J. Jacob HudsonAlpha Angela L. Jones HudsonAlpha Jozef Lazar HudsonAlpha Shawn E. Levy HudsonAlpha J. Scott Newberry HudsonAlphahttp://dx.doi.org/10.1038/gim.2017.128HudsonAlpha
2017O'Daniel, JM;McLaughlin, HM;Amendola, LM;Bale, SJ;Berg, JS;Bick, D;Bowling, KM;Chao, EC;Chung, WK;Conlin, LK;Cooper, GM;Das, S;Deignan, JL;Dorschner, MO;Evans, JP;Ghazani, AA;Goddard, KA;Gornick, M;Farwell Hagman, KD;Hambuch, T;Hegde, M;Hindorff, LA;Holm, IA;Jarvik, GP;Knight Johnson, A;Mighion, L;Morra, M;Plon, SE;Punj, S;Richards, CS;Santani, A;Shirts, BH;Spinner, NB;Tang, S;Weck, KE;Wolf, SM;Yang, Y;Rehm, HL;A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratoriesGenet. Med.575-58219527811861While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews. Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data. This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016.82,0Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.++Laboratory for Molecular Medicine, Partners Healthcare Personalized Medicine, Cambridge, Massachusetts, USA.++Division of Medical Genetics, University of Washington, Seattle, Washington, USA.++GeneDx, Inc., Gaithersburg, Maryland, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.++Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++Ambry Genetics, Aliso Viejo, California, USA.++Department of Pediatrics, Columbia University, New York, New York, USA.++Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and Perelman School of Medicine at The University of Pennsylvania, Philadelphia, Pennsylvania, USA.++HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.++Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.++Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.++Department of Genome Sciences, University of Washington, Seattle, Washington, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.++Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.++Center for Health Research, Kaiser Permanente Northwest, Portland, Oregon, USA.++Department of Internal Medicine, Center for Bioethics Social Science and Medicine, University of Michigan, Ann Arbor, Michigan, USA.++Ambry Genetics, Aliso Viejo, California, USA.++Illumina, Inc., San Diego, California, USA.++Emory Genetics Laboratory, Department of Human Genetics, Emory University, Atlanta, Georgia, USA.++Division of Genomic Medicine, National Human Genome Research Institute, Bethesda, Maryland, USA.++Division of Genetics and Genomics and Manton Center for Orphan Diseases Research, Boston Children's Hospital, Boston, Massachusetts, USA.++Division of Medical Genetics, University of Washington, Seattle, Washington, USA.++Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.++Emory Genetics Laboratory, Department of Human Genetics, Emory University, Atlanta, Georgia, USA.++Personalis, Inc., Menlo Park, California, USA.++Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.++Department of Molecular and Medical Genetics, Oregon Health &Science University, Portland, Oregon, USA.++Department of Molecular and Medical Genetics, Oregon Health &Science University, Portland, Oregon, USA.++Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and Perelman School of Medicine at The University of Pennsylvania, Philadelphia, Pennsylvania, USA.++Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.++Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and Perelman School of Medicine at The University of Pennsylvania, Philadelphia, Pennsylvania, USA.++Ambry Genetics, Aliso Viejo, California, USA.++Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.++Consortium on Law and Values in Health, Environment &the Life Sciences, Law School, Medical School, University of Minnesota, Minneapolis, Minnesota, USA.++Department of Molecular and Human Genetics, Baylor College of Medicine and Baylor Miraca Genetics Laboratories, Houston, Texas, USA.++The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.http://dx.doi.org/10.1038/gim.2016.152HudsonAlpha
2017Rydzak, T;Garcia, D;Stevenson, DM;Sladek, M;Klingeman, DM;Holwerda, EK;Amador-Noguez, D;Brown, SD;Guss, AM;Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellumMetab. Eng.182-1914128400329Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum. Copyright © 2017. Published by Elsevier Inc.81,0; diluted. Sequencing was completed using a SR50 sequencing protocol and V4 chemistry on an Illumina HiSeq. 2500 platform (HudsonAlpha Genomic Services Laboratory; Huntsville, AL). 2.7. RNA-Seq analysis. Raw readshttps://www.sciencedirect.com/science/article/pii/S1096717616301306HudsonAlpha
2015Grabek, KR;Diniz Behn, C;Barsh, GS;Hesselberth, JR;Martin, SL;Enhanced stability and polyadenylation of select mRNAs support rapid thermogenesis in the brown fat of a hibernatorElife425626169During hibernation, animals cycle between torpor and arousal. These cycles involve dramatic but poorly understood mechanisms of dynamic physiological regulation at the level of gene expression. Each cycle, Brown Adipose Tissue (BAT) drives periodic arousal from torpor by generating essential heat. We applied digital transcriptome analysis to precisely timed samples to identify molecular pathways that underlie the intense activity cycles of hibernator BAT. A cohort of transcripts increased during torpor, paradoxical because transcription effectively ceases at these low temperatures. We show that this increase occurs not by elevated transcription but rather by enhanced stabilization associated with maintenance and/or extension of long poly(A) tails. Mathematical modeling further supports a temperature-sensitive mechanism to protect a subset of transcripts from ongoing bulk degradation instead of increased transcription. This subset was enriched in a C-rich motif and genes required for BAT activation, suggesting a model and mechanism to prioritize translation of key proteins for thermogenesis.77,0Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, United States.++Department of Applied Math and Statistics, Colorado School of Mines, Golden, United States.++Department of Research, HudsonAlpha Institute for Biotechnology, Huntsville, United States.++Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, United States.++Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, United States.http://dx.doi.org/10.7554/eLife.04517HudsonAlpha
2017Tigue, NJ;Bamber, L;Andrews, J;Ireland, S;Hair, J;Carter, E;Sridharan, S;Jovanović, J;Rees, DG;Springall, JS;Solier, E;Li, YM;Chodorge, M;Perez-Martinez, D;Higazi, DR;Oberst, M;Kennedy, M;Black, CM;Yan, L;Schwickart, M;Maguire, S;Cann, JA;de Haan, L;Young, LL;Vaughan, T;Wilkinson, RW;Stewart, R;MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potentialOncoimmunologye12806456328405505Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.77,0... xP3 Tissue samples from 76 cancer patients, with NSCLC, colorectal cancer (CRC), bladder cancer, or squamous cell cancer of the head and neck (SCCHN), were obtained from commercial sources (Asterand, ConversantBio, ProteoGenex, National Disease Research Interchange (NDRI) or ILSBio) and used in accordance with the ethical principles originating in the Declaration of Helsinki and in compliance withhttps://www.tandfonline.com/doi/abs/10.1080/2162402X.2017.1280645"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2016Hu, X;Liu, X;Moisan, J;Wang, Y;Lesch, CA;Spooner, C;Morgan, RW;Zawidzka, EM;Mertz, D;Bousley, D;Majchrzak, K;Kryczek, I;Taylor, C;Van Huis, C;Skalitzky, D;Hurd, A;Aicher, TD;Toogood, PL;Glick, GD;Paulos, CM;Zou, W;Carter, LL;Synthetic RORγ agonists regulate multiple pathways to enhance antitumor immunityOncoimmunologye125485451228123897RORγt is the key transcription factor controlling the development and function of CD4+ Th17 and CD8+ Tc17 cells. Across a range of human tumors, about 15% of the CD4+ T cell fraction in tumor-infiltrating lymphocytes are RORγ+ cells. To evaluate the role of RORγ in antitumor immunity, we have identified synthetic, small molecule agonists that selectively activate RORγ to a greater extent than the endogenous agonist desmosterol. These RORγ agonists enhance effector function of Type 17 cells by increasing the production of cytokines/chemokines such as IL-17A and GM-CSF, augmenting expression of co-stimulatory receptors like CD137, CD226, and improving survival and cytotoxic activity. RORγ agonists also attenuate immunosuppressive mechanisms by curtailing Treg formation, diminishing CD39 and CD73 expression, and decreasing levels of co-inhibitory receptors including PD-1 and TIGIT on tumor-reactive lymphocytes. The effects of RORγ agonists were not observed in RORγ-/- T cells, underscoring the selective on-target activity of the compounds. In vitro treatment of tumor-specific T cells with RORγ agonists, followed by adoptive transfer to tumor-bearing mice is highly effective at controlling tumor growth while improving T cell survival and maintaining enhanced IL-17A and reduced PD-1 in vivo. The in vitro effects of RORγ agonists translate into single agent, immune system-dependent, antitumor efficacy when compounds are administered orally in syngeneic tumor models. RORγ agonists integrate multiple antitumor mechanisms into a single therapeutic that both increases immune activation and decreases immune suppression resulting in robust inhibition of tumor growth. Thus, RORγ agonists represent a novel immunotherapy approach for cancer.77,0... ion. After 5 days, cytokine levels in the media were determined using a luminex panel (R&D Systems). Cells were collected for flow cytometry analysis. PBMCs from cancer patients were purchased from Conversant Bio and activated with anti-CD3/28 beads and differentiated as described above. PD-L1 inhibition assay Splenocytes were differentiated in Type 17 conditions as above. 5 days after differehttps://www.tandfonline.com/doi/abs/10.1080/2162402X.2016.1254854"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2019Angelova, E;Audette, C;Kovtun, Y;Daver, N;Wang, SA;Pierce, S;Konoplev, SN;Khogeer, H;Jorgensen, JL;Konopleva, M;Zweidler-McKay, PA;Medeiros, LJ;Kantarjian, HM;Jabbour, EJ;Khoury, JD;CD123 expression patterns and selective targeting with a CD123-targeted antibody-drug conjugate (IMGN632) in acute lymphoblastic leukemiaHaematologica749-755104430361418The potential of CD123-targeted therapies in acute lymphoblastic leukemia/lymphoma remains largely unexplored. We examined CD123 expression levels in a large cohort of patients with acute lymphoblastic leukemia/lymphoma and assessed the in vitro impact of IMGN632, a conjugate of CD123-binding antibody with a novel DNA-alkylating payload. CD123 expression on leukemic blasts was surveyed using multicolor/multiparameter flow cytometry. The in vitro effect of IMGN632 was evaluated on B acute lymphoblastic leukemia/lymphoma cell lines and primary B acute lymphoblastic leukemia/lymphoma blasts. The study cohort (n=213) included 183 patients with B acute lymphoblastic leukemia/lymphoma and 30 with T acute lymphoblastic leukemia/lymphoma. CD123 expression was more prevalent in B acute lymphoblastic leukemia/lymphoma than in T acute lymphoblastic leukemia/lymphoma (164/183, 89.6% versus 13/30, 43.3%; P<0.0001), and within B acute lymphoblastic leukemia/lymphoma CD123 expression was more prevalent in Philadelphia chromosome-positive patients than in Philadelphia chromosome-negative patients (96.6% versus 86.3%; P=0.033). In T acute lymphoblastic leukemia/lymphoma, 12/13 (92.3%) patients with CD123-positive blasts had either early T precursor (ETP) or early non-ETP immunophenotype. IMGN632 was highly cytotoxic to B acute lymphoblastic leukemia/lymphoma cell lines, with half maximal inhibitory concentrations (IC50) between 0.6 and 20 pM. In five of eight patients' samples, low picomolar concentrations of IMGN632 eliminated more than 90% of the B acute lymphoblastic leukemia/lymphoma blast population, sparing normal lymphocytes. In conclusion, CD123 expression is prevalent across acute lymphoblastic leukemia/lymphoma subtypes, and the CD123-targeted antibody-drug conjugate IMGN632 demonstrates promising selective activity in preclinical models of B acute lymphoblastic leukemia/lymphoma. Copyright© 2019 Ferrata Storti Foundation.77,0... In vitro evaluation of primary B-cell acute lymphoblastic leukemia samples Bone marrow mononuclear cells from 11 newly diagnosed and 10 relapsed/refractory B-ALL patients were obtained from MDACC or ConversantBio. The number of CD123 antibody-binding sites per cell (ABC) was quantified by the BD QuantiBRITE™ Fluorescence Quantitation Kit (BD Biosciences) using G4723A conjugated to R-phycoerythrhttp://www.haematologica.org/content/104/4/749.abstract"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2016Lieberman, LA;Zeng, W;Singh, C;Wang, W;Otipoby, KL;Loh, C;Plavina, T;Gorelik, L;Ransohoff, RM;Cahir-McFarland, E;CD62L is not a reliable biomarker for predicting PML risk in natalizumab-treated R-MS patientsNeurology375-38186426718566To assess if the percentage of CD3(+)CD4(+)CD62L(+) cells in cryopreserved peripheral blood mononuclear cells (PBMCs) (here termed %CD62L) can predict risk of developing progressive multifocal leukoencephalopathy (PML) and better inform the physician for benefit-risk assessment of natalizumab treatment decisions in a global setting. Cryopreserved PBMCs from 21 natalizumab-treated patients who developed PML and 104 matched natalizumab-treated patients with multiple sclerosis (MS) without PML collected as a part of Biogen clinical trials were retrospectively examined for CD3, CD4, CCR7, CD45RA, and CD62L by flow cytometry. In this cohort, %CD62L in natalizumab-treated patients did not predict PML risk. Natalizumab-treated patients with MS without PML showed highly variable %CD62L upon serial sampling. In the STRATA study, the distribution of %CD62L in samples collected more than 6 months before a PML diagnosis, at diagnosis, and in natalizumab-treated patients without PML overlapped. No statistical threshold for risk could be determined. In addition, we demonstrated that lymphocyte viability strongly affects %CD62L, supporting previous reports that %CD62L is inherently unstable following cryopreservation and is sensitive to sample collection. Data from this well-controlled cohort of natalizumab-treated patients indicate that %CD62L is not a biomarker of PML risk. © 2015 American Academy of Neurology.76,0Thirty-four frozen PBMC samples were purchased from Conversant Bio (Huntsville, AL) to test the effects of other medical conditions on the % CD62L determination. Twenty-one matched healthy donor samples were collected. Eight samples were collected from hospitalized patients who had at least 24 hours bedrest (5 surgery patients). Five samples were collected from methicillin-resistant Staphylococcus aureus (MRSA)–positive patients who had a fever above 38°C. Influenza vaccination PBMC samples and corresponding healthy donors were collected and cryopreserved at Biogen (Cambridge, MA).https://n.neurology.org/content/86/4/375.short"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2014Rosenfeldt, MT;Bell, LA;Long, JS;O'Prey, J;Nixon, C;Roberts, F;Dufès, C;Ryan, KM;E2F1 drives chemotherapeutic drug resistance via ABCG2Oncogene4164-4172333224276245Multidrug resistance is a major barrier against successful chemotherapy, and this has been shown in vitro to be often caused by ATP-binding cassette (ABC) transporters. These transporters are frequently overexpressed in human cancers and confer an adverse prognosis in many common malignancies. The genetic factors, however, that initiate their expression in cancer are largely unknown. Here we report that the major multidrug transporter ABCG2 (BCRP/MXR) is directly and specifically activated by the transcription factor E2F1--a factor perturbed in the majority of human cancers. E2F1 regulates ABCG2 expression in multiple cell systems, and, importantly, we have identified a significant correlation between elevated E2F1 and ABCG2 expression in human lung cancers. We show that E2F1 causes chemotherapeutic drug efflux both in vitro and in vivo via ABCG2. Furthermore, the E2F1-ABCG2 axis suppresses chemotherapy-induced cell death that can be restored by the inhibition of ABCG2. These findings therefore identify a new axis in multidrug resistance and highlight a radical new function of E2F1 that is relevant to tumor therapy.75,0Biotechnology (St Louis, MO, USA). The ChIP Assay Kit was from Millipore (Watford, UK) and the human lung cancer tissue microarray was from Folio Biosciences (Columbus, OH, USA). Plasmids. pTRE-E2F1 constructs were generatedhttps://www.nature.com/articles/onc2013470"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2011Ahmad, I;Morton, JP;Singh, LB;Radulescu, SM;β-Catenin activation synergizes with PTEN loss to cause bladder cancer formationOncogeneAs the Uroplakin Cre recombinase is expressed throughout development of the urothelium, it is difficult to assess whether the upregulation of PTEN is a direct consequence of β-catenin accumulation. Therefore, we next investigated the consequence of inducibly activating Wnt signalling in the adult urothelium. To perform this, we used mice carrying the cytochrome p450-inducible _AhCreER__T_ transgene. Following administration of both β-naphthoflavone and tamoxifen, this yields _Cre_-mediated recombination within the urothelium of the bladder (as well as the intestine). Figures 2a and b show recombination in the bladder as evidenced by GFP signalling identified by OV100 imaging (_ex vivo_) and IHC for GFP in _AhCreER__T__Z/EG_ reporter mice 7 days following induction. To investigate the effect of acutely activating Wnt signalling in the adult bladder, we inter-crossed AhCreERT mice with mice carrying the inducible knockout _Apc__580S_ allele from hereafter known as Apcfl (Shibata et al., 1997 [/articles/onc2010399#ref43]). Significantly, examination of the bladders from _AhCreER__T__APC__fl/fl_ mice 7 days following induction revealed development of urothelial lesions that phenocopied those from our _UroIICRE_+ _β-catenin__exon3/exon3_ mice (Figure 2c). Consistent with the activation of Wnt signalling, lesions showed a high level of nuclear β-catenin and again an upregulation in BrdU compared with the surrounding urothelium (Figures 2e and g). Importantly, once again very high levels of PTEN were observed within these lesions (Figure 2i) with minimal upregulation of pAKT (Figure 2k). To confirm that this was because of increased levels of Wnt signalling, we also activated Wnt signalling by deleting both copies of GSK3. Bladders from induced _AhCreER__T_ _GSK3_α_fl/fl_β_fl/fl_ mice showed similar lesions to the _AhCreER__T_ _APC__fl/fl_ mice, with the accumulation of nuclear β-catenin, BrdU and PTEN (Supplementary Figures 3A–D [/articles/onc2010399#s1]) (Kemp et al., 2004 [/articles/onc2010399#ref18]; MacAulay et al., 2007 [/articles/onc2010399#ref23]; Patel et al., 2008 [/articles/onc2010399#ref37]). Using this induction regime, it was found that mice developed hyperplastic intestinal epithelium, which precluded long-term tumour experiments; however, in mice aged up to 4 months bladder lesions still remained small and did not progress to cancer, suggesting PTEN was once again blocking tumorigenesis.75,0invasive bladder carcinoma. We next looked at human bladder UCC using a tissue microarray of 80 cases, 60 UCC (transitional cell carcinomas) and 20 benign controls (Folio Biosciences, Columbus, OH, USA). Using the histoscorehttps://www.nature.com/articles/onc2010399"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2017Liu, Z;Zhang, C;Skamagki, M;Khodadadi-Jamayran, A;Zhang, W;Kong, D;Chang, CW;Feng, J;Han, X;Townes, TM;Li, H;Kim, K;Zhao, R;Elevated p53 Activities Restrict Differentiation Potential of MicroRNA-Deficient Pluripotent Stem CellsStem Cell Reports1604-16179529141234Pluripotent stem cells (PSCs) deficient for microRNAs (miRNAs), such as Dgcr8-/- or Dicer-/- embryonic stem cells (ESCs), contain no mature miRNA and cannot differentiate into somatic cells. How miRNA deficiency causes differentiation defects remains poorly understood. Here, we report that miR-302 is sufficient to enable neural differentiation of differentiation-incompetent Dgcr8-/- ESCs. Our data showed that miR-302 directly suppresses the tumor suppressor p53, which is modestly upregulated in Dgcr8-/- ESCs and serves as a barrier restricting neural differentiation. We demonstrated that direct inactivation of p53 by SV40 large T antigen, a short hairpin RNA against Trp53, or genetic ablation of Trp53 in Dgcr8-/- PSCs enables neural differentiation, while activation of p53 by the MDM2 inhibitor nutlin-3a in wild-type ESCs inhibits neural differentiation. Together, we demonstrate that a major function of miRNAs in neural differentiation is suppression of p53 and that modest activation of p53 blocks neural differentiation of miRNA-deficient PSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.73,0Total RNA was extracted from Dgcr8−/−-302 cells and submitted to the Genomic Services Lab at the HudsonAlpha Institute. The library was constructed by the standard miRNA library construction protocol (Illumina) and 15 million, 50 bp single-end reads were acquired. Adapters were removed from the reads using cutadapt (v.1.8.1) (Anders et al., 2015). All the reads were mapped to the mouse reference genome (GRCm38.74/mm10) using STAR aligner guided by a Gene Transfer File (Ensembl GTF version GRCm38.74) (Dobin et al., 2013).http://dx.doi.org/10.1016/j.stemcr.2017.10.006HudsonAlpha
2015Li, C;Gowan, S;Anil, A;Beck, BH;Thongda, W;Kucuktas, H;Kaltenboeck, L;Peatman, E;Discovery and validation of gene-linked diagnostic SNP markers for assessing hybridization between Largemouth bass (Micropterus salmoides) and Florida bass (M. floridanus)Mol Ecol Resour395-40415225047482Efforts to improve recreational fisheries have included widespread stocking of Micropterus floridanus outside its native range of peninsular Florida. Hybridization of Florida bass (M. floridanus) with largemouth bass (Micropterus salmoides) has now dramatically expanded beyond a naturally occurring intergrade zone in the southeast U.S. In recent years, there has been growing interest in protecting the genetic integrity of native basses and assessing the impact and nature of M. salmoides/M. floridanus introgression from the standpoint of hatchery and sport-fishery managers, fish biologists, ecologists and evolutionary biologists. Here, we conducted RNA-seq-based sequencing of the transcriptomes of M. salmoides, M. floridanus and their F1 hybrid and identified a set of 3674 SNP markers with fixed-allelic differences from 2112 unique genes. We then developed a subset of 25 of these markers into a single diagnostic multiplex assay and validated its capacity for assessing integrity and hybridization in hatchery and wild populations of largemouth and Florida bass. The availability of this resource, high-quality transcriptomes and a large set of gene-linked SNPs, should greatly facilitate functional and population genomics studies in these key species and allow the identification of traits and processes under selection during introgressive hybridization. © 2014 John Wiley & Sons Ltd.73,0; of 110-140 nm. After KAPA quantitation and dilution, based on included DNA standards (1-6), the libraries were sequenced in a single lane on an Illumina HiSeq 2000 instrument with 100 bp paired‐end (PE) reads at HudsonAlpha Genomic Services Lab (Huntsville, AL, USA)https://onlinelibrary.wiley.com/doi/abs/10.1111/1755-0998.12308HudsonAlpha
2017Bowling, KM;Thompson, ML;Amaral, MD;Finnila, CR;Hiatt, SM;Engel, KL;Cochran, JN;Brothers, KB;East, KM;Gray, DE;Kelley, WV;Lamb, NE;Lose, EJ;Rich, CA;Simmons, S;Whittle, JS;Weaver, BT;Nesmith, AS;Myers, RM;Barsh, GS;Bebin, EM;Cooper, GM;Genomic diagnosis for children with intellectual disability and/or developmental delayGenome Med439128554332Developmental disabilities have diverse genetic causes that must be identified to facilitate precise diagnoses. We describe genomic data from 371 affected individuals, 309 of which were sequenced as proband-parent trios. Whole-exome sequences (WES) were generated for 365 individuals (127 affected) and whole-genome sequences (WGS) were generated for 612 individuals (244 affected). Pathogenic or likely pathogenic variants were found in 100 individuals (27%), with variants of uncertain significance in an additional 42 (11.3%). We found that a family history of neurological disease, especially the presence of an affected first-degree relative, reduces the pathogenic/likely pathogenic variant identification rate, reflecting both the disease relevance and ease of interpretation of de novo variants. We also found that improvements to genetic knowledge facilitated interpretation changes in many cases. Through systematic reanalyses, we have thus far reclassified 15 variants, with 11.3% of families who initially were found to harbor a VUS and 4.7% of families with a negative result eventually found to harbor a pathogenic or likely pathogenic variant. To further such progress, the data described here are being shared through ClinVar, GeneMatcher, and dbGaP. Our data strongly support the value of large-scale sequencing, especially WGS within proband-parent trios, as both an effective first-choice diagnostic tool and means to advance clinical and research progress related to pediatric neurological disease.71,0Methods" (see Additional file 1). Whole-exome and whole-genome sequencing. Blood samples were sent for sequencing at the HudsonAlpha Genomic Services Laboratory (http://gsl.hudsonalpha.org). Genomic DNA was isolatedhttps://genomemedicine.biomedcentral.com/articles/10.1186/s13073-017-0433-1"HudsonAlpha Genomic Services"
2013Sah, S;Chen, L;Houghton, J;Kemppainen, J;Marko, AC;Zeigler, R;Latham, GJ;Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsiesGenome Med775824001039The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay ('QFI-PCR') that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing.71,0Seventy-six FFPE blocks containing thyroid tumor biopsies were purchased from Asterand (Detroit, MI, USA). The remaining 39 FFPE tissue specimens were processed from colorectal cancer resections purchased from Folio Biosciences (Columbus, OH, USA) [14]https://genomemedicine.biomedcentral.com/articles/10.1186/gm481"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2018Abraham, B;Pharma, GUCB;Sankyo, HD;Disclosure of relevant financial relationshipsJournal of the American Academy of DermatologyAB315-AB42079370,0Abramovits, William; Grants - AbbVie, Allergan, Amgen, Anacor Pharm, Aqua Pharma, Celgene, Centocor, Conversant Bio, Dermavant Abramovits, William; Honoraria - AbbVie, Allergan, Amgen, Anacor Pharm, Aqua Pharma, Celgene, Centocor, Conversant Bio, Dermavanthttps://www.jaad.org/article/S0190-9622(18)32043-7/abstract"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2017Harden, ME;Prasad, N;Griffiths, A;Munger, K;Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 OncoproteinsMBio8128049151The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. High-risk human papillomaviruses (HPVs) are the causative agents of almost all cervical cancers and many other cancers, including anal, vaginal, vulvar, penile, and oropharyngeal cancers. Despite the availability of efficacious HPV vaccines, it is critical to determine how HPVs cause cancer, as many people remain unvaccinated and the vaccine does not prevent cancer development in individuals who are already infected. Two HPV proteins, E6 and E7, are the major drivers of cancer development, and much remains to be learned about how the expression of these viral proteins reprograms infected cells, ultimately resulting in cancer development. Small, noncoding human RNAs, termed microRNAs (miRs), regulate gene expression and have been implicated in almost all human cancers, including HPV-associated cancers. Our study provides a comprehensive analysis of how E6 and E7 alter the expression of human miRs and how this potentially impacts cellular gene expression, which may contribute to cancer development. Copyright © 2017 Harden et al.70,0;... million 50-bp PE reads) was performed with the Illumina MiSeq Sequencing System (Illumina). miR-seq data analysis. Postprocessing of the miR-seq reads from each sample was performed according to the HudsonAlpha Genomic Services Laboratory (GSL) unique in-house pipeline as previously described (62). The differential expression of miRs was calculated on the basis of the difference (cutoff, ±3.0-fohttps://mbio.asm.org/content/8/1/e02170-16.shortHudsonAlpha
2014Wildsmith, KR;Schauer, SP;Smith, AM;Arnott, D;Zhu, Y;Haznedar, J;Kaur, S;Mathews, WR;Honigberg, LA;Identification of longitudinally dynamic biomarkers in Alzheimer's disease cerebrospinal fluid by targeted proteomicsMol Neurodegener22924902845Alzheimer's disease (AD) is the leading cause of dementia affecting greater than 26 million people worldwide. Although cerebrospinal fluid (CSF) levels of Aβ42, tau, and p-tau181 are well established as diagnostic biomarkers of AD, there is a need for additional CSF biomarkers of neuronal function that continue to change during disease progression and could be used as pharmacodynamic measures in clinical trials. Multiple proteomic discovery experiments have reported a range of CSF biomarkers that differ between AD and control subjects. These potential biomarkers represent multiple aspects of the disease pathology. The performance of these markers has not been compared with each other, and their performance has not been evaluated longitudinally. We developed a targeted-proteomic, multiple reaction monitoring (MRM) assay for the absolute quantitation of 39 peptides corresponding to 30 proteins. We evaluated the candidate biomarkers in longitudinal CSF samples collected from aged, cognitively-normal control (n = 10), MCI (n = 5), and AD (n = 45) individuals (age > 60 years). We evaluated each biomarker for diagnostic sensitivity, longitudinal consistency, and compared with CSF Aβ42, tau, and p-tau181. Four of 28 quantifiable CSF proteins were significantly different between aged, cognitively-normal controls and AD subjects including chitinase-3-like protein 1, reproducing published results. Four CSF markers demonstrated significant longitudinal change in AD: Amyloid precursor protein, Neuronal pentraxin receptor, NrCAM and Chromogranin A. Robust correlations were observed within some subgroups of proteins including the potential disease progression markers. Using a targeted proteomics approach, we confirmed previous findings for a subset of markers, defined longitudinal performance of our panel of markers, and established a flexible proteomics method for robust multiplexed analyses.68,0... ap in clinical biomarker development. Methods Source of CSF CSF was purchased from Bioreclamation, LLC (Hicksville, NY) (pooled cynomolgus monkey and pooled human CSF used in discovery experiments), Folio Biosciences (Columbus, OH) (individual longitudinal AD only) and PrecisionMed, Inc. (San Diego, CA) (individual longitudinal Control, MCI and AD). Details on sample collection were provided by t ... ap in clinical biomarker development. Methods Source of CSF CSF was purchased from Bioreclamation, LLC (Hicksville, NY) (pooled cynomolgus monkey and pooled human CSF used in discovery experiments), Folio Biosciences (Columbus, OH) (individual longitudinal AD only) and PrecisionMed, Inc. (San Diego, CA) (individual longitudinal Control, MCI and AD). Details on sample collection were provided by t ... ap in clinical biomarker development. Methods Source of CSF CSF was purchased from Bioreclamation, LLC (Hicksville, NY) (pooled cynomolgus monkey and pooled human CSF used in discovery experiments), Folio Biosciences (Columbus, OH) (individual longitudinal AD only) and PrecisionMed, Inc. (San Diego, CA) (individual longitudinal Control, MCI and AD). Details on sample collection were provided by thttps://molecularneurodegeneration.biomedcentral.com/articles/10.1186/1750-1326-9-22"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2019Bao, L;Tian, C;Liu, S;Zhang, Y;Elaswad, A;Yuan, Z;Khalil, K;Sun, F;Yang, Y;Zhou, T;Li, N;Tan, S;Zeng, Q;Liu, Y;Li, Y;Li, Y;Gao, D;Dunham, R;Davis, K;Waldbieser, G;Liu, Z;The Y chromosome sequence of the channel catfish suggests novel sex determination mechanisms in teleost fishBMC Biol.617130683095Sex determination mechanisms in teleost fish broadly differ from mammals and birds, with sex chromosomes that are far less differentiated and recombination often occurring along the length of the X and Y chromosomes, posing major challenges for the identification of specific sex determination genes. Here, we take an innovative approach of comparative genome analysis of the genomic sequences of the X chromosome and newly sequenced Y chromosome in the channel catfish. Using a YY channel catfish as the sequencing template, we generated, assembled, and annotated the Y genome sequence of channel catfish. The genome sequence assembly had a contig N50 size of 2.7 Mb and a scaffold N50 size of 26.7 Mb. Genetic linkage and GWAS analyses placed the sex determination locus within a genetic distance less than 0.5 cM and physical distance of 8.9 Mb. However, comparison of the channel catfish X and Y chromosome sequences showed no sex-specific genes. Instead, comparative RNA-Seq analysis between females and males revealed exclusive sex-specific expression of an isoform of the breast cancer anti-resistance 1 (BCAR1) gene in the male during early sex differentiation. Experimental knockout of BCAR1 gene converted genetic males (XY) to phenotypic females, suggesting BCAR1 as a putative sex determination gene. We present the first Y chromosome sequence among teleost fish, and one of the few whole Y chromosome sequences among vertebrate species. Comparative analyses suggest that sex-specific isoform expression through alternative splicing may underlie sex determination processes in the channel catfish, and we identify BCAR1 as a potential sex determination gene.68,0;... Bioanalyzer. Equal amounts of RNA from each 5-day period were pooled from 10 to 14 dpf and 15 to 19 dpf for male and female, respectively. These four pooled samples were outsourced for RNA-Seq at the HudsonAlpha Genomic Services Lab (Huntsville, AL, USA). The RNA-Seq libraries were prepared following the standard TruSeq protocols and were sequenced on an Illumina HiSeq 2500 instrument for 100 bphttps://bmcbiol.biomedcentral.com/articles/10.1186/s12915-019-0627-7HudsonAlpha
2019Rieker, C;Migliavacca, E;Vaucher, A;Mayer, FC;Baud, G;Marquis, J;Charpagne, A;Hegde, N;Guignard, L;McLachlan, M;Pooler, AM;Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial CellsArterioscler. Thromb. Vasc. Biol.ATVBAHA11831226131315437The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aβ (amyloid-β) 40 and 42, increased release of cytokines, and overexpression of the blood clotting protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.66,0Immunohistochemistry Analysis of Postmortem Human Brain Tissue Human hippocampal brain tissue from AD (Braak stage V-VI; both male and female) individuals was purchased from Folio Biosciences, genotyped for ApoE, and processed for cryo- sectioninghttps://www.ahajournals.org/doi/abs/10.1161/ATVBAHA.118.312261"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2017Weiss, G;Schlegel, A;Kottwitz, D;König, T;Tetzner, R;Validation of the SHOX2/PTGER4 DNA Methylation Marker Panel for Plasma-Based Discrimination between Patients with Malignant and Nonmalignant Lung DiseaseJ Thorac Oncol77-8412127544059Low-dose computed tomography (LDCT) is used for screening for lung cancer (LC) in high-risk patients in the United States. The definition of high risk and the impact of frequent false-positive results of low-dose computed tomography remains a challenge. DNA methylation biomarkers are valuable noninvasive diagnostic tools for cancer detection. This study reports on the evaluation of methylation markers in plasma DNA for LC detection and discrimination of malignant from nonmalignant lung disease. Circulating DNA was extracted from 3.5-mL plasma samples, treated with bisulfite using a commercially available kit, purified, and assayed by real-time polymerase chain reaction for assessment of DNA methylation of short stature homeobox 2 gene (SHOX2), prostaglandin E receptor 4 gene (PTGER4), and forkhead box L2 gene (FOXL2). In three independent case-control studies these assays were evaluated and optimized. The resultant assay, a triplex polymerase chain reaction combining SHOX2, PTGER4, and the reference gene actin, beta gene (ACTB), was validated using plasma from patients with and without malignant disease. A panel of SHOX2 and PTGER4 provided promising results in three independent case-control studies examining a total of 330 plasma specimens (area under the receiver operating characteristic curve = 91%-98%). A validation study with 172 patient samples demonstrated significant discriminatory performance in distinguishing patients with LC from subjects without malignancy (area under the curve = 0.88). At a fixed specificity of 90%, sensitivity for LC was 67%; at a fixed sensitivity of 90%, specificity was 73%. Measurement of SHOX2 and PTGER4 methylation in plasma DNA allowed detection of LC and differentiation of nonmalignant diseases. Development of a diagnostic test based on this panel may provide clinical utility in combination with current imaging techniques to improve LC risk stratification. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.66,0The patient samples were either material collected over the course of several months by commercial partners (ProteoGenex Inc., Sofia Bio LLC) or leftover material from previously conducted clinical trials in colon cancer.21, 22 More specifically, patients with lung disease (LChttps://www.sciencedirect.com/science/article/pii/S1556086416308425"Sofia Bio"
2012Polisetty, RV;Gautam, P;Sharma, R;Harsha, HC;Nair, SC;Gupta, MK;Uppin, MS;Challa, S;Puligopu, AK;Ankathi, P;Purohit, AK;Chandak, GR;Pandey, A;Sirdeshmukh, R;LC-MS/MS analysis of differentially expressed glioblastoma membrane proteome reveals altered calcium signaling and other protein groups of regulatory functionsMol. Cell ProteomicsM111.01356511622219345Membrane proteins play key roles in the development and progression of cancer. We have studied differentially expressed membrane proteins in glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, by high resolution LC-MS/MS mass spectrometry and quantitation by iTRAQ. A total of 1834 membrane proteins were identified with high confidence, of which 356 proteins were found to be altered by 2-fold change or more (198 up- and 158 down-regulated); 56% of them are known membrane proteins associated with major cellular processes. Mass spectrometry results were confirmed for representative proteins on individual specimens by immunohistochemistry. On mapping of the differentially expressed proteins to cellular pathways and functional networks, we notably observed many calcium-binding proteins to be altered, implicating deregulation of calcium signaling and homeostasis in GBM, a pathway also found to be enriched in the report (Dong, H., Luo, L., Hong, S., Siu, H., Xiao, Y., Jin, L., Chen, R., and Xiong, M. (2010) Integrated analysis of mutations, miRNA and mRNA expression in glioblastoma. BMC Syst. Biol. 4, 163) based on The Cancer Genome Atlas analysis of GBMs. Annotations of the 356 proteins identified by us with The Cancer Genome Atlas transcriptome data set indicated overlap with 295 corresponding transcripts, which included 49 potential miRNA targets; many transcripts correlated with proteins in their expression status. Nearly 50% of the differentially expressed proteins could be classified as transmembrane domain or signal sequence-containing proteins (159 of 356) with potential of appearance in cerebrospinal fluid or plasma. Interestingly, 75 of them have been already reported in normal cerebrospinal fluid or plasma along with other proteins. This first, in-depth analysis of the differentially expressed membrane proteome of GBM confirms genes/proteins that have been implicated in earlier studies, as well as reveals novel candidates that are being reported for the first time in GBM or any other cancer that could be investigated further for clinical applications.65,0... tion [http://www.mcponline.org/cgi/content/full/M111.013565/DC1]. IMMUNOHISTOCHEMISTRY Formalin-fixed paraffin-embedded individual GBM tissue sections or commercially available tissue microarrays (Folio Biosciences, Columbus, OH) with 35 GBM tissue cores and five cases of normal brain in duplicate were used for the IHC studies. The following primary antibodies were used: anti-ANXA2 mouse monocl ... tion [http://www.mcponline.org/cgi/content/full/M111.013565/DC1]. IMMUNOHISTOCHEMISTRY Formalin-fixed paraffin-embedded individual GBM tissue sections or commercially available tissue microarrays (Folio Biosciences, Columbus, OH) with 35 GBM tissue cores and five cases of normal brain in duplicate were used for the IHC studies. The following primary antibodies were used: anti-ANXA2 mouse monocl ... tion [http://www.mcponline.org/cgi/content/full/M111.013565/DC1]. IMMUNOHISTOCHEMISTRY Formalin-fixed paraffin-embedded individual GBM tissue sections or commercially available tissue microarrays (Folio Biosciences, Columbus, OH) with 35 GBM tissue cores and five cases of normal brain in duplicate were used for the IHC studies. The following primary antibodies were used: anti-ANXA2 mouse monoclhttps://www.mcponline.org/content/11/6/M111.013565.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2016Cataisson, C;Michalowski, AM;Shibuya, K;Ryscavage, A;Klosterman, M;Wright, L;Dubois, W;Liu, F;Zhuang, A;Rodrigues, KB;Hoover, S;Dwyer, J;Simpson, MR;Merlino, G;Yuspa, SH;MET signaling in keratinocytes activates EGFR and initiates squamous carcinogenesisSci Signalra62943327330189The receptor tyrosine kinase MET is abundant in many human squamous cell carcinomas (SCCs), but its functional significance in tumorigenesis is not clear. We found that the incidence of carcinogen-induced skin squamous tumors was substantially increased in transgenic MT-HGF (mouse metallothionein-hepatocyte growth factor) mice, which have increased abundance of the MET ligand HGF. Squamous tumors also erupted spontaneously on the skin of MT-HGF mice that were promoted by wounding or the application of 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C. Carcinogen-initiated tumors had Ras mutations, but spontaneous tumors did not. Cultured keratinocytes from MT-HGF mice and oncogenic RAS-transduced keratinocytes shared phenotypic and biochemical features of initiation that were dependent on autocrine activation of epidermal growth factor receptor (EGFR) through increased synthesis and release of EGFR ligands, which was mediated by the kinase SRC, the pseudoproteases iRhom1 and iRhom2, and the metallopeptidase ADAM17. Pharmacological inhibition of EGFR caused the regression of MT-HGF squamous tumors that developed spontaneously in orthografts of MT-HGF keratinocytes combined with dermal fibroblasts and implanted onto syngeneic mice. The global gene expression profile in MET-transformed keratinocytes was highly concordant with that in RAS-transformed keratinocytes, and a core RAS/MET coexpression network was activated in precancerous and cancerous human skin lesions. Tissue arrays revealed that many human skin SCCs have abundant HGF at both the transcript and protein levels. Thus, through the activation of EGFR, MET activation parallels a RAS pathway to contribute to human and mouse cutaneous cancers. Copyright © 2016, American Association for the Advancement of Science.65,0This procedure was performed by a commercial vendor (Phylogeny, Inc.) according to the published method (88). FFPE TMAs from US Biomax were deparaffinized for 5 minutes in xylene, immersed in 100% ethanol for 5 minutes then air-dried. Treatment was with Bond Epitope Retrieval Solution 2 from Leica (AR9640) for 30 minutes. LNA probes HGF-1–1, HGF-1–2 and Scramble-miR, were prepared according to the manufacturer’s recommended conditions (Exiqon) and each was labeled at the 5’ end with digoxigenin.https://stke.sciencemag.org/content/9/433/ra62?intcmp=trendmd-stke"Phylogeny Inc"
2018Menard, LC;Fischer, P;Kakrecha, B;Linsley, PS;Wambre, E;Liu, MC;Rust, BJ;Lee, D;Penhallow, B;Manjarrez Orduno, N;Nadler, SG;Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4+CD8+ T Cells With Expression of Exhaustion MarkersFront Immunol2728930534127Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.64,0Samples for the discovery and replication cohorts were provided by Folio-Conversant Bio (AL), MT Group (CA), Rutgers-CINJ or the Benaroya Research Institute. All patients gave written informed consent at time of sample collection according to IRB protocols of each providerhttps://www.frontiersin.org/articles/10.3389/fimmu.2018.02728/abstract"Folio Conversant";"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2015Nguyen, D;Rubinstein, L;Takebe, N;Miele, L;Tomaszewski, JE;Ivy, P;Doroshow, JH;Yang, SX;Notch1 phenotype and clinical stage progression in non-small cell lung cancerJ Hematol Oncol9825653136Notch1 transmembrane receptor is activated through ligand-binding- triggered proteolytic cleavages and, upon release, the intracellular domain (N1-ICD) translocates into the nucleus and modulates target gene transcriptions. Notch activation has been implicated in tumorigenesis in an increasing number of human malignancies including non-small cell lung cancer (NSCLC). However, Notch1 in distinct expression patterns and activation status with tumor progression remains to be defined in NSCLC. Notch1 and activated Notch1, N1-ICD, were examined by immunohistochemistry in 58 cases of stage I to IV NSCLC tumors. Association between Notch1 or N1-ICD expression and clinicopathological factors was assessed via correlation coefficient r statistics. P-values are two-sided. Detectable tumor Notch1, predominantly localized to the membrane and cytoplasm, was observed in 29 cases (50%, 95% Blyth-Still-Casella confidence interval 37 - 63%). It was negatively associated with stage (r=- 0.43, P<0.001) and nodal status (r=- 0.33, P = 0.01), but not tumor size. In contrast, nuclear N1-ICD expression level was low and found in 12% of NSCLC patients, neither significantly associated with stage nor nodal status. Upon Notch1 activation in vitro, a mostly extra-nuclear staining was substantially turned into the nuclear signal in cancer cells. Notch1 in the largely inactivated phenotype is inversely associated with clinical stage progression in NSCLC. Notch1, rather than activated N1-ICD, may be a context-dependent restrictive factor to nodal metastasis.63,0Lung cancer tissue microarrays and clinicopathological data were obtained from Cybrdi, Incorporation (Rockville, MD), and FOLIO Biosciences (Powell, OH). Approval of the biomarker study on de-identified human tissues was obtained from the Office of Human Research Protections, National Institutes of Health, Bethesda, Maryland. Tumor presence and histology were confirmed on HE stained sections.https://jhoonline.biomedcentral.com/articles/10.1186/s13045-014-0104-2"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2017Park, KC;Lee, M;Jeon, Y;Jeon, R;Baek, SH;Lee, H;Kim, KI;Skin-Specific Deletion of Mis18α Impedes Proliferation and Stratification of Epidermal KeratinocytesJ. Invest. Dermatol.414-421137227670610The Mis18 proteins (Mis18α, Mis18β, and M18BP1) are pivotal to the deposition of CENP-A at the centromere during cell cycle progression and are indispensable for embryonic development. Here, we show that Mis18α is critical for the proliferation of keratinocytes and stratification of the epidermis. Mice lacking Mis18α in the epidermis died shortly after birth, showing skin abnormalities like thin and translucent skin and defective skin barrier functions. The epidermis of newborn Mis18α-deficient mice lacked distinct stratification and mature hair follicles, with a reduction in the number of proliferating cells and increased cell death in the basal layer. Earlier expression of the Cre recombinase from keratin-14 promoter in the ventral region resulted in earlier keratinocyte death in the ventral part compared with the dorsal part in the absence of Mis18α, leading to more severe malformation of the ventral epidermal layers. As observed in Mis18α-deficient mouse keratinocytes, knockdown of Mis18α in HaCaT cells caused marked loss of centromeric CENP-A dots and chromosomal misalignment. Overall, we propose that Mis18α is important for epidermal cell proliferation and stratification, because it is required for the deposition of CENP-A at the centromeric nucleosomes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.63,0Materials and Methods. In situ hybridization. All procedures of in situ hybridization, except probe design and cloning, were performed by Phylogeny Inc. (Columbus, OH). Briefly, the whole bodies of C57BL/6 mice were frozen and cut into 10-μm sectionshttps://www.sciencedirect.com/science/article/pii/S0022202X1632454X"Phylogeny Inc"
2014Farber, CR;Reich, A;Barnes, AM;Becerra, P;Rauch, F;Cabral, WA;Bae, A;Quinlan, A;Glorieux, FH;Clemens, TL;Marini, JC;A novel IFITM5 mutation in severe atypical osteogenesis imperfecta type VI impairs osteoblast production of pigment epithelium-derived factorJ. Bone Miner. Res.1402-141129624519609Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5'-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization.63,0Exome sequencing was performed by the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA). Briefly, gDNA (1 to 2 μg) was fragmented and subjected to exome enrichment using the Nimblegen SeqCap EZ Human Exome Library v2.0 kit (Roche Nimblegen, Madison, WI, USA). The enriched libraries were barcoded and 100-bp paired-end reads were generated on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA).http://dx.doi.org/10.1002/jbmr.2173HudsonAlpha
2013Rafaelsen, SH;Raeder, H;Fagerheim, AK;Knappskog, P;Carpenter, TO;Johansson, S;Bjerknes, R;Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcificationJ. Bone Miner. Res.1378-138528623325605Fibroblast growth factor 23 (FGF23) plays a crucial role in renal phosphate regulation, exemplified by the causal role of PHEX and DMP1 mutations in X-linked hypophosphatemic rickets and autosomal recessive rickets type 1, respectively. Using whole exome sequencing we identified compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) in two siblings referred for hypophosphatemia and severe dental demineralization disease. FAM20C mutations were not found in other undiagnosed probands of a national Norwegian population of familial hypophosphatemia. Our results demonstrate that mutations in FAM20C provide a putative new mechanism in human subjects leading to dysregulated FGF23 levels, hypophosphatemia, hyperphosphaturia, dental anomalies, intracerebral calcifications and osteosclerosis of the long bones in the absence of rickets. Copyright © 2013 American Society for Bone and Mineral Research.63,0Genomic DNA was purified from blood using the QiaSymphony system (Qiagen, Hilden, Germany). Whole‐genome single‐nucleotide polymorphism (SNP) genotyping was performed with Genome‐Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA, USA). Whole‐exome capture using Roche‐Nimblegen's SeqCap EZ Exome v2 and sequencing on the Illumina HiSeq was performed at the HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA) to a median coverage of 154× according to the manufacturer's protocol.http://dx.doi.org/10.1002/jbmr.1850HudsonAlpha
2016Bryant, SA;Herdy, JR;Amemiya, CT;Smith, JJ;Characterization of Somatically-Eliminated Genes During Development of the Sea Lamprey (Petromyzon marinus)Mol. Biol. Evol.2337-234433927288344The sea lamprey (Petromyzon marinus) is a basal vertebrate that undergoes developmentally programmed genome rearrangements (PGRs) during early development. These events facilitate the elimination of ∼20% of the genome from the somatic cell lineage, resulting in distinct somatic and germline genomes. Thus far only a handful of germline-specific genes have been definitively identified within the estimated 500 Mb of DNA that is deleted during PGR, although a few thousand germline-specific genes are thought to exist. To improve our understanding of the evolutionary/developmental logic of PGR, we generated computational predictions to identify candidate germline-specific genes within a new transcriptomic dataset derived from adult germline and the early embryonic stages during which PGR occurs. Follow-up validation studies identified 44 germline-specific genes and further characterized patterns of transcription and DNA loss during early embryogenesis. Expression analyses reveal that many of these genes are differentially expressed during early embryogenesis and presumably function in the early development of the germline. Ontology analyses indicate that many of these germline-specific genes play known roles in germline development, pluripotency, and oncogenesis (when misexpressed). These studies provide support for the theory that PGR serves to segregate molecular functions related to germline development/pluripotency in order to prevent their potential misexpression in somatic cells. This larger set of eliminated genes also allows us to extend the evolutionary/developmental breadth of this theory, as some deleted genes (or their gnathostome homologs) appear to be associated with the early development of somatic lineages, perhaps through the evolution of novel functions within gnathostome lineages. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.62,0Total RNA was isolated from lamprey embryos using Trizol extraction. RNA quality was assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies) and samples with RNA Integrity Number (RIN) 8 were sent to the HudsonAlpha Genomic Services Lab (HudsonAlpha, Huntsville, AL) for paired-end sequencing on the Illumina platform. Raw reads were assembled using Trinity (trinityrnaseq_r2013-02-25) using default parameters and integrated quality clipping with Trimmomatic (Bolger et al. 2014).https://academic.oup.com/mbe/article-abstract/33/9/2337/2579241"HudsonAlpha Genomic Services"
2017Steffen, MM;Davis, TW;McKay, RML;Bullerjahn, GS;Krausfeldt, LE;Stough, JMA;Neitzey, ML;Gilbert, NE;Boyer, GL;Johengen, TH;Gossiaux, DC;Burtner, AM;Palladino, D;Rowe, MD;Dick, GJ;Meyer, KA;Levy, S;Boone, BE;Stumpf, RP;Wynne, TT;Zimba, PV;Gutierrez, D;Wilhelm, SW;Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OHEnviron. Sci. Technol.6745-6755511228535339Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for >2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of reoccurring Lake Erie blooms and thus may reoccur in the future.62,0RNA was stored at −80 °C until sent to HudsonAlpha Institute for Biotechnology (Huntsville, AL) for sequencing. Total RNA concentrations and quality were assessed fluorometrically via RiboGreen (Life Technologies, Carlsbad, CA) followed by integrity measurement via Bioanalysis (Agilent Technologies, Santa Clara, CA). Ribosomal RNA reduction was done using the Illumina Ribo-Zero Epidemiology rRNA removal kit (San Diego, CA) followed by first- and second-strand cDNA synthesis (New England Biolabs, Ipswitch, MA) and library preparation (Kapa Biosystems, Wilmington, MA).https://pubs.acs.org/doi/abs/10.1021/acs.est.7b00856HudsonAlpha
2018Klemann, CJHM;Xicoy, H;Poelmans, G;Bloem, BR;Martens, GJM;Visser, JE;Physical Exercise Modulates L-DOPA-Regulated Molecular Pathways in the MPTP Mouse Model of Parkinson's DiseaseMol. Neurobiol.5639-565755729019056Parkinson's disease (PD) is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), resulting in motor and non-motor dysfunction. Physical exercise improves these symptoms in PD patients. To explore the molecular mechanisms underlying the beneficial effects of physical exercise, we exposed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP)-treated mice to a four-week physical exercise regimen, and subsequently explored their motor performance and the transcriptome of multiple PD-linked brain areas. MPTP reduced the number of DA neurons in the SNpc, whereas physical exercise improved beam walking, rotarod performance, and motor behavior in the open field. Further, enrichment analyses of the RNA-sequencing data revealed that in the MPTP-treated mice physical exercise predominantly modulated signaling cascades that are regulated by the top upstream regulators L-DOPA, RICTOR, CREB1, or bicuculline/dalfampridine, associated with movement disorders, mitochondrial dysfunction, and epilepsy-related processes. To elucidate the molecular pathways underlying these cascades, we integrated the proteins encoded by the exercise-induced differentially expressed mRNAs for each of the upstream regulators into a molecular landscape, for multiple key brain areas. Most notable was the opposite effect of physical exercise compared to previously reported effects of L-DOPA on the expression of mRNAs in the SN and the ventromedial striatum that are involved in-among other processes-circadian rhythm and signaling involving DA, neuropeptides, and endocannabinoids. Altogether, our findings suggest that physical exercise can improve motor function in PD and may, at the same time, counteract L-DOPA-mediated molecular mechanisms. Further, we hypothesize that physical exercise has the potential to improve non-motor symptoms of PD, some of which may be the result of (chronic) L-DOPA use.62,0pooled for RNAseq analysis. RNA Sequencing and Data Processing. All RNA samples were subjected to RNA sequencing (RNAseq; HudsonAlpha Genomic Services Lab, Huntsville, AL) as performed before [37]. In short, totalhttps://link.springer.com/article/10.1007/s12035-017-0775-0"HudsonAlpha Genomic Services"
2018Van Laar, R;Lincoln, M;Van Laar, B;Development and validation of a plasma-based melanoma biomarker suitable for clinical useBr. J. Cancer857-866118629360813This corrects the article DOI: 10.1038/bjc.2017.85.62,0expression profiles of individuals with and without melanoma, 0.5-2 ml of cell-free plasma was obtained from 16 healthy controls and 32 Caucasian individuals at the time of their diagnosis with cutaneous melanoma (Cureline Inc., CA, USA and Folio Biosciences, OH, USA)https://www.nature.com/articles/bjc2017477"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2014Shipitsin, M;Small, C;Choudhury, S;Giladi, E;Friedlander, S;Nardone, J;Hussain, S;Hurley, AD;Ernst, C;Huang, YE;Chang, H;Nifong, TP;Rimm, DL;Dunyak, J;Loda, M;Berman, DM;Blume-Jensen, P;Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling errorBr. J. Cancer1201-1212111625032733Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.62,0Acquisition, processing, quality control, and annotation of FFPE prostate cancer tissue blocks. A set of FFPE human prostate cancer tissue blocks with clinical annotations and long-term patient outcome information was acquired from Folio Biosciences (Powell, OH, USA)https://www.nature.com/articles/bjc2014396"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2012Gonzalez de Castro, D;Angulo, B;Gomez, B;Mair, D;Martinez, R;Suarez-Gauthier, A;Shieh, F;Velez, M;Brophy, VH;Lawrence, HJ;Lopez-Rios, F;A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimensBr. J. Cancer345-351107222713664KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.62,0Materials: FFPET specimens from CRC tumours were purchased from US commercial vendors: Discovery Life Sciences, Inc. (Los Osos, CA, USA); BioServe (Beltsville, MD, USA); ProteoGenex (Culver City, CA, USA); CureLine, Inchttps://www.nature.com/articles/bjc2012259"Discovery Life Sciences Inc"
2017Blanchet, P;Bebin, M;Bruet, S;Cooper, GM;Thompson, ML;Duban-Bedu, B;Gerard, B;Piton, A;Suckno, S;Deshpande, C;Clowes, V;Vogt, J;Turnpenny, P;Williamson, MP;Alembik, Y;, ;, ;Glasgow, E;McNeill, A;MYT1L mutations cause intellectual disability and variable obesity by dysregulating gene expression and development of the neuroendocrine hypothalamusPLoS Genet.e100695713828859103Deletions at chromosome 2p25.3 are associated with a syndrome consisting of intellectual disability and obesity. The smallest region of overlap for deletions at 2p25.3 contains PXDN and MYT1L. MYT1L is expressed only within the brain in humans. We hypothesized that single nucleotide variants (SNVs) in MYT1L would cause a phenotype resembling deletion at 2p25.3. To examine this we sought MYT1L SNVs in exome sequencing data from 4, 296 parent-child trios. Further variants were identified through a genematcher-facilitated collaboration. We report 9 patients with MYT1L SNVs (4 loss of function and 5 missense). The phenotype of SNV carriers overlapped with that of 2p25.3 deletion carriers. To identify the transcriptomic consequences of MYT1L loss of function we used CRISPR-Cas9 to create a knockout cell line. Gene Ontology analysis in knockout cells demonstrated altered expression of genes that regulate gene expression and that are localized to the nucleus. These differentially expressed genes were enriched for OMIM disease ontology terms "mental retardation". To study the developmental effects of MYT1L loss of function we created a zebrafish knockdown using morpholinos. Knockdown zebrafish manifested loss of oxytocin expression in the preoptic neuroendocrine area. This study demonstrates that MYT1L variants are associated with syndromic obesity in humans. The mechanism is related to dysregulated expression of neurodevelopmental genes and altered development of the neuroendocrine hypothalamus.61,0Département de Génétique Médicale, Hôpital Arnaud de Villeneuve, Montpellier Cedex 5, France.++Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, United States of America.++Service de génétique médicale, Hôpitaux Universitaires de Clermont-Ferrand, Clermont-Ferrand, France.++Human Genetics and Genomics, HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, United States of America.++Human Genetics and Genomics, HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, United States of America.++Centre de génétique chromosomique, 51 Boulevard de Belfort, Lille, France.++Laboratoire de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.++Laboratoire de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France.++Service de neuropédiatrie, Hôpital Saint Vincent de Paul, Lille, France.++South East Thames Regional Genetics Service, Guy's Hospital, London, United Kingdom.++North West Thames Regional Genetics Centre, Northwick Park Hospital, Harrow, United Kingdom.++Clinical Genetics Unit, Birmingham Women's Hospital, Birmingham, United Kingdom.++Peninsula Clinical Genetics, Royal Devon & Exeter Hospital (Heavitree), Exeter, United Kingdom.++Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, United Kingdom.++Department of Medical Genetics, CHU Hautepierre, Strasbourg, France.++++++Department of Medicine, Georgetown University, Washington, DC, United States of America.++Sheffield Institute for Translational Neuroscience, The University of Sheffield, Sheffield, United Kingdom.https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006957&rev=2"HudsonAlpha Genomic Services"
2016Jiang, L;Huang, J;Higgs, BW;Hu, Z;Xiao, Z;Yao, X;Conley, S;Zhong, H;Liu, Z;Brohawn, P;Shen, D;Wu, S;Ge, X;Jiang, Y;Zhao, Y;Lou, Y;Morehouse, C;Zhu, W;Sebastian, Y;Czapiga, M;Oganesyan, V;Fu, H;Niu, Y;Zhang, W;Streicher, K;Tice, D;Zhao, H;Zhu, M;Xu, L;Herbst, R;Su, X;Gu, Y;Li, S;Huang, L;Gu, J;Han, B;Jallal, B;Shen, H;Yao, Y;Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung CancerPLoS Genet.e100589512427093186Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.61,0... s >70%; the tumor content in each NAT was< 3%. The Caucasian SCLC patient cohort consisted of 25 FFPE lung tumor tissue specimens with matched normal adjacent tissue pairs, which were purchased from Conversant Biologics, Inc (Huntsville, AL) (S11 Table). The diagnosis of SCLC was confirmed by two independent pathologists in Medimmune by H&E staining. All samples were treatment naïve surgical sam ... s >70%; the tumor content in each NAT was< 3%. The Caucasian SCLC patient cohort consisted of 25 FFPE lung tumor tissue specimens with matched normal adjacent tissue pairs, which were purchased from Conversant Biologics, Inc (Huntsville, AL) (S11 Table). The diagnosis of SCLC was confirmed by two independent pathologists in Medimmune by H&E staining. All samples were treatment naïve surgical samhttps://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1005895"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2014Hermetz, KE;Newman, S;Conneely, KN;Martin, CL;Ballif, BC;Shaffer, LG;Cody, JD;Rudd, MK;Large inverted duplications in the human genome form via a fold-back mechanismPLoS Genet.e100413910124497845Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. For half of the rearrangements in our study, we sequenced at least one breakpoint junction. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted sequences are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a "fold-back" intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the fold-back loop. This process can lead to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes.61,0SureSelect capture and Illumina HiSeq sequencing were performed at Hudson Alpha Genomic Services Lab (http://www.hudsonalpha.org/gsl/). After NGS, we aligned 100-bp paired-end reads from fastq files to the GRC37/hg19 reference genome using Burrows-Wheeler Alignment (BWA) tool 0.5.9 [66] and identified misaligned pairs using the SAMTools 0.1.18 filter function [67].http://dx.doi.org/10.1371/journal.pgen.1004139HudsonAlpha
2011Kelly, RK;Olson, DL;Sun, Y;Wen, D;Wortham, KA;Antognetti, G;Cheung, AE;Orozco, OE;Yang, L;Bailly, V;Sanicola, M;An antibody-cytotoxic conjugate, BIIB015, is a new targeted therapy for Cripto positive tumoursEur. J. Cancer1736-1746471121458984BIIB015 is an immunoconjugate created for the treatment of solid tumours and is currently in Phase I of clinical evaluation. BIIB015 consists of a humanised monoclonal antibody against the Cripto protein carrying a payload, via a hindered disulphide linker, of the maytansinoid derivative, DM4. Cripto is a GPI-linked protein required for signal transduction of the TGF-beta ligand, Nodal. Cripto has been previously described as an oncogene and fits the classic pattern of an embryonic gene that is re-expressed in a transformed tumour cell. Cripto expression is highly prevalent on a number of solid tumours, including greater than 75% of breast, lung, and colorectal tumours. Our report documents for the first time that targeting the cell surface Cripto protein with an anti-Cripto antibody-cytotoxic conjugate is an effective means of inhibiting or regressing growth of Cripto positive tumours. BIIB015 which utilises a 'cleavable' linker containing a disulphide bond exhibits superior activity when compared to huB3F6 mAb conjugates with different linker systems, including one with a 'non-cleavable' linker. BIIB015 displays specificity for Cripto in both in vitro and in vivo experiments. In human xenograft models originating from lung (Calu-6), colon (CT-3), testicular (NCCIT) and breast (MDA-MB-231) tumour samples, BIIB015 shows robust activity with results ranging from >50% tumour inhibition to complete tumour regression. The efficacy seen in the MDA-MB-231 model, a triple negative (-HER2, -ER, and -PR) tumour, is particularly exciting since there is currently no approved therapy for this indication. In addition, BIIB015 can be combined with standard of care chemotherapeutics for enhanced efficacy. Copyright © 2011 Elsevier Ltd. All rights reserved.60,02.3. Immunohistochemistry. Human breast, colon, lung and ovarian formalin-fixed paraffin-embedded tumour tissue arrays (Imgenex, San Diego, CA, Folio Biosciences, Columbus, OH and Zymed, San Francisco, CA) were stained as described previouslyhttps://www.sciencedirect.com/science/article/pii/S0959804911001432"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2012Lenehan, PF;Boardman, LA;Riegert-Johnson, D;De Petris, G;Fry, DW;Ohrnberger, J;Heyman, ER;Gerard, B;Almal, AA;Worzel, WP;Generation and external validation of a tumor-derived 5-gene prognostic signature for recurrence of lymph node-negative, invasive colorectal carcinomaCancer5234-52441182122605513One in 4 patients with lymph node-negative, invasive colorectal carcinoma (CRC) develops recurrent disease after undergoing curative surgery, and most die of advanced disease. Predicting which patients will develop a recurrence is a significantly growing, unmet medical need. Archival formalin-fixed, paraffin-embedded (FFPE) primary adenocarcinoma tissues obtained at surgery were retrieved from 74 patients with CRC (15 with stage I disease and 59 with stage II disease) for Training/Test Sets. In addition, FFPE tissues were retrieved from 49 patients with stage I CRC and 215 patients with stage II colon cancer for an External Validation (EV) Set (n = 264) from 18 hospitals in 4 countries. No patients had received neoadjuvant/adjuvant therapy. Proprietary genetic programming analysis of expression profiles for 225 prespecified tumor genes was used to create a 36-month recurrence risk signature. Using reverse transcriptase-polymerase chain reaction, a 5-gene rule correctly classified 62 of 92 recurrent patients and 87 of 172 nonrecurrent patients in the EV Set (sensitivity, 0.67; specificity, 0.51). "High-risk" patients had a greater probability of 36-month recurrence (42%) than "low-risk" patients (26%; hazard ratio, 1.80; 95% confidence interval, 1.19-2.71; P = .007; Cox regression) independent of T-classification, the number of lymph nodes examined, histologic grade/subtype, anatomic location, age, sex, or race. The rule outperformed (P = .021) current National Comprehensive Cancer Network Guidelines (hazard ratio, 0.897). The same rule also differentiated the risk of recurrence (hazard ratio, 1.63; P = .031) in a subset of patients from the EV Set who had stage I/II colon cancer only (n = 251). To the authors' knowledge, the 5-gene rule (OncoDefender-CRC) is the first molecular prognostic that has been validated in both stage I CRC and stage II colon cancer. It outperforms standard clinicopathologic prognostic criteria and obviates the need to retrieve ≥12 lymph nodes for accurate prognostication. It identifies those patients most likely to develop recurrent disease within 3 years after curative surgery and, thus, those most likely to benefit from adjuvant treatment. Copyright © 2012 American Cancer Society.60,0(South San Francisco, Calif), Mayo Clinic (Rochester, Minn; Jacksonville, Fla; and Scottsdale, Ariz), Folio Biosciences (Columbus, Ohio), Trans‐Hit Biomarkers, Inc. (Montreal, Quebec, Canada), BioServe (Laurel, Md), and Amp‐Tec (Hamburg, Germany)https://onlinelibrary.wiley.com/doi/abs/10.1002/cncr.27628"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2019McCoy, CR;Glover, ME;Flynn, LT;Simmons, RK;Cohen, JL;Ptacek, T;Lefkowitz, EJ;Jackson, NL;Akil, H;Wu, X;Clinton, SM;Altered DNA Methylation in the Developing Brains of Rats Genetically Prone to High versus Low AnxietyJ. Neurosci.3144-3158391630683683There is growing evidence of abnormal epigenetic processes playing a role in the neurobiology of psychiatric disorders, although the precise nature of these anomalies remains largely unknown. To study neurobiological (including epigenetic) factors that influence emotionality, we use rats bred for distinct behavioral responses to novelty. Rats bred for low novelty response (low responder [LR]) exhibit high levels of anxiety- and depressive-like behavior compared with high novelty responder (HR) rats. Prior work revealed distinct limbic brain development in HR versus LR rats, including altered expression of genes involved in DNA methylation. This led us to hypothesize that DNA methylation differences in the developing brain drive the disparate HR/LR neurobehavioral phenotypes. Here we report altered DNA methylation markers (altered DNA methyltransferase protein levels and increased global DNA methylation levels) in the early postnatal amygdala of LR versus HR male rats. Next-generation sequencing methylome profiling identified numerous differentially methylated regions across the genome in the early postnatal HR/LR amygdala. We also contrasted methylation profiles of male HRs and LRs with a control rat strain that displays an intermediate behavioral phenotype relative to the HR/LR extremes; this revealed that the LR amygdalar methylome was abnormal, with the HR profile more closely resembling that of the control group. Finally, through two methylation manipulations in early life, we found that decreasing DNA methylation in the developing male and female amygdala improves adult anxiety- and depression-like behavior. These findings suggest that inborn DNA methylation differences play important roles in shaping brain development and lifelong emotional behavior.SIGNIFICANCE STATEMENT Epigenetic changes are biological mechanisms that regulate the expression and function of genes throughout the brain and body. DNA methylation, one type of epigenetic mechanism, is known to be altered in brains of psychiatric patients, which suggests a role for DNA methylation in the pathogenesis of psychiatric disorders, such as depression and anxiety. The present study examines brains of rats that display high versus low levels of anxiety- and depression-like behavior to investigate how neural DNA methylation levels differ in these animals and how such differences shape their emotional behavioral differences. Studying how epigenetic processes affect emotional behavior may improve our understanding of the neurobiology of psychiatric disorders and lead to improved treatments. Copyright © 2019 the authors.60,0Starting material for the enrichment protocol was 1g of sonicated DNA, and captured fragments were eluted using 2500 mM NaCl per the manufacturer’s instructions and as described previously (Brinkman et al., 2010). Captured material was purified using QIAquick PCR purification spin columns (QIAGEN) and quantified using Quant-it high sensitivity DNA Assay Kit (Invitrogen, Q-33120) and a 2100 Bioanalyzer high sensitivity chip kit (Agilent Technologies). Samples were sent to HudsonAlpha Genomic Services Laboratory (Huntsville, AL; http://gsl.hudsonalpha.org) for next-generation sequencing. Barcoded DNA fragment libraries were created, checked for quality, and quantified with the Kapa Library Quant Kithttp://www.jneurosci.org/content/39/16/3144.abstract"HudsonAlpha Genomic Services"
2010Yelamanchili, SV;Chaudhuri, AD;Chen, LN;Xiong, H;Fox, HS;MicroRNA-21 dysregulates the expression of MEF2C in neurons in monkey and human SIV/HIV neurological diseaseCell Death Dise77121170291MicroRNAs (miRNAs) play important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both HIV associated dementia in humans and its monkey model SIV encephalitis we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. MiR-21 can be induced in neurons by prolonged N-methyl-D-aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease but also as a crucial effector of HIV induced neuronal dysfunction and neurodegeneration.60,0miRNA in situ hybridization. Following necropsy, monkey caudate and hippocampus were formalin fixed, and embedded in paraffin, and 5 μm sections cut onto glass slides. The in situ hybridization was performed at Phylogeny Inc. (Columbus, OH, USA) as described previouslyhttps://www.nature.com/articles/cddis201056"Phylogeny Inc"
2018Buatois, V;Johnson, Z;Salgado-Pires, S;Papaioannou, A;Hatterer, E;Chauchet, X;Richard, F;Barba, L;Daubeuf, B;Cons, L;Broyer, L;D'Asaro, M;Matthes, T;LeGallou, S;Fest, T;Tarte, K;Clarke Hinojosa, RK;Genescà Ferrer, E;Ribera, JM;Dey, A;Bailey, K;Fielding, AK;Eissenberg, L;Ritchey, J;Rettig, M;DiPersio, JF;Kosco-Vilbois, MH;Masternak, K;Fischer, N;Shang, L;Ferlin, WG;Preclinical Development of a Bispecific Antibody that Safely and Effectively Targets CD19 and CD47 for the Treatment of B-Cell Lymphoma and LeukemiaMol. Cancer Ther.1739-175117829743205CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal "don't eat me" signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701-induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20-targeted therapy. Mol Cancer Ther; 17(8); 1739-51. ©2018 AACR. ©2018 American Association for Cancer Research.58,0Cells were cultured at 37°C and 5% CO2. Primary samples were collected from Conversant Biologics (Huntsville, USA), the Leukemia Research Center (Glasgow, UK), the Geneva University Hospital (Geneva, Switzerland) ;... tometry and finally selected as the most silenced stable clone by quantification of CD47 receptors using QIFIKIT® (DAKO). Cells were cultured at 37°C and 5% CO2. Primary samples were collected from Conversant Biologics (Huntsville, USA), the Leukemia Research Center (Glasgow, UK), the Geneva University Hospital (Geneva, Switzerland), the Josep Carreras Leukaemia Research Institute (Barcelona, Sp ... tometry and finally selected as the most silenced stable clone by quantification of CD47 receptors using QIFIKIT® (DAKO). Cells were cultured at 37°C and 5% CO2. Primary samples were collected from Conversant Biologics (Huntsville, USA), the Leukemia Research Center (Glasgow, UK), the Geneva University Hospital (Geneva, Switzerland), the Josep Carreras Leukaemia Research Institute (Barcelona, Sphttp://dx.doi.org/10.1158/1535-7163.MCT-17-1095"Conversant Biologics" OR "Conversant Bio" OR "ConversantBio"
2015Huynh, H;Hao, HX;Chan, SL;Chen, D;Ong, R;Soo, KC;Pochanard, P;Yang, D;Ruddy, D;Liu, M;Derti, A;Balak, MN;Palmer, MR;Wang, Y;Lee, BH;Sellami, D;Zhu, AX;Schlegel, R;Huang, A;Loss of Tuberous Sclerosis Complex 2 (TSC2) Is Frequent in Hepatocellular Carcinoma and Predicts Response to mTORC1 Inhibitor EverolimusMol. Cancer Ther.1224-123514525724664Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide and hyperactivation of mTOR signaling plays a pivotal role in HCC tumorigenesis. Tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2, functions as a negative regulator of mTOR signaling. In the current study, we discovered that TSC2 loss-of-function is common in HCC. TSC2 loss was found in 4 of 8 HCC cell lines and 8 of 28 (28.6%) patient-derived HCC xenografts. TSC2 mutations and deletions are likely to be the underlying cause of TSC2 loss in HCC cell lines, xenografts, and primary tumors for most cases. We further demonstrated that TSC2-null HCC cell lines and xenografts had elevated mTOR signaling and, more importantly, were significantly more sensitive to RAD001/everolimus, an mTORC1 inhibitor. These preclinical findings led to the analysis of TSC2 status in HCC samples collected in the EVOLVE-1 clinical trial of everolimus using an optimized immunohistochemistry assay and identified 15 of 139 (10.8%) samples with low to undetectable levels of TSC2. Although the sample size is too small for formal statistical analysis, TSC2-null/low tumor patients who received everolimus tended to have longer overall survival than those who received placebo. Finally, we performed an epidemiology survey of more than 239 Asian HCC tumors and found the frequency of TSC2 loss to be approximately 20% in Asian HBV(+) HCC. Taken together, our data strongly argue that TSC2 loss is a predictive biomarker for the response to everolimus in HCC patients. ©2015 American Association for Cancer Research.58,0HCC cell lines and patient-derived xenografts were collected, fixed with formalin and processed for paraffin embedding. HCC tissue microarrays were purchased from BioChain (catalog # Z7020056 and Z7020057, unrelated HCC TMAs) and FolioBio (catalog# ARY-HH0225).http://mct.aacrjournals.org/content/14/5/1224.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2014Campbell, VT;Nadesan, P;Ali, SA;Wang, CY;Whetstone, H;Poon, R;Wei, Q;Keilty, J;Proctor, J;Wang, LW;Apte, SS;McGovern, K;Alman, BA;Wunder, JS;Hedgehog pathway inhibition in chondrosarcoma using the smoothened inhibitor IPI-926 directly inhibits sarcoma cell growthMol. Cancer Ther.1259-126913524634412Hedgehog (Hh) pathway inhibition in cancer has been evaluated in both the ligand-independent and ligand-dependent settings, where Hh signaling occurs either directly within the cancer cells or within the nonmalignant cells of the tumor microenvironment. Chondrosarcoma is a malignant tumor of cartilage in which there is ligand-dependent activation of Hh signaling. IPI-926 is a potent, orally delivered small molecule that inhibits Hh pathway signaling by binding to Smoothened (SMO). Here, the impact of Hh pathway inhibition on primary chondrosarcoma xenografts was assessed. Mice bearing primary human chondrosarcoma xenografts were treated with IPI-926. The expression levels of known Hh pathway genes, in both the tumor and stroma, and endpoint tumor volumes were measured. Gene expression profiling of tumors from IPI-926-treated mice was conducted to identify potential novel Hh target genes. Hh target genes were studied to determine their contribution to the chondrosarcoma neoplastic phenotype. IPI-926 administration results in downmodulation of the Hh pathway in primary chondrosarcoma xenografts, as demonstrated by evaluation of the Hh target genes GLI1 and PTCH1, as well as inhibition of tumor growth. Chondrosarcomas exhibited autocrine and paracrine Hh signaling, and both were affected by IPI-926. Decreased tumor growth is accompanied by histopathologic changes, including calcification and loss of tumor cells. Gene profiling studies identified genes differentially expressed in chondrosarcomas following IPI-926 treatment, one of which, ADAMTSL1, regulates chondrosarcoma cell proliferation. These studies provide further insight into the role of the Hh pathway in chondrosarcoma and provide a scientific rationale for targeting the Hh pathway in chondrosarcoma.58,0Formalin-fixed, paraffin-embedded human chondrosarcoma tissue microarrays (TMA) were purchased from Folio Biosciences for cilia staining. Before staining, all slides were placed in a 60 C oven and baked for 1 hour. Sections were deparaffinized in xylene (3 5 minutes) and rehydrated through a graded series of ethanols for 3 minutes each. Heat-induced epitope retrieval was carried out in the DIVA Decloaker solution using the Decloaking Chamber from Biocare Medicalhttp://mct.aacrjournals.org/content/13/5/1259.short"Folio Biosciences" OR "Folio Bioscience" OR "FolioBio" OR "Folio Bio"
2015Mezache, L;Paniccia, B;Nyinawabera, A;Nuovo, GJ;Enhanced expression of PD L1 in cervical intraepithelial neoplasia and cervical cancersMod. Pathol.1594-1602281226403783Programmed death ligand 1 (PD L1) expression can reduce the immune response in both infectious diseases and cancers. We thus examined PD L1 expression in cervical intraepithelial neoplasias (CINs) and cancers since they each reflect infection by human papillomavirus (HPV). PD L1 protein was not evident by immunohistochemistry in histologically normal cervical epithelia (0/55) even when adjacent to CIN or cancer. PD L1 expression was much increased in CINs (20/21=95%) and cervical squamous cell cancer (56/70=80%) and localized to the dysplastic/neoplastic squamous cells and mononuclear cells, respectively. There was also a significant increase (each P<0.001) in PD L1 detection in mononuclear cells when comparing cervical squamous cell cancers to endometrial (22/115=19%) and ovarian adenocarcinomas (5/40=13%). Co-expression