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HIST1H2BB and MAGI2 Methylation and Somatic Mutations As Precision Medicine Biomarkers for Diagnosis and Prognosis of High-Grade Serous Ovarian Cancer Blanca L

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HIST1H2BB and MAGI2 Methylation and Somatic Mutations As Precision Medicine Biomarkers for Diagnosis and Prognosis of High-Grade Serous Ovarian Cancer Blanca L Author Manuscript Published OnlineFirst on June 24, 2020; DOI: 10.1158/1940-6207.CAPR-19-0412 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. HIST1H2BB and MAGI2 methylation and somatic mutations as precision medicine biomarkers for diagnosis and prognosis of high-grade serous ovarian cancer Blanca L. Valle1, Sebastian Rodriguez-Torres2,3, Elisabetta Kuhn4,5, Teresa Díaz-Montes6, Edgardo Parrilla-Castellar7, Fahcina P. Lawson1, Oluwasina Folawiyo1, Carmen Ili-Gangas8, Priscilla Brebi-Mieville8, James R. Eshleman9, James Herman3, Ie-Ming Shih4, David Sidransky1, Rafael Guerrero-Preston1, 10,11* 1Otolaryngology Department, Head and Neck Cancer Research Division, The Johns Hopkins University, School of Medicine, Baltimore, MD 2Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA 3Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA 4Division of Pathology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico; Department of Biomedical, Surgical, and Dental Sciences, University of Milan, Italy 5Departments of Pathology, Gynecology and Obstetrics, The Johns Hopkins University, School of Medicine, Baltimore, MD 6The Lya Segall Ovarian Cancer Institute, Mercy Medical Center, Baltimore, MD 7Department of Pathology, University of Washington, Seattle, WA 8Laboratory Integrative Biology (LIBi), Center for Excellence in Translational Medicine- Scientific and Technological Bioresources Nucleus (CEMT-BIOREN), Universidad de La Frontera, Temuco, Chile 9Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA 10University of Puerto Rico School of Medicine, Department of Obstetrics and Gynecology, San Juan, Puerto Rico 11LifeGene Biomarks Inc., San Juan, Puerto Rico Conflicts of Interests: There are no conflicts of interests *Corresponding author: Rafael Guerrero-Preston, DrPH, MPH University of Puerto Rico School of Medicine San Juan, PR 00927 [email protected] Keywords: HIST1H2BB, MAGI2, HGSC, ovarian carcinoma methylation and somatic mutation markers, precision medicine 1 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 24, 2020; DOI: 10.1158/1940-6207.CAPR-19-0412 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Molecular alterations that contribute to long-term (LT) and short-term (ST) survival in ovarian High-Grade Serous Carcinoma (HGSC) may be used as precision medicine biomarkers. DNA promoter methylation is an early event in tumorigenesis, which can be detected in blood and urine, making it a feasible companion biomarker to somatic mutations for early detection and targeted treatment workflows. We compared the methylation profile in 12 high-grade serous ovarian cancer (HGSC) tissue samples to 30 fallopian tube epithelium samples, using the Infinium Human Methylation 450K Array. We also used 450K methylation arrays to compare methylation among HGSCs long-term survivors (more than 5 years) and short-term survivors (less than 3 years). We verified the array results using bisulfite sequencing and Methylation Specific PCR (qMSP). in another cohort of HGSC patient samples (n=35). Immunoblot and clonogenic assays after pharmacologic unmasking show that HIST1H2BB and MAGI2 promoter methylation down regulates mRNA expression levels in ovarian cancer cells. We then used qMSP in paired tissue, ascites, plasma/serum, vaginal swabs and urine from a third cohort of HGSC cancer patients (n=85) to test the clinical potential of HIST1H2BB and MAGI2 in precision medicine workflows. We also performed next-generation exome sequencing of 50 frequently mutated in human cancer genes, using the Ion AmpliSeqCancer Hotspot Panel, to show that the somatic mutation profile found in tissue and plasma can be quantified in paired urine samples from HGSC patients. Our results suggest that HIST1H2BB and MAGI2 have growth-suppressing roles and can be used as HGSC precision medicine biomarkers. Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 24, 2020; DOI: 10.1158/1940-6207.CAPR-19-0412 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Ovarian cancer is the fifth cause of cancer deaths among women, and the most lethal gynecological malignancy[1]. Clinical and molecular factors that contribute to long-term (LT) and short-term (ST) survival in ovarian high-grade serous cancer (HGSC) are lacking and only a few molecular alterations of response to therapy have been identified. Somatic mutations are rare in HGSC [2], BRCA1/2 germline mutations, and homologous repair deficiency in HGSC are among the few validated molecular predictors of response to platinum therapy and poly-ADP polymerase (PARP) inhibitors [3-6]. DNA promoter methylation is an early event in tumorigenesis, and can be detected in blood and other body fluids, making it a feasible biomarker for early detection of tumors.[7-9] In addition, DNA methylation has potential as a prognostic biomarker. For instance, the FDA has recently approved EpiColon, a blood-based test for diagnosis of colorectal cancer based on methylation of septin 9.[10] Detection of promoter methylation in tissues and biofluids represents a potential biomarker strategy for ovarian cancer diagnosis and therapeutic management within precision medicine workflows. Until recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells [11]. However, recent studies suggest that many of HGSCs arise from lesions in the fallopian tubes[12-17]. In this study, we sought to identify genes differentially methylated between fallopian tube tissue and HGSC and test whether ovarian cancer associated-DNA methylation and somatic mutations measured in tissue can be reproducibly measured in urine samples. Materials and Methods 3 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 24, 2020; DOI: 10.1158/1940-6207.CAPR-19-0412 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Patient samples The study population consists of samples from three patient cohorts (n=77) and publicly available data from 1,742 patients: a retrospective cohort of HGSC Formalin Fixed and Paraffin Embedded (FFPE) samples selected from Johns Hopkins Pathology Department tumor bank (n=12); a cohort of women who were seen in the Ohio State University School of Medicine Obstetrics and Gynecology Department (n=30); a cohort of HGSC patients who were seen in Mercy Medical Center in Baltimore, Maryland (n=35); and data from the Cancer Genome Atlas Project (TCGA). The inclusion criterion was to have a clinical diagnosis of HGCS (ICD9-CM code 183), all determined by pathologists at two different institutions Hopkins and Mercy Medical Center. The Institutional Review Boards of Ohio State School of Medicine, Mercy Medical Center and Johns Hopkins School of Medicine (NA_00020633) approved the research protocols. Informed written consent was obtained from all patients included in the study. Cancer Genome Atlas Project (TCGA) data TCGA data was downloaded and analyzed for DNA methylation alterations using the minfi package. Somatic mutation and expression data were downloaded from the CBioPortal (http://www.cbioportal.org/). DNA extraction DNA was extracted from frozen normal fallopian tube epithelium, FFPE HGSC tissue samples, as well as from normal and ovarian cancer cell lines. Biofluids DNA was extracted as previously described.[11, 18] The protocol for trans-renal DNA extraction reduces the possibility of contamination from urinary tract DNA, by selecting for small and extra cellular DNA. Samples 4 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 24, 2020; DOI: 10.1158/1940-6207.CAPR-19-0412 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. were digested using 1% SDS and 20 ug/ml proteinase K for 48-72 hours at 48°C, followed by phenol/chloroform extraction and ethanol precipitation. Discovery with Next Generation Sequencing We examined the Ion AmpliSeq™Cancer Hotspot Panel v2 to profile 50 frequently mutated in human cancer genes in FPPE tissue DNA from four patients with short-term survival and four patients with long-term survival, the eight of which looked identical under the microscope in a pathology laboratory. The cancer Hotspot Panel was also examined paired tissue, plasma and urine samples from two HGSC patients. Libraries for the discovery cohort were generated using the Ion AmpliSeq Library kit 2.0 according to the manufacturer’s instructions (Life Technologies, Carlsbad, USA). Included in this panel were primers for 207 amplicons covering 2800 Catalog of Somatic Mutations in Cancer of 50 genes with known cancer associations: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, JAK2, JAK3, IDH2, KDR, KIT, KRAS, MET, MLH1,
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