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• W2808833299 abstract "BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1 promoter hypermethylation and pedigree analysis were also performed. In the training cohort, 45 of 46 germline BRCA1/2 variants were detected (sensitivity, 98%). In the prospective cohort (n = 62), all six germline variants were identified (sensitivity, 100%), together with five somatic BRCA1/2 variants and eight cases with BRCA1 promoter hypermethylation. In four BRCA1/2 variant–negative patients, surveillance or prophylactic management options were offered on the basis of positive family histories. We conclude that BRCA1/2 formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2 variants. When taking family history of BRCA1/2 variant–negative patients into account, tumor BRCA1/2 variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counseling and simultaneously selects patients who benefit most from targeted treatment. BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1 promoter hypermethylation and pedigree analysis were also performed. In the training cohort, 45 of 46 germline BRCA1/2 variants were detected (sensitivity, 98%). In the prospective cohort (n = 62), all six germline variants were identified (sensitivity, 100%), together with five somatic BRCA1/2 variants and eight cases with BRCA1 promoter hypermethylation. In four BRCA1/2 variant–negative patients, surveillance or prophylactic management options were offered on the basis of positive family histories. We conclude that BRCA1/2 formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2 variants. When taking family history of BRCA1/2 variant–negative patients into account, tumor BRCA1/2 variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counseling and simultaneously selects patients who benefit most from targeted treatment. Germline BRCA1/2 pathogenic variants confer elevated lifetime risks for epithelial ovarian cancer (EOC), and especially for high-grade serous ovarian, fallopian tube, and primary peritoneal cancers (HGSCs).1Alsop K. Fereday S. Meldrum C. deFazio A. Emmanuel C. George J. Dobrovic A. Birrer M.J. Webb P.M. Stewart C. Friedlander M. Fox S. Bowtell D. Mitchell G. BRCA mutation frequency and patterns of treatment response in BRCA mutation-positive women with ovarian cancer: a report from the Australian Ovarian Cancer Study Group.J Clin Oncol. 2012; 30: 2654-2663Crossref PubMed Scopus (863) Google Scholar, 2King M.C. Marks J.H. Mandell J.B. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2.Science. 2003; 302: 643-646Crossref PubMed Scopus (1818) Google Scholar, 3Norquist B.M. Harrell M.I. Brady M.F. Walsh T. Lee M.K. Gulsuner S. Bernards S.S. Casadei S. Yi Q. Burger R.A. Chan J.K. Davidson S.A. Mannel R.S. DiSilvestro P.A. Lankes H.A. Ramirez N.C. King M.C. Swisher E.M. Birrer M.J. Inherited mutations in women with ovarian carcinoma.JAMA Oncol. 2016; 2: 482-490Crossref PubMed Scopus (450) Google Scholar Analysis of 489 HGSCs by The Cancer Genome Atlas Research Network demonstrated that germline BRCA1/2 variants, somatic BRCA1/2 variants, and epigenetic silencing of BRCA1 via promoter hypermethylation are frequent events, found in approximately 16%, 7%, and 11% of cases, respectively.4The Cancer Genome Atlas Research NetworkIntegrated genomic analyses of ovarian carcinoma.Nature. 2011; 474: 609-615Crossref PubMed Scopus (5388) Google Scholar Other studies reported comparable rates of BRCA1/2 defects.1Alsop K. Fereday S. Meldrum C. deFazio A. Emmanuel C. George J. Dobrovic A. Birrer M.J. Webb P.M. Stewart C. Friedlander M. Fox S. Bowtell D. Mitchell G. BRCA mutation frequency and patterns of treatment response in BRCA mutation-positive women with ovarian cancer: a report from the Australian Ovarian Cancer Study Group.J Clin Oncol. 2012; 30: 2654-2663Crossref PubMed Scopus (863) Google Scholar, 3Norquist B.M. Harrell M.I. Brady M.F. Walsh T. Lee M.K. Gulsuner S. Bernards S.S. Casadei S. Yi Q. Burger R.A. Chan J.K. Davidson S.A. Mannel R.S. DiSilvestro P.A. Lankes H.A. Ramirez N.C. King M.C. Swisher E.M. Birrer M.J. Inherited mutations in women with ovarian carcinoma.JAMA Oncol. 2016; 2: 482-490Crossref PubMed Scopus (450) Google Scholar, 5Berchuck A. Heron K.A. Carney M.E. Lancaster J.M. Fraser E.G. Vinson V.L. Deffenbaugh A.M. Miron A. Marks J.R. Futreal P.A. Frank T.S. Frequency of germline and somatic BRCA1 mutations in ovarian cancer.Clin Cancer Res. 1998; 4: 2433-2437PubMed Google Scholar, 6Baldwin R.L. Nemeth E. Tran H. Shvartsman H. Cass I. Narod S. Karlan B.Y. BRCA1 promoter region hypermethylation in ovarian carcinoma: a population-based study.Cancer Res. 2000; 60: 5329-5333PubMed Google Scholar, 7Esteller M. Silva J.M. Dominguez G. Bonilla F. Matias-Guiu X. Lerma E. Bussaglia E. Prat J. Harkes I.C. Repasky E.A. Gabrielson E. Schutte M. Baylin S.B. Herman J.G. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.J Natl Cancer Inst. 2000; 92: 564-569Crossref PubMed Scopus (963) Google Scholar, 8Martincorena I. Campbell P.J. Somatic mutation in cancer and normal cells.Science. 2015; 349: 1483-1489Crossref PubMed Scopus (671) Google Scholar The high prevalence of pathogenic germline BRCA1/2 variants in EOC patients led to the generally accepted recommendation that all women diagnosed with EOC should receive genetic counseling and be offered genetic testing, with some slight differences observed between countries.9Lancaster J.M. Powell C.B. Chen L.M. Richardson D.L. Society of Gynecologic Oncology statement on risk assessment for inherited gynecologic cancer predispositions.Gynecol Oncol. 2015; 136: 3-7Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 10Vergote I. Banerjee S. Gerdes A.M. van Asperen C. Marth C. Vaz F. Ray-Coquard I. Stoppa-Lyonnet D. Gonzalez-Martin A. Sehouli J. Colombo N. Current perspectives on recommendations for BRCA genetic testing in ovarian cancer patients.Eur J Cancer. 2016; 69: 127-134Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar In the Netherlands, BRCA1/2 variant screening is recommended for every EOC patient, irrespective of family history, age, and histologic subtype.10Vergote I. Banerjee S. Gerdes A.M. van Asperen C. Marth C. Vaz F. Ray-Coquard I. Stoppa-Lyonnet D. Gonzalez-Martin A. Sehouli J. Colombo N. Current perspectives on recommendations for BRCA genetic testing in ovarian cancer patients.Eur J Cancer. 2016; 69: 127-134Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar BRCA1 and BRCA2 have multiple roles in maintaining genome integrity and are crucial for high-fidelity repair of DNA double-strand breaks via homologous recombination–mediated repair.11Lord C.J. Ashworth A. BRCAness revisited.Nat Rev Cancer. 2016; 16: 110-120Crossref PubMed Scopus (766) Google Scholar, 12Roy R. Chun J. Powell S.N. BRCA1 and BRCA2: different roles in a common pathway of genome protection.Nat Rev Cancer. 2012; 12: 68-78Crossref Scopus (918) Google Scholar BRCA1/2-deficient tumors show specific genomic aberrations associated with this homologous recombination repair deficiency.13Marquard A.M. Eklund A.C. Joshi T. Krzystanek M. Favero F. Wang Z.C. Richardson A.L. Silver D.P. Szallasi Z. Birkbak N.J. Pan-cancer analysis of genomic scar signatures associated with homologous recombination deficiency suggests novel indications for existing cancer drugs.Biomark Res. 2015; 3: 9Crossref PubMed Google Scholar, 14Davies H. Glodzik D. Morganella S. Yates L.R. Staaf J. Zou X. Ramakrishna M. Martin S. Boyault S. Sieuwerts A.M. Simpson P.T. King T.A. Raine K. Eyfjord J.E. Kong G. Borg A. Birney E. Stunnenberg H.G. van de Vijver M.J. Borresen-Dale A.L. Martens J.W. Span P.N. Lakhani S.R. Vincent-Salomon A. Sotiriou C. Tutt A. Thompson A.M. Van Laere S. Richardson A.L. Viari A. Campbell P.J. Stratton M.R. Nik-Zainal S. HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.Nat Med. 2017; 23: 517-525Crossref PubMed Scopus (514) Google Scholar, 15Naipal K.A. Verkaik N.S. Ameziane N. van Deurzen C.H. Ter Brugge P. Meijers M. Sieuwerts A.M. Martens J.W. O'Connor M.J. Vrieling H. Hoeijmakers J.H. Jonkers J. Kanaar R. de Winter J.P. Vreeswijk M.P. Jager A. van Gent D.C. Functional ex vivo assay to select homologous recombination-deficient breast tumors for PARP inhibitor treatment.Clin Cancer Res. 2014; 20: 4816-4826Crossref PubMed Scopus (114) Google Scholar The platinum sensitivity frequently observed in HGSC is thought to be related to the underlying homologous recombination repair deficiency, because homologous recombination repair is involved in the repair of DNA damage induced by these agents.13Marquard A.M. Eklund A.C. Joshi T. Krzystanek M. Favero F. Wang Z.C. Richardson A.L. Silver D.P. Szallasi Z. Birkbak N.J. Pan-cancer analysis of genomic scar signatures associated with homologous recombination deficiency suggests novel indications for existing cancer drugs.Biomark Res. 2015; 3: 9Crossref PubMed Google Scholar, 16Konstantinopoulos P.A. Ceccaldi R. Shapiro G.I. D'Andrea A.D. Homologous recombination deficiency: exploiting the fundamental vulnerability of ovarian cancer.Cancer Discov. 2015; 5: 1137-1154Crossref PubMed Scopus (493) Google Scholar, 17Kelland L. The resurgence of platinum-based cancer chemotherapy.Nat Rev Cancer. 2007; 7: 573-584Crossref PubMed Scopus (3648) Google Scholar Another group of drugs that exploit the presence of homologous recombination repair deficiency in tumor cells are the poly (ADP-ribose) polymerase (PARP) inhibitors. By increasing the burden on homologous recombination repair, these drugs induce synthetic lethality in tumor cells with acquired homologous recombination repair deficiency.11Lord C.J. Ashworth A. BRCAness revisited.Nat Rev Cancer. 2016; 16: 110-120Crossref PubMed Scopus (766) Google Scholar, 18O'Sullivan Coyne G. Chen A.P. Meehan R. Doroshow J.H. PARP inhibitors in reproductive system cancers: current use and developments.Drugs. 2017; 77: 113-130Crossref PubMed Scopus (39) Google Scholar Multiple studies have shown that PARP inhibitors improve progression-free survival (PFS) in platinum-sensitive recurrent EOC.19Ledermann J. Harter P. Gourley C. Friedlander M. Vergote I. Rustin G. Scott C.L. Meier W. Shapira-Frommer R. Safra T. Matei D. Fielding A. Spencer S. Dougherty B. Orr M. Hodgson D. Barrett J.C. Matulonis U. Olaparib maintenance therapy in patients with platinum-sensitive relapsed serous ovarian cancer: a preplanned retrospective analysis of outcomes by BRCA status in a randomised phase 2 trial.Lancet Oncol. 2014; 15: 852-861Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 20Mirza M.R. Monk B.J. Herrstedt J. Oza A.M. Mahner S. Redondo A. Fabbro M. Ledermann J.A. Lorusso D. Vergote I. Ben-Baruch N.E. Marth C. Madry R. Christensen R.D. Berek J.S. Dorum A. Tinker A.V. du Bois A. Gonzalez-Martin A. Follana P. Benigno B. Rosenberg P. Gilbert L. Rimel B.J. Buscema J. Balser J.P. Agarwal S. Matulonis U.A. Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer.N Engl J Med. 2016; 375: 2154-2164Crossref PubMed Scopus (1478) Google Scholar, 21Pujade-Lauraine E. Ledermann J.A. Selle F. Gebski V. Penson R.T. Oza A.M. Korach J. Huzarski T. Poveda A. Pignata S. Friedlander M. Colombo N. Harter P. Fujiwara K. Ray-Coquard I. Banerjee S. Liu J. Lowe E.S. Bloomfield R. Pautier P. Olaparib tablets as maintenance therapy in patients with platinum-sensitive, relapsed ovarian cancer and a BRCA1/2 mutation (SOLO2/ENGOT-Ov21): a double-blind, randomised, placebo-controlled, phase 3 trial.Lancet Oncol. 2017; 18: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 22Ledermann J.A. Harter P. Gourley C. Friedlander M. Vergote I. Rustin G. Scott C. Meier W. Shapira-Frommer R. Safra T. Matei D. Fielding A. Spencer S. Rowe P. Lowe E. Hodgson D. Sovak M.A. Matulonis U. Overall survival in patients with platinum-sensitive recurrent serous ovarian cancer receiving olaparib maintenance monotherapy: an updated analysis from a randomised, placebo-controlled, double-blind, phase 2 trial.Lancet Oncol. 2016; 17: 1579-1589Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 23Swisher E.M. Lin K.K. Oza A.M. Scott C.L. Giordano H. Sun J. Konecny G.E. Coleman R.L. Tinker A.V. O'Malley D.M. Kristeleit R.S. Ma L. Bell-McGuinn K.M. Brenton J.D. Cragun J.M. Oaknin A. Ray-Coquard I. Harrell M.I. Mann E. Kaufmann S.H. Floquet A. Leary A. Harding T.C. Goble S. Maloney L. Isaacson J. Allen A.R. Rolfe L. Yelensky R. Raponi M. McNeish I.A. Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 part 1): an international, multicentre, open-label, phase 2 trial.Lancet Oncol. 2017; 18: 75-87Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar Although recent studies also reported a significantly longer PFS of patients with relapsed platinum-sensitive BRCA1/2 wild-type HGSC receiving niraparib20Mirza M.R. Monk B.J. Herrstedt J. Oza A.M. Mahner S. Redondo A. Fabbro M. Ledermann J.A. Lorusso D. Vergote I. Ben-Baruch N.E. Marth C. Madry R. Christensen R.D. Berek J.S. Dorum A. Tinker A.V. du Bois A. Gonzalez-Martin A. Follana P. Benigno B. Rosenberg P. Gilbert L. Rimel B.J. Buscema J. Balser J.P. Agarwal S. Matulonis U.A. Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer.N Engl J Med. 2016; 375: 2154-2164Crossref PubMed Scopus (1478) Google Scholar or olaparib19Ledermann J. Harter P. Gourley C. Friedlander M. Vergote I. Rustin G. Scott C.L. Meier W. Shapira-Frommer R. Safra T. Matei D. Fielding A. Spencer S. Dougherty B. Orr M. Hodgson D. Barrett J.C. Matulonis U. Olaparib maintenance therapy in patients with platinum-sensitive relapsed serous ovarian cancer: a preplanned retrospective analysis of outcomes by BRCA status in a randomised phase 2 trial.Lancet Oncol. 2014; 15: 852-861Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar compared with placebo treatment, most of the PFS benefit was observed for patients with pathogenic BRCA1/2 variants. Therefore, identification of patients with either a somatic or a germline BRCA1/2 variant would significantly improve the selection of patients who benefit most from PARP inhibition.19Ledermann J. Harter P. Gourley C. Friedlander M. Vergote I. Rustin G. Scott C.L. Meier W. Shapira-Frommer R. Safra T. Matei D. Fielding A. Spencer S. Dougherty B. Orr M. Hodgson D. Barrett J.C. Matulonis U. Olaparib maintenance therapy in patients with platinum-sensitive relapsed serous ovarian cancer: a preplanned retrospective analysis of outcomes by BRCA status in a randomised phase 2 trial.Lancet Oncol. 2014; 15: 852-861Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 20Mirza M.R. Monk B.J. Herrstedt J. Oza A.M. Mahner S. Redondo A. Fabbro M. Ledermann J.A. Lorusso D. Vergote I. Ben-Baruch N.E. Marth C. Madry R. Christensen R.D. Berek J.S. Dorum A. Tinker A.V. du Bois A. Gonzalez-Martin A. Follana P. Benigno B. Rosenberg P. Gilbert L. Rimel B.J. Buscema J. Balser J.P. Agarwal S. Matulonis U.A. Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer.N Engl J Med. 2016; 375: 2154-2164Crossref PubMed Scopus (1478) Google Scholar, 23Swisher E.M. Lin K.K. Oza A.M. Scott C.L. Giordano H. Sun J. Konecny G.E. Coleman R.L. Tinker A.V. O'Malley D.M. Kristeleit R.S. Ma L. Bell-McGuinn K.M. Brenton J.D. Cragun J.M. Oaknin A. Ray-Coquard I. Harrell M.I. Mann E. Kaufmann S.H. Floquet A. Leary A. Harding T.C. Goble S. Maloney L. Isaacson J. Allen A.R. Rolfe L. Yelensky R. Raponi M. McNeish I.A. Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 part 1): an international, multicentre, open-label, phase 2 trial.Lancet Oncol. 2017; 18: 75-87Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar Although pathogenic germline BRCA1/2 variants are relatively common in EOC patients, most (approximately 85%) do not have a BRCA1/2 variant. Referring all women with EOC for genetic counseling is, therefore, inefficient and causes unnecessary distress. This problem could be overcome by the integration of a reliable tumor screening test in the care pathway of ovarian cancer patients. A test for genetic variants in BRCA1/2 should be capable of detecting both germline and somatic variants using tumor DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue. Initial use of a tumor DNA test, followed by referral of only those patients with a BRCA1/2 variant (somatic or germline) for genetic counseling, would avoid an estimated 80% of referrals. The analysis of BRCA1/2 in low-quality, highly fragmented FFPE-derived tumor DNA is technically challenging because BRCA1/2 are both large genes with a wide mutation spectrum.24Wallace A.J. New challenges for BRCA testing: a view from the diagnostic laboratory.Eur J Hum Genet. 2016; 24: S10-S18Crossref PubMed Scopus (63) Google Scholar, 25Endris V. Stenzinger A. Pfarr N. Penzel R. Mobs M. Lenze D. Darb-Esfahani S. Hummel M. Sabine Merkelbach B. Jung A. Lehmann U. Kreipe H. Kirchner T. Buttner R. Jochum W. Hofler G. Dietel M. Weichert W. Schirmacher P. NGS-based BRCA1/2 mutation testing of high-grade serous ovarian cancer tissue: results and conclusions of the first international round robin trial.Virchows Arch. 2016; 468: 697-705Crossref PubMed Scopus (20) Google Scholar, 26Do H. Dobrovic A. 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Mobs M. Lenze D. Darb-Esfahani S. Hummel M. Sabine Merkelbach B. Jung A. Lehmann U. Kreipe H. Kirchner T. Buttner R. Jochum W. Hofler G. Dietel M. Weichert W. Schirmacher P. NGS-based BRCA1/2 mutation testing of high-grade serous ovarian cancer tissue: results and conclusions of the first international round robin trial.Virchows Arch. 2016; 468: 697-705Crossref PubMed Scopus (20) Google Scholar, 29Shin S. Kim Y. Oh S.C. Yu N. Lee S.T. Choi J.R. Lee K.A. Validation and optimization of the Ion Torrent S5 XL sequencer and Oncomine workflow for BRCA1 and BRCA2 genetic testing.Oncotarget. 2017; 8: 34858-34866Crossref PubMed Scopus (25) Google Scholar, 30Loman N.J. Misra R.V. Dallman T.J. Constantinidou C. Gharbia S.E. Wain J. Pallen M.J. Performance comparison of benchtop high-throughput sequencing platforms.Nat Biotechnol. 2012; 30: 434-439Crossref PubMed Scopus (1013) Google Scholar, 31Trujillano D. Weiss M.E. Schneider J. Koster J. Papachristos E.B. Saviouk V. Zakharkina T. Nahavandi N. 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The aim of this study was to investigate the performance of BRCA1/2 variant analysis in DNA isolated from FFPE tumor tissue in comparison with sequencing of leukocyte DNA (currently the gold standard in BRCA1/2 variant screening). On the basis of the results, we recommend integrating tumor screening within the care pathway of ovarian cancer patients. The 50 patients in the retrospective training cohort were collected as follows. First, 67 patients were randomly selected who fulfilled the following selection criteria: previously identified germline BRCA1/2 pathogenic variants at the Laboratory for Diagnostic Genome Analysis of the Leiden University Medical Center and breast or gynecologic malignancy. From this cohort, 33 samples were selected by expert clinical molecular geneticists (J.T.W. and N.v.d.S.) for pathogenic variants that were potentially challenging to detect, including deletions, insertions, and variants in flanking introns and homopolymer regions. An additional 17 cases with pathogenic germline variants were randomly selected (not based on type of variant) to reach a total of 50 cases (Figure 1A). For the prospective clinical implementation of BRCA1/2 screening on ovarian tumor tissue (COBRA) cohort, women were recruited in seven participating hospitals in the southwestern region of the Netherlands from February 2016 to June 2017. Women with (a history of) EOC and not previously screened for germline BRCA1/2 variants were eligible for inclusion. The cohort was enriched for HGSCs. After inclusion, leukocyte DNA was used for routine germline analysis at the Department of Clinical Genetics. Simultaneously, FFPE tumor tissue blocks were collected for parallel tumor BRCA1/2 screening at the Department of Pathology, thus allowing detection of both somatic and germline variants (Figure 1B). The study was approved by the medical ethics committee of the Leiden University Medical Center (reference number P16.009). Sixty-six women gave signed informed consent and were included. Routine germline BRCA1/2 screening and tumor BRCA1/2 screening were requested simultaneously, either directly by the treating physician (gynecologist or medical oncologist) or by the clinical geneticist. Histopathology slides from all cases were revised by an expert gynecopathologist (T.B.) in line with the most recent (2014) World Health Organization classification system. Pedigrees including first-, second-, and third-degree relatives were constructed on the basis of questionnaires. The pedigrees were evaluated by expert clinical geneticists (C.J.v.A., M.N.) for tumor types and age of onset. All family histories of BRCA1/2-negative cases were classified on the basis of the presence or absence of an indication for extra surveillance or management options for first-degree relatives, according to current national guidelines. Tumor DNA was isolated from FFPE blocks from routine diagnostics. In most cases, the tumor tissue underwent at least overnight fixation in formalin. For isolation, either three 0.6-mm tissue cores or the microdissected tumor areas from five 10-μm tissue sections were used. For the purposes of optimization, DNA from paired normal FFPE tissue was isolated and analyzed for a subset of cases in both the training cohort and the COBRA cohort. The mean tumor percentage was 61% (range, 30% to 90%) for the training cohort and 65% (range, 10% to 95%) for the COBRA cohort. For NGS and methylation-specific multiplex ligation-dependent probe amplification (MLPA), DNA was isolated using the automated Tissue Preparation System (Siemens Healthcare Diagnostics, Erlangen, Germany), as described previously.35van Eijk R. Stevens L. Morreau H. van Wezel T. Assessment of a fully automated high-throughput DNA extraction method from formalin-fixed, paraffin-embedded tissue for KRAS, and BRAF somatic mutation analysis.Exp Mol Pathol. 2013; 94: 121-125Crossref PubMed Scopus (58) Google Scholar For copy number variant (CNV) MLPA, crude DNA was manually isolated using overnight proteinase K digestion. FFPE tissue cores did not undergo deparaffinization. For microdissected samples, deparaffinization in xylene was performed, followed by rehydration through a graded ethanol series and staining with hematoxylin. Also, 20 μL of 20% chelex was added during overnight proteinase K digestion. After overnight incubation in a heat block at 56°C, samples were heated for 10 minutes at 99°C and centrifuged at 13,000 × g at 4°C, after which the chelex was removed from the microdissected samples. DNA was quantified using the Qubit dsDNA HS Assay Kit, according to manufacturer's instructions (Qubit 2.0 Fluorometer; Life Technologies, Carlsbad, CA). BRCA1 and BRCA2 AmpliSeq NGS libraries were prepared using the Oncomine BRCA Research panel (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer's protocol. The panel contains 265 amplicons and covers 100% of the coding sequences of BRCA1 and BRCA2, and it also includes flanking intronic sequences (average, 64 bases in 5′ and 3′ direction). Insert sizes (ie, the amplicon minus the primers) range from 65 to 138 bp. NGS libraries were equimolary pooled to 60 pmol/L, and the final library pool was loaded on an Ion PI Chip (Thermo Fisher Scientific) using an Ion Chef instrument (Thermo Fisher Scientific). Sequencing was performed in an Ion Proton system (Thermo Fisher Scientific). CNV-MLPA was performed using the SALSA MLPA probe mix P002 BRCA1 (MRC-Holland, Amsterdam, the Netherlands) on approximately 37.5 ng of DNA in a 20-μL reaction, according to manufacturer's protocol, with small adaptations. Briefly, the SALSA probe mix and MLPA buffer were added to a solution containing approximately 37.5 ng of DNA, and the mix was denatured for 10 minutes at 95°C, followed by hybridization at 60°C for 16 to 20 hours. Next, for ligation, the Master mix (ligase buffer A, ligase buffer B, and Ligase-65 enzyme) was added at 54°C and samples were heated for 20 minutes at 54°C, followed by 5 minutes at 98°C. The PCR master mix (including SALSA primer mix and SALSA polymerase) was then added, and the following PCR was performed for 35 cycles: 30 seconds at 95°C, 30 seconds at 60°C, and 60 seconds at 72°C, followed by incubation for 20 minutes at 72°C. For the training cohort, CNV-MLPA was only performed for cases in which no variant was identified via NGS data analysis. In the COBRA cohort, CNV-MLPA was performed in all cases for which sufficient tumor tissue was available. Methylation-specific MLPA using the SALSA MLPA ME001 tumor suppressor mix (MRC-Holland) was performed, according to the manufacturer's protocol, with some adaptations. After denaturation of approximately 75 ng of DNA for 5 minutes at 98°C, the SALSA probe mix and MLPA buffer were added and samples were incubated for 1 minute at 98°C, followed by hybridization at 60°C for 16 to 20 hours. Then, ligase buffer A was added at room temperature, and the samples were heated for 2 minutes at 48°C. Samples were then split and ligated for 30 minutes at 48°C (ligase buffer B and Ligase-65 enzyme, with or without the addition of HhaI enzyme), followed by heating for 5 minutes at 98°C. After the master mix was added" @default.
• W2808833299 created "2018-06-29" @default.
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• W2808833299 date "2018-09-01" @default.
• W2808833299 modified "2023-10-16" @default.
• W2808833299 title "Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue" @default.
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• W2808833299 doi "https://doi.org/10.1016/j.jmoldx.2018.05.005" @default.
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