PLoS ONE

PLoS One. 12(10): e0186043

Prevalence of deleterious germline variants in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1)

http://orcid.org/0000-0002-9663-5005Philipp Harter, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – original draft¹*, Jan Hauke, Data curation, Formal analysis, Writing – review & editing²³, Florian Heitz, Data curation, Investigation, Writing – review & editing¹, Alexander Reuss, Conceptualization, Data curation, Formal analysis, Software, Writing – review & editing⁴, Stefan Kommoss, Data curation, Investigation, Writing – review & editing⁵, Frederik Marmé, Data curation, Investigation, Writing – review & editing⁶, André Heimbach, Data curation, Investigation, Writing – review & editing⁷, Katharina Prieske, Data curation, Investigation, Writing – review & editing⁸, Lisa Richters, Data curation, Investigation, Writing – review & editing²³, Alexander Burges, Data curation, Investigation, Writing – review & editing⁹, Guido Neidhardt, Data curation, Investigation, Writing – review & editing²³, Nikolaus de Gregorio, Data curation, Investigation, Writing – review & editing¹⁰, Ahmed El-Balat, Data curation, Investigation, Writing – review & editing¹¹, Felix Hilpert, Data curation, Investigation, Writing – review & editing¹²¹³, Werner Meier, Conceptualization, Investigation, Writing – review & editing¹⁴, Rainer Kimmig, Data curation, Formal analysis, Writing – review & editing¹⁵, Karin Kast, Data curation, Investigation, Writing – review & editing¹⁶¹⁷¹⁸, Jalid Sehouli, Data curation, Investigation, Writing – review & editing¹⁹, Klaus Baumann, Conceptualization, Investigation, Writing – review & editing²⁰²¹, Christian Jackisch, Conceptualization, Investigation, Writing – review & editing²², Tjoung-Won Park-Simon, Data curation, Investigation, Writing – review & editing²³, Lars Hanker, Data curation, Investigation, Writing – review & editing²⁴, Sandra Kröber, Data curation, Investigation, Writing – review & editing²³, Jacobus Pfisterer, Data curation, Investigation, Writing – review & editing²⁵, Heidrun Gevensleben, Data curation, Investigation, Writing – review & editing²⁶, Andreas Schnelzer, Data curation, Investigation²⁷, Dimo Dietrich, Data curation, Investigation, Writing – review & editing²⁸, Tanja Neunhöffer, Data curation, Investigation, Writing – review & editing²⁹, Mathias Krockenberger, Data curation, Investigation, Writing – review & editing³⁰, Sara Y. Brucker, Data curation, Investigation, Writing – review & editing⁵, Peter Nürnberg, Data curation, Investigation, Writing – review & editing³¹³²³³, Holger Thiele, Data curation, Investigation, Writing – review & editing³², Janine Altmüller, Data curation, Investigation, Writing – review & editing³¹³², Josefin Lamla, Conceptualization, Methodology, Project administration, Writing – review & editing³⁴, Gabriele Elser, Conceptualization, Project administration, Supervision, Writing – review & editing³⁴, Andreas du Bois, Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing – review & editing¹, Eric Hahnen, Conceptualization, Investigation, Methodology, Writing – review & editing²³, Rita Schmutzler, Conceptualization, Investigation, Methodology, Writing – review & editing²³

Alvaro Galli, Editor

1. Department of Gynecology & Gynecologic Oncology, Kliniken Essen-Mitte, Essen, Germany

2. Center for Hereditary Breast and Ovarian Cancer, University Hospital Cologne, Medical Faculty, Cologne, Germany

3. Center for Integrated Oncology (CIO), University Hospital Cologne, Medical Faculty, Cologne, Germany

4. Coordinating Center for Clinical Trials, Philipps-University of Marburg, Marburg, Germany

5. Department of Women’s Health, Tübingen University Hospital, Tübingen, Germany

6. National Center for Tumor Disease/Department of Gynecology, University of Heidelberg, Heidelberg, Germany

7. Institute of Human Genetics, University of Bonn, Bonn, Germany

8. Department of Gynecology and Gynecologic Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

9. Department of Gynecology, University Hospital Munich-Großhadern. Munich, Germany

10. Department of Gynecology, University of Ulm, Ulm, Germany

11. Department of Gynecology, University of Frankfurt, Frankfurt, Germany

12. Onkologisches Therapiezentrum, Krankenhaus Jerusalem, Hamburg, Germany/ Department of Gynecology, University of Kiel, Kiel, Germany

13. Department of Gynecology, University of Kiel, Kiel, Germany

14. Department of Gynecology, Evangelisches Krankenhaus, Duesseldorf, Germany

15. Department of Gynecology, University of Essen, Essen, Germany

16. Department of Gynecology and Obstetrics, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

17. National Center for Tumor Diseases (NCT), Partner Site Dresden, Dresden, Germany

18. German Cancer Consortium (DKTK), Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany

19. Department of Gynecology and Gynecological Oncology, Charité, Campus Virchow, Berlin, Germany

20. Department of Gynecology and Obstetrics, Klinikum Ludwigshafen, Ludwigshafen, Germany

21. Department of Gynecology, Gynecologic Endocrinology & Oncology, Philipps-University Marburg, Marburg, Germany

22. Department of Gynecology and Obstetrics, Sana Klinikum, Offenbach, Germany

23. Department of Gynecology, Medizinische Hochschule Hannover, Hannover, Germany

24. Department of Gynecology & Obstetrics, University Hospital Schleswig-Holstein, Campus Lübeck, Lübeck, Germany

25. Center of Gynecological Oncology, Kiel, Germany

26. Institute of Pathology, University Hospital Bonn, Bonn, Germany

27. Department of Gynecology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

28. University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

29. Department of Gynecology, Dr. Horst-Schmidt-Kliniken, Wiesbaden, Germany

30. Department of Gynecology, University of Würzburg, Würzburg, Germany

31. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

32. Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany

33. Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

34. AGO Study Group, Wiesbaden, Germany

CNR, ITALY

Competing Interests: The competing interests do not alter our policies on sharing data and materials as described below (#3): Philipp Harter, Consulting or Advisory Role—AstraZeneca; Roche/Genentech; Tesaro; Clovis; Pharmamar, Lilly, Sotio, Research Funding—AstraZeneca (Inst), Travel, Accommodations, Expenses—Medac. Stefan Kommoss, Honoraria: Astra Zeneca, Roche, Consulting or Advisory role: Roche. Alexander Burges, Honoraria – AstraZeneca, Roche, Consulting or Advisory Role – AstraZeneca, Roche, Response to Reviewers. Katharina Prieske, Travel, Research funding- Medac Oncology, Honoraria- Astra Zeneca, Roche. Nikolaus de Gregorio,, Consulting or Advisory Role—Roche Pharma AG, Astra Zeneca, Tesaro, Travel, Accommodations, Expenses—Astra Zeneca, Tesaro. Rainer Kimmig, Honoraria—AstraZeneca; Intuitive Surgical; Prostrakan; RIEMSER, Consulting or Advisory Role—Medtronic; Roche; Teva, Travel, Accommodations, Expenses—Cambridge Medical Robotics. Jalid Sehouli, Honoraria—AstraZeneca; PharmaMar; Roche; Tesaro, Consulting or Advisory Role—AstraZeneca; Clovis Oncology; Novocure; Roche; Tesaro, Research Funding—Amgen (Inst); Bayer (Inst); Lilly (Inst); Novartis (Inst). Ahmed El-Balat, Consulting or Advisory Role—AstraZeneca; Roche; Pharmamar; Olympus, Honoraria—Olympus, Travel, Accommodations, Expenses—Pharmamar; Olympus. Jacobus Pfisterer, Honoraria—Roche Pharma AG, Consulting or Advisory Role—Roche Pharma AG, Research Funding—Roche Pharma AG (Inst); Roche Pharma AG (Inst); Roche, Pharma AG (Inst), Travel, Accommodations, Expenses—Roche Pharma AG. Andreas Schnelzer, Honoraria: TEVA, Consulting or Advisory Role: AstraZeneca, Roche. Dimo Dietrich, Consulting or Advisory Role - Bayer; MDxHealth; r-biopharm, Research Funding—Metanomics (Inst), Patents, Royalties, Other Intellectual Property—Epigenomics AG. Andreas Du Bois, Consulting or Advisory Role—AstraZeneca; Pfizer; Pharmamar; Roche/Genentech, Advaxis, Clovis. Rita Schmutzler, Honoraria—AstraZeneca, Consulting or Advisory Role—AstraZeneca, Research Funding—AstraZeneca (Inst), Patents, Royalties, Other Intellectual Property—University of Cologne. Eric Hahnen, Honoraria—AstraZeneca, Consulting or Advisory Role—AstraZeneca, Research Funding—AstraZeneca (Inst). Jan Hauke, Florian Heitz, Alexander Reuss, Frederik Marme, Andre Heimbach, Lisa Richters, Guido Neidhardt,, Felix Hilpert, Werner Meier, Karin Kast, Klaus Baumann, Christian Jackisch, Tjoung-Won Park-Simon, Lars Hanker, Sandra Kroeber, Heidrun Gevensleben, Tanja Neunhöffer, Peter Nürnberg, Holger Thiele, Janine Altmüller, Mathias Krockenberger, Sara Y Brucker, Josefin Lamla, Gabriele Elser, No Relationships to Disclose.

* E-mail: p.harter@gmx.de

© 2017 Harter et al

DOI: 10.1371/journal.pone.0186043

Published online: 20 October 2017

Abstract

Background

Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated.

Methods

Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history.

Results

In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16–93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients <60 years was 30.2% (33.2%) versus 10.6% (18.9%) in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants of 31.6% (36.0%) versus 11.4% (17.6%) and histologic subtype of high grade serous ovarian cancer versus other showed a prevalence of deleterious variants of 23.2% (29.1%) and 10.2% (14.8%), respectively. Testing only for BRCA1/2 would miss in our series more than 5% of the patients with a deleterious variant in established risk genes.

Conclusions

26.4% of all patients harbor at least one deleterious variant in established risk genes. The threshold of 10% mutation rate which is accepted for reimbursement by health care providers in Germany was observed in all subgroups analyzed and neither age at primary diagnosis nor histo-type or family history sufficiently enough could identify a subgroup not eligible for genetic counselling and testing. Genetic testing should therefore be offered to every patient with invasive epithelial ovarian cancer and limiting testing to BRCA1/2 seems to be not sufficient.

Introduction

Ovarian cancer is the leading cause of death of all gynecologic cancers both in the European Union and the United States [1]. In the US, 22,280 patients were diagnosed in 2016 for ovarian cancer and 14,240 died [2]. Despite improvements in chemotherapy [3,4,5] and surgery [6,7] still most patients relapse and finally die of disease. A decade ago, it was estimated that about 10–15% of all patients have a hereditary risk for ovarian cancer [8,9]. The first identified predisposition genes were BRCA1 and BRCA2 with a lifetime penetrance regarding ovarian cancer of 35–59% (BRCA1) and 11–17% (BRCA2), respectively [10,11,12,13]. Unfortunately, several trials which tried to establish a successful screening for ovarian cancer failed [14]. The development of a serous tubal intraepithelial carcinoma (STIC) as a precursor lesion in the fallopian tube with the potential to metastasize within the peritoneal cavity before a solid tumor in the pelvis can be detected, might be one reason for the trial failures [15,16]. Consequently, the majority of patients is diagnosed at an advanced stage with the above-mentioned mortality. So far, the only effective method to reduce the mortality is risk-reducing surgery including bilateral salpingo-oophorectomy (rrBSO) [17]. As rrBSO cannot be recommend to the general population, identification of population at risk might help to identify those women in whom the benefit of rrBSO may outweight it’s burden. Identifying families with deleterious BRCA1/2 variants might provide this opportunity.

So far, we did not know how many family members must be affected before we can assume an elevated risk for their relatives and especially, if one affected member already qualifies for genetic testing. A further hurdle is the decreasing number of family members in Germany over decades and more recently the increase of so-called blended families. In Germany and in most other countries, genetic testing of patients with ovarian cancer is limited to patients with a family history of breast and/or ovarian cancer. The aim of this study was to investigate the prevalence of genetic risk factors in consecutive patients with ovarian cancer irrespective of family history and histologic subtype treated in centers of the Arbeitsgemeinschaft Gynäkologische Onkologie (AGO) study group. In addition, we wanted to evaluate patient satisfaction with gynecological oncology counseling.

Methods

Patients

The protocol was approved by the ethical committee of the Landesaerztekammer Nordrhein (Nr. 2014340) and registered (NCT02222883); all patients gave written informed consent prior to any study related procedure. Patients aged at least 18 years with primary diagnosis or platinum-sensitive relapse of invasive epithelial ovarian cancer in Germany were tested and counselled in 20 centers of the AGO study group in Germany. Platinum sensitive disease was defined as relapse at least 6 months after prior platinum based chemotherapy. It was allowed to include patients up to 6 months after last platinum dose and patients with more than one prior platinum line. We excluded patients with non-epithelial ovarian malignancy and those with a platinum-free interval of less than 6 months. Demographic data, disease characteristics, family history, and medical history were documented. Further follow up of the patients is planned for 5 years and will be reported later. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history. This means, that also patients with the diagnosis of breast cancer in personal history and now the additional diagnosis of ovarian cancer were classified as having a positive family history.

Gene selection

Germline testing was centrally performed at the Center of Hereditary Breast and Ovarian Cancer at the University of Cologne, Germany. Testing included 25 established and candidate risk genes related to ovarian and/or breast cancer or rare cancer predisposition syndromes (ATM, BARD1, BRCA1, BRCA2, BRIP1, BUB1B, CDH1, CHEK1, CHEK2, FAM175A, FANCM, MLH1, MSH2, MSH6, MRE11A, NBN, PMS2, PTEN, PALB2, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2). Among these 25 genes, 16 were defined as as established cancer “risk genes”, including the Lynch syndrome-associated genes (MLH1, MSH2, MSH6, PMS2), genes causative for rare cancer predisposition syndromes (CDH1, PTEN, STK11, TP53), and genes known to contribute to hereditary breast and/or ovarian cancer risk, namely ATM, BRCA1, BRCA2, CHEK2, NBN, PALB2, RAD51C and RAD51D [18,19,20,21,22]. For the remaining 9 genes, only limited evidence is available so far for BUB1B, BARD1, BRIP1, CHEK1, FANCM, FAM175A, MRE11A, RAD50 and XRCC2 [23,24,25,26,27,28]. BUB1B and CHEK1 were included because deleterious variants in both genes have been described in ovarian cancer patients [29]. We classified the analyzed genes in 3 different categories: BRCA1/BRCA2 (group A), 16 risk genes (group B) and all 25 genes (group C).

Genetic analyses

Genomic DNA was isolated from venous blood samples using standard methods. All samples were screened for gross genomic aberrations in the BRCA1/2 genes by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA® MLPA® probemixes P002 (BRCA1) and P045 (BRCA2) (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturers protocol. Data was analysed using the Coffalyzer.Net v.140429.1057 software (MRC-Holland). All BRCA1/2 deletions/duplications were verified using the SALSA® MLPA® probemixes P087 (BRCA1) or P077 (BRCA2), respectively (MRC-Holland). All samples were subsequently analyzed by next generation sequencing (NGS) covering the entire coding regions and exon-flanking sequences (±15nt) of the 25 above-mentioned genes. For NGS, we employed a customer-tailored SureSelect gene panel (Agilent, Santa Clare, U.S.) using the XT Target Enrichment for Illumina Paired-End Multiplexed Sequencing protocol optimized for 200ng of genomic DNA (Agilent). Sequencing was performed on MiSeq or HiSeq4000 devices (Illumina, San Diego, U.S.). Bioinformatic analyses were carried out using the VARBANK version 2.10 pipeline of the Cologne Center for Genomics. In addition, the obtained NGS data were utilized to identify LGRs (large genomic rearrangements) in non BRCA1/2 genes using an in silico CNV-Tool incorporated in the Sophia DDM® platform (Sophia Genetics). Conspicuous regions indicative for a duplication or deletion were verified using an appropriate SALSA® MLPA® probemix, if available (MRC-Holland) or by aCGH (Array comparative genomic hybridization) using a customized probe set covering the regions of interest (Agilent).

Variant classification

Variant classification was performed in accordance with the regulations of the international ENIGMA consortium (Evidence-based Network for the Interpretation of Germline Mutant Alleles; https://enigmaconsortium.org; version 1.1: 26th of March 2015). All genetic variants were classified using a 5-tier variant classification system as proposed by the International Agency for Research on Cancer (IARC) Unclassified Genetic Variants Working Group, namely, deleterious = class 5, likely deleterious = class 4, variant of uncertain significance (VUS) = class 3, likely benign = class 2, and benign = class 1 [30]. Variants reported to occur in large outbred control reference groups at an allele frequency ≥1% (MAF ≥ 0.01) are generally considered to be benign (class 1). Class 4/5 variants are subsequently denominated as ´deleterious variants´. All deleterious variants were validated by Sanger sequencing.

Counselling

Counselling was performed by gynecologic oncologists according to local standard and we evaluated the patient perspectives and satisfaction regarding testing and counseling by a survey. Patients had a BRCA elucidation before testing by their treating gynecologic oncologist and in case of a positive result a subsequent counselling by a gynecologic oncologist or geneticist depending on the local standard. The patients were asked the following questions after testing:

Finden Sie es gut, dass Ihnen eine Beratung und Testung bzgl. ihres familiären Risikos angeboten wurde? (Did you appreciate that counselling and testing regarding your family risk was offered to you?)

Waren Sie mit der Beratung zufrieden? (Were you satisfied with the counselling?)

Waren Sie mit der Ergebnismitteilung zufrieden? (Were you satisfied with how the result was communicated?)

Statistical methods

The protocol stated a target sample size of 500 patients. No formal power analysis was performed. The primary objectives were to assess the prevalence of deleterious germline variants in the investigated genes. All analyses are merely descriptive; no confirmatory hypothesis testing was done. The prevalence was calculated as number of patients with at least one deleterious variant in the respective genes or gene categories divided by all tested patients. SAS version 9.4 was used for all statistical analyses. Further details are provided in the study protocol (S1 study protocol and S1 Trend Statement Checklist)

Results

In total, 525 patients were registered and blood was sent for testing in 523 cases including 281 patients with primary ovarian cancer and 242 with relapsed platinum-sensitive ovarian cancer between 3/2015 and 7/2015 (Table 1). The majority of the patients were of White/Caucasian/European heritage (96.8%) followed by White/Arabic/North African heritage with 1.3%. Other ethnicities were more infrequent (< 0.5%). The mean age at primary diagnosis of all patients was 58 years, 43% had a positive family history and 77.6% showed a high-grade serous ovarian cancer (Table 1). In total, 146 of 523 patients (27.9%) showed a deleterious variant in at least one of the investigated genes (S1 Table), of which 9 patients carried two deleterious germline variants (S2 Table). In the overall sample, 109 patients carried a deleterious variant in BRCA1, BRCA2, or both genes (Table 2, S1 and S2 Tables). In this subgroub of 109 patients with deleterious BRCA1/2 variants, 6 carried deleterious variants in further genes investigated in this study (Table 2). Deleterious germline variants in BRCA1 were most abundant (81 patients, 15.5%), followed by BRCA2 (29 patients, 5.5%), RAD51C (13 patients, 2.5%) and PALB2 (6 patients, 1.1%). Deleterious variants in all other genes analyzed were identified in less than 1% of the patients each (Table 2). The combined analysis showed the following prevalence of deleterious veriants for the respective categories: BRCA1/2: 20.8% (group A, 109 patients); risk genes: 26.4% (group B, 138 patients); all genes: 27.9% (group C, 146 patients). The number of patients with at least one deleterious vatiant in any gene (group C) was slightly though not statistically significantly higher in the relapsed versus the primary diagnosis cohort (21.4% [76 of 242] versus 24.9% [70 of 281]; two-sided chi-square p = 0.1).

See full table Table 1. Patient characteristics.

See full tableTable 2. Prevalence of deleterious variants in the selected genes.No deleterious variants were observed in BARD1, CDH1, MLH1, PMS2, PTEN, STK11 and TP53.

Furthermore, we analyzed the prevalence of deleterious variants by different subgroups regarding age (< versus ≥ 60 years), family history (positive versus negative) and histologic subtypes (high-grade serous versus others). All analyses in all subgroups showed a prevalence of deleterious BRCA1/2 variants above 10.0%. The highest prevalence of deleterious BRCA1/2 variants was 30.2% in patients below the age of 60 years at primary diagnosis and 31.6% with a positive family history, respectively. Of note, 33/109 patients (30.3%) with deleterious BRCA1/2 variants would have been missed by using the classical criteria family history. This rate increases to missing 51/138 patients (37%) regarding the 16 risk genes as defined in our series. However, the threshold frequency of 10% that qualifies for reimbursement of consultation and testing was observed in every subgroup analyzed and even analysis per decade of patients’ age did not identify any subgroup with a lower rate of deleterious variants—unfortunately, we were not able to analyze patients in their octogenarium who are underrepresented in our trial with 11 patients only.

The prevalence of deleterious variants in the 16 risk genes ranged from 14.8% (patients with a histologic subtype other than HGS, Table 3) to 36.0% (patients with a positive family history, Table 3) and the prevalence of deleterious variants in any of the investigated genes from 16.7 (patients with a histologic subtype other than HGS, Table 3) to 36.4% (patients with a positive family history, Table 3). The number of patients with non high-grade serous histologic subtypes was limited, however, deleterious BRCA1/2 variants were also detected in non high grade serous histo-types. Twenty-three patients had high-grade endometrioid histology. This subtype showed a prevalence of deleterious variants in the BRCA1/2 genes of 13%, 21.7% had a deleterious variant in at least one of the 16 risk genes. Furthermore, we had 18 patients with low-grade serous histology (deleterious variant in BRCA1/2: 5.6%, risk gene: 11.1%, any gene: 16.7%). All other known histologic subtypes included less than 10 patients (Table 4). The rate of deleterious variants in the Lynch-associated genes (MSH2, MSH6, MLH1, PMS2) was 0.6% (3 patients; 2 MSH2-positive patients, 1 with high grade serous and 1 with high-grade endometrial subtype; 1 MSH6-positive patient with high-grade serous subtype).

See full table Table 3. Prevalence of deleterious variants within subgroups.

See full table Table 4. Histologic subtypes and prevalence of deleterious variants in patients with serous, endometrioid, mucinous or clear cell ovarian cancer.

The compliance with the survey was high and the rate of responders for each of the questions was 80.9% each (“Did you appreciate that counselling and testing regarding your family risk was offered to you?” and “Were you satisfied with the counselling?”) and 66.2% (“Were you satisfied with how the result was communicated?”). Question 1 (Did you appreciate that counselling and testing regarding your family risk was offered to you?) was answered with “yes” by 98.4%, question 2 (Were you satisfied with the counselling?) by 93.9% and question 3 (Were you satisfied with how the result was communicated?) by 97.4% of the participating patients. The corresponding rates for “No” were 0.5%, 3.2%, and 1.2%, respectively. The rates of “I don’t know” were 1.2%, 2.9% and 1.2%, respectively.

Discussion

Our data indicate that a genetic background regarding the development of ovarian cancer is present in a higher proportion of patients than anticipated in the past. The strength of our trial is the testing of consecutive unselected patients with ovarian cancer in a multicenter setting. The rate of patients with deleterious variants in BRCA1/2 was about 21% without any meaningful difference between patients with first diagnosis or relapsed disease. This is in line with other recent publications reporting a rate of BRCA1/2 carriers of about 16–19% [23, 31,32]. One of the main questions for the future is therefore, how we could prevent about 1/5 of all ovarian cancer cases. According to the National Institute for Health and Care Excellence (NICE), genetic BRCA1/2 testing is generally recommended when the combined probability to detect a deleterious BRCA1 and BRCA2 variant is ≥ 10% [27]. Thus, age at onset and family history were the main selection criteria to identify suitable index patients for genetic testing to identify families at risk. However, this resulted in multiple different guidelines regarding selection criteria differing between the countries and medical societies [33]. Family history and age at onset are only of limited value to identify patients at genetic risk. This was already reported by other authors [34,35,36,37,38]. Using traditional criteria might miss about one third of all patients with a hereditary risk regarding BRCA1/2. In our cohort, 33/109 patients (30.28%) with deleterious variants in BRCA1/2 would have been missed by using only classical criteria like family history. This rate increases to missing 51/138 patients (37%) regarding the 16 risk genes as defined in our series. Disease modifiers that are independent from BRCA1/2, de novo mutations, or limited family structures may explain the finding that a subgroup of BRCA1/2-positive patients with ovarian cancer did not show a positive family history.

A limitation of our series is the limited number of patients with other histologic subtypes than high-grade serous ovarian cancer. Unfortunately, we have only less than 10 patients with a mucinous or clear cell histologic subtype. Therefore, we are not able to give an appropriate answer regarding the rate of deleterious variants in this subtype. Of note, we reported already the finding of deleterious BRCA1/2 variants in patients with clear cell and mucinous ovarian cancer within an international multicenter first line therapy trial [31]. Whether this finding was by chance or revealed indeed the driver alterations for ovarian cancers remains an open question. In addition, the changed classification of the histologic subtypes in ovarian cancer in 2014 has to be kept in mind as further potential factor. Within a large multicenter study, it was shown that reviewing the pathologic samples by an expert gynecologic pathologist using the old and new classification system results in diagnosing the same histologic subtype again in only 54% [39]. In accordance to another recent presentation, we could also show that gynecologic oncologist led testing shows a high level of satisfaction in patients with ovarian cancer [40].

In conclusion, the limited value of any supporting instruments to identify families at risk of ovarian cancer highlights the necessity to offer germline testing to all patients with ovarian cancer. This is so far the only option we have, to identify families at risk for the usually fatal course of the disease of ovarian cancer. Offering prophylactic surgery in BRCA1/2-positive women is the only effective option to decrease ovarian cancer mortality. Therefore, Scotland established offering genetic testing to all patients with non-mucinous histologies of ovarian cancer already in 2013 [41]. The results of our study were already discussed with German health care providers and the Medizinische Dienst der Krankenkassen (MDK), which led to offering testing to all patients with ovarian cancer up to the age of 80 years who were covered by the Verband der Ersatzkassen in Germany in October 2016. Negotiations with further health care providers are ongoing.

Supplementary Material

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Acknowledgments

We would like to thank Astra Zeneca Germany for the support of the trial, the team of Peter Schantl (Schantl Pharmacy Service, Wiesbaden, Germany) for the monitoring, the team of Carmen Schade-Brittinger (Coordinating Center for Clinical trials, University of Marburg, Marburg, Germany) for the statistical analysis and the team of Gabriele Elser (AGO Study Group, Wiesbaden/Essen, Germany) for data management.

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References

Sant M, Chirlaque Lopez MD, Agresti R, Sanchéz Pérez M, Holleczeck B, Bielska Lasota M, et al Survival of women with cancers of breast and genital organs in Europe 1999–2007: results of the EUROCARE-5 study. Eur J Cancer. 2015 9 6.

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016 7; 66(4):271–8927253694

du Bois A, Lück HJ, Meier W, Adams HP, Möbus V, Costa S, et al A randomized clinical trial of cisplatin/paclitaxel versus carboplatin/paclitaxel as first-line treatment of ovarian cancer. J Natl Cancer Inst. 2003 9 3; 95(17):1320–9. 12953086

Heitz F, Harter P, Barinoff J, Beutel B, Kannisto P, Grabowski JP, et al Bevacizumab in the treatment of ovarian cancer. Adv Ther. 2012 9;29(9):723–35 doi: 10.1007/s12325-012-0041-9 22941523

Hanker LC, Loibl S, Burchardi N, Pfisterer J, Meier W, Pujade-Lauraine E, et al The impact of second to sixth line therapy on survival of relapsed ovarian cancer after primary taxane/platinum-based therapy. Ann Oncol. 2012 10;23(10):2065–12.22589332

Harter P, Muallem ZM, Buhrmann C, Lorenz D, Kaub C, Hils R, et al Impact of a structured quality management program on surgical outcome in primary advanced ovrian cancer. Gynecol Oncol. 2011 6 1;121(3):615–9. doi: 10.1016/j.ygyno.2011.02.014 21414656

Harter P, du Bois A, Hahmann M, Hasenburg A, Burges A, Loibl S, et al Surgery in recurrent ovarian cancer: the Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) DESKTOP OVAR trial. Ann Surg Oncol. 2006 12;13(12):1702–10. doi: 10.1245/s10434-006-9058-0 17009163

Stratton JF, Gayther SA, Russell P, Dearden J, Gore M, Plake P, et al Contribution of BRCA 1 mutations to ovarian cancer. N Engl J Med. 1997 4 17;336(16):1125–30. doi: 10.1056/NEJM199704173361602 9099656

Risch HA, MCLaughlin JR, Cole DE, Rosen B, Bradley L, Kwan E, et al Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer. Am J Hum Genet. 2001 3;68(3):700–10. doi: 10.1086/318787 11179017

Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, et al Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet. 2003 5;72(5):1117–30. doi: 10.1086/375033 12677558

King MC, Marks JH, Mandell JB, New York Breast Cancer Study G: Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 2003, 302(5645):643–646. doi: 10.1126/science.1088759 14576434

Antoniou AC, Cunningham AP, Peto J, Evans DG, Lalloo F, Narod SA, et al: The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions. Br J Cancer 2008, 98(8):1457–1466 doi: 10.1038/sj.bjc.6604305 18349832

Mavaddat N, Peock S, Frost D, Ellis S, Platte R, Fineberg E, et al Cancer risks for BRCA1 and BRCA2 mutation carriers: results from prospective analysis of EMBRACE. J Natl Cancer Inst 2013, 105(11):812–822. doi: 10.1093/jnci/djt095 23628597

Buys SS, Partridge E, Black A, Johnson CC, Lamerato L, Isaacs C, et al Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovaran (PLCO) Cancer Screening Randomized Controlled Trial. JAMA. 2011 6 8;305(22):2295–303. doi: 10.1001/jama.2011.766 21642681

Shih leM, Kurman RJ. Ovarian tumorgenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol. 2004 5;164(5):1511–8. 15111296

Schneider S, Heikaus S, Harter P, Heitz F, Grimm C, Ataseven B, et al Serous Tubal Intraepithelial Cacrinoma Associated With Extraovarian Metastases. Int J Geynecol Cancer. 2017 3;27(3):444–451.

Müller D, Danner M, Rhiem K, Stollenwerk B, Engel C, Rasche L, et al Cost-effectiveness of different strategies to prevent breast and ovarian cancer in German women with a BRCA 1or 2 mutation. Eur J Health Econ. 2017 4 5 doi: 10.1007/s10198-017-0887-5 [Epub ahead of print] 28382503

Easton DF, Pharoah PD, Antoniou AC, Tischkowitz M, Tavtigian SV, Nathanson KL, et al Gene-Panel Sequencing and the Prediction of Breast-Cancer Risk. N Engl J Med 2015; 372(23):2243–57 doi: 10.1056/NEJMsr1501341 26014596

Walsh CS. Two decades beyond BRCA1/2: Homologous recombination, hereditary cancer risk and a target for ovarian cancer therapy. Gynecol Oncol 2015;137(2):343–350. doi: 10.1016/j.ygyno.2015.02.017 25725131

Meindl A, Hellebrand H, Wiek C, Erven V, Wappenschmidt B, Niederacher D et al Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene. Nat Genet 2010;42(5):410–414. doi: 10.1038/ng.569 20400964

Ramus SJ, Song H, Dicks E, Tyrer JP, Rosenthal AN, Intermaggio MP, et al Germline Mutations in the BRIP1, BARD1, PALB2, and NBN Genes in Women With Ovarian Cancer. J Natl Cancer Inst 2015;107(11).

Kiiski JI, Pelttari LM, Khan S, Freysteinsdottir ES, Reynisdottir I, Hart SN, et al Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer. Proc Natl Acad Sci U S A. 10 21 2014;111(42):15172–15177. doi: 10.1073/pnas.1407909111 25288723

Easton DF, Pharoah PD, Antoniou AC, Tischkowitz M, Tavtigian SV, Nathanson KL, et al Gene-Panel Sequencing and the Prediction of Breast-Cancer Risk. N Engl J Med 2015; 372(23):2243–57 doi: 10.1056/NEJMsr1501341 26014596

Walsh CS. Two decades beyond BRCA1/2: Homologous recombination, hereditary cancer risk and a target for ovarian cancer therapy. Gynecol Oncol 2015;137(2):343–350. doi: 10.1016/j.ygyno.2015.02.017 25725131

Meindl A, Hellebrand H, Wiek C, Erven V, Wappenschmidt B, Niederacher D et al Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene. Nat Genet 2010;42(5):410–414. doi: 10.1038/ng.569 20400964

Ramus SJ, Song H, Dicks E, Tyrer JP, Rosenthal AN, Intermaggio MP, et al Germline Mutations in the BRIP1, BARD1, PALB2, and NBN Genes in Women With Ovarian Cancer. J Natl Cancer Inst 2015;107(11).

Kiiski JI, Pelttari LM, Khan S, Freysteinsdottir ES, Reynisdottir I, Hart SN, et al Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer. Proc Natl Acad Sci U S A. 10 21 2014;111(42):15172–15177. doi: 10.1073/pnas.1407909111 25288723

Norquist BM, Harrel MI, Brady MF, Walsh T, Lee MK, Gulsuner S, et al Inherited mutations in woman with ovarian carcinoma. JAMA Oncol 2016;2(4): 482–490 doi: 10.1001/jamaoncol.2015.5495 26720728

Pennington KP, Walsh T, Harrell MI, Lee MK, Pennil CC, Rendi MH, et al Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas. Clin Cancer Res. 2014 2 1;20(3):764–75. doi: 10.1158/1078-0432.CCR-13-2287 24240112

Plon SE, Eccles DM, Easton D, Foulkes WD, Genuardi M, Greenblatt MS, et al Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results. Hum Mutat. 2008 11;29(11):1282–91 doi: 10.1002/humu.20880 18951446

Maistro S, Teixeira N, Encinas G, Katayama ML, Niewiadonski VD, Cabral LG, et al Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil. BMC Cancer. 2016 12 3;16(1):934 doi: 10.1186/s12885-016-2966-x 27914478

Konstantinopoulos PA, Ceccaldi R, Shapiro GI, D'Andrea AD. Homologous Recombination Deficiency: Exploiting the Fundamental Vulnerability of Ovarian Cancer. Cancer Discov. 2015 11;5(11):1137–54 doi: 10.1158/2159-8290.CD-15-0714 26463832

Eccles DM, Balmaña J, Clune J, Ehlken B, Gohlke A, Hirst Cet al Selecting Patients with Ovarian Cancer for Germline BRCA Mutation Testing: Findings from Guidelines and a Systematic Literature Review. Adv Ther. 2016 2;33(2):129–50 doi: 10.1007/s12325-016-0281-1 26809252

Walsh T, Casadei S, Lee MK, Pennil CC, Nord AS, Thornton AM, et al Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci U S A 2011;108:18032–18037 doi: 10.1073/pnas.1115052108 22006311

Soegaard M, Kjaer SK, Cox M, Wozniak E, Høgdall E, Høgdall C, et al BRCA1 and BRCA2 mutation prevalence and clinical characteristics of a population-based series of ovarian cancer cases from Denmark. Clin Cancer Res 2008;14:3761–3767 doi: 10.1158/1078-0432.CCR-07-4806 18559594

Malander S, Ridderheim M, Måsbäck A, Loman N, Kristoffersson U, Olsson H, et al One in 10 ovarian cancer patients carry germ line BRCA1 or BRCA2 mutations: results of a prospective study in Southern Sweden. Eur J Cancer 2004;40:422–428 14746861

Alsop K, Fereday S, Meldrum C, deFazio A, Emmanuel C, George J, et al 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;30:2654–2663 doi: 10.1200/JCO.2011.39.8545 22711857

Harter P, Johnson T, Berton-Rigaud D, Park SY, Friedlander M, Del Campo JM, et al BRCA1/2 mutations associated with progression-free survival in ovarian cancer patients in the AGO-OVAR 16 study. Gynecol Oncol. 2016 3;140(3):443–9. doi: 10.1016/j.ygyno.2015.12.027 26740259

Kommoss S, Gilks CB, du Bois A, Kommoss F. Ovarian carcinoma diagnosis: the clinical impact of 15 years of change. Br J Cancer. 2016 10 11;115(8):993–999. doi: 10.1038/bjc.2016.273 27632374

Scambia G, Chalas E, Huang G, Grana Suarez B, González-Santiago S, Colombo N, et al. A novel oncologist-lead BRCA1/2 germline mutation (gBRCAm) testing and counselling model for patients with ovarian cancer: Interim results from the ENGAGE (NCT02406235) study. Presented at Annual Meeting of the European Society of Medical Oncologist 2016 # LBA34.

Scottish Intercollegiate Guidelines Network (SIGN). Management of epithelial ovarian cancer. Edinburgh: SIGN; 2013 (SIGN publication no. 135). [November 2013]

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Table 1. Patient characteristics.

Variable | All (n = 523)
Age (mean, range) [years] | 58 (16–93)
Positive family history (%) | 225 (43.0)
Histologic subtype (%) |
 High grade serous | 406 (77.6)
 High grade endometrioid | 23 (4.4)
 Low grade serous | 18 (3.4)
 Low grade endometrioid | 7 (1.3)
 Clear cell | 6 (1.1)
 Mucinous | 9 (1.7)
 Others/unspecified | 45 (8.6)
 Missing | 9 (1.7)
Timepoint of inclusion |
 Primary ovarian cancer | 281 (53.7)
 Recurrent ovarian cancer | 242 (46.3)

DOI: 10.1371/journal.pone.0186043.t001

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Table 2. Prevalence of deleterious variants in the selected genes.

No deleterious variants were observed in BARD1, CDH1, MLH1, PMS2, PTEN, STK11 and TP53.

Genes / gene categories | Patients, (% of all; n = 523)
BRCA1/2 (group A) | 109 (20.8)
risk genes (group B) | 138 (26.4)
any gene (group C) | 146 (27.9)
Group A (109 patients) |
BRCA1 only | 78 (15.0)
BRCA1 and NBN | 1 (0.2)
BRCA1 and XRCC2 | 1 (0.2)
BRCA1 and BRCA2 | 1 (0.2)
BRCA2 only | 24 (4.6)
BRCA2 and FANCM | 2 (0.4)
BRCA2 and RAD50 | 1 (0.2)
BRCA2 and BUB1B | 1 (0.2)
Group B (138 patients) |
BRCA1/2 carriers | 109 (20.8)
RAD51C only | 13 (2.5)
PALB2 only | 5 (1.0)
PALB2 and ATM | 1 (0.2)
ATM only | 1 (0.2)
RAD51D only | 3 (0.6)
MSH2 only | 2 (0.4)
CHEK2 and BRIP1 | 1 (0.2)
CHEK2 only | 1 (0.2)
NBN only | 1 (0.2)
MSH6 only | 1 (0.2)
Group C (146 patients) |
risk genes (group B) | 138 (25.8)
FANCM only | 3 (0.6)
MRE11A only | 2 (0.4)
CHEK1 only | 1 (0.2)
FAM175A only | 1 (0.2)
BRIP1 only | 1 (0.2)

DOI: 10.1371/journal.pone.0186043.t002

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Table 3. Prevalence of deleterious variants within subgroups.

Subgroup
n
BRCA1/2 positive
n (%)
risk genes
n (%)
any gene
n (%)

All (%)
523
109 (20.8)
138 (26.4)
146 (27.9)

Age*

 < 60 years
268
81 (30.2)
89 (33.2)
94 (35.1)

 ≥ 60 years
254
27 (10.6)
48 (18.9)
51 (20.1)

Family history

 Positive
225
71 (31.6)
81 (36.0)
82 (36.4)

 Negative
289
33 (11.4)
51 (17.6)
58 (20.1)

Histologic subtype*

 High grade serous (HGS)
406
94 (23.2)
118 (29.1)
123 (30.3)

 other
108
11 (10.2)
16 (14.8)
18 (16.7)

Within subset of HGS

 Primary diagnosis
203
46 (22.7)
54 (26.6)
57 (28.1)

 Relapse
203
48 (23.6)
64 (31.5)
66 (32.5)

DOI: 10.1371/journal.pone.0186043.t003

* Age class was missing for 1 patient; histologic subtype was missing for 9 patients.

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Table 4. Histologic subtypes and prevalence of deleterious variants in patients with serous, endometrioid, mucinous or clear cell ovarian cancer.

Histologic subtype
n
BRCA1/2 positive
n (%)
risk gene positive
n (%)
any gene
n (%)

High grade serous
406
94 (23.2)
118 (29.1)
123 (30.3)

Low grade serous
18
1 (5.6)
2 (11.1)
3 (16.7)

High grade endometrioid
23
3 (13.0)
5 (21.7)
5 (21.7)

Low grade endometrioid
7
1 (14.3)
1 (14.3)
1 (14.3)

Mucinous
9
0
0
1 (11.11)

Clear cell
6
0
0
0

DOI: 10.1371/journal.pone.0186043.t004

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