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Supplementary Table 2. Results of the statistical analyses in BRCA mutation carriers.

Supplementary Table 1. The 3,248 SNPs included in the study

Summary results for all SNPs at the 4q24 locus associated with breast cancer risk at P

Background: A recent association study identified a common variant (rs9790517) at 4q24 to be associated with breast cancer risk. Independent association signals and potential functional variants in this locus have not been explored.Methods: We conducted a fine-mapping analysis in 55,540 breast cancer cases and 51,168 controls from the Breast Cancer Association Consortium.Results: Conditional analyses identified two independent association signals among women of European ancestry, represented by rs9790517 [conditional P = 2.51 × 10−4; OR, 1.04; 95% confidence interval (CI), 1.02–1.07] and rs77928427 (P = 1.86 × 10−4; OR, 1.04; 95% CI, 1.02–1.07). Functional annotation using data from the Encyclopedia of DNA Elements (ENCODE) project revealed two putative functional variants, rs62331150 and rs73838678 in linkage disequilibrium (LD) with rs9790517 (r2 ≥ 0.90) residing in the active promoter or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor–binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150 was associated with level of expression of TET2 in breast normal and tumor tissue.Conclusion: Our study identified two independent association signals at 4q24 in relation to breast cancer risk and suggested that observed association in this locus may be mediated through the regulation of TET2.Impact: Fine-mapping study with large sample size warranted for identification of independent loci for breast cancer risk. Cancer Epidemiol Biomarkers Prev; 24(11); 1680–91. ©2015 AACR.

Supplementary Table 3. Results of the statistical analyses of SNPs from project 12 in BRCA-mutation carriers affected or non-affected with breast cancer and according to their estrogen receptor status.

Figure S1: BCRA2 p.Y3035S Pedigrees. Figure S2: Western blot of ectopically expressed full-length wildtype and mutant hBRCA2 protein in VC8 DR-GFP cells subjected to HDR assay. Figure S3: Purification of wildtype and mutant BRCA2 from human cells. Figure S4: Position of BRCA2 DNA binding domain (DBD) variants on a ribbon diagram of the murine DBD crystal structure.

Background:BRCA1 and BRCA2 mutation carriers are at substantially increased risk for developing breast and ovarian cancer. The incomplete penetrance coupled with the variable age at diagnosis in carriers of the same mutation suggests the existence of genetic and nongenetic modifying factors. In this study, we evaluated the putative role of variants in many candidate modifier genes.Methods: Genotyping data from 15,252 BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n = 3,248) located within or around 445 candidate genes, were available through the iCOGS custom-designed array. Breast and ovarian cancer association analysis was performed within a retrospective cohort approach.Results: The observed P values of association ranged between 0.005 and 1.000. None of the variants was significantly associated with breast or ovarian cancer risk in either BRCA1 or BRCA2 mutation carriers, after multiple testing adjustments.Conclusion: There is little evidence that any of the evaluated candidate variants act as modifiers of breast and/or ovarian cancer risk in BRCA1 or BRCA2 mutation carriers.Impact: Genome-wide association studies have been more successful at identifying genetic modifiers of BRCA1/2 penetrance than candidate gene studies. Cancer Epidemiol Biomarkers Prev; 24(1); 308–16. ©2014 AACR.

Table S1: Description of the BCAC studies contributing to COGS. Table S2: Predicted effects of BRCA1 and BRCA2 variants included in the iCOGS array on protein function. Table S3: Frequency of BRCA1 and BRCA2 variants from iCOGS in breast cancer cases and controls. Table S4: Family studies of Y3035S showing scores for each family by constant relative risk and 75% penetrance. Supplementary References.

Breast cancer risks conferred by many germline missense variants in the BRCA1 and BRCA2 genes, often referred to as variants of uncertain significance (VUS), have not been established. In this study, associations between 19 BRCA1 and 33 BRCA2 missense substitution variants and breast cancer risk were investigated through a breast cancer case–control study using genotyping data from 38 studies of predominantly European ancestry (41,890 cases and 41,607 controls) and nine studies of Asian ancestry (6,269 cases and 6,624 controls). The BRCA2 c.9104A>C, p.Tyr3035Ser (OR = 2.52; P = 0.04), and BRCA1 c.5096G>A, p.Arg1699Gln (OR = 4.29; P = 0.009) variant were associated with moderately increased risks of breast cancer among Europeans, whereas BRCA2 c.7522G>A, p.Gly2508Ser (OR = 2.68; P = 0.004), and c.8187G>T, p.Lys2729Asn (OR = 1.4; P = 0.004) were associated with moderate and low risks of breast cancer among Asians. Functional characterization of the BRCA2 variants using four quantitative assays showed reduced BRCA2 activity for p.Tyr3035Ser compared with wild-type. Overall, our results show how BRCA2 missense variants that influence protein function can confer clinically relevant, moderately increased risks of breast cancer, with potential implications for risk management guidelines in women with these specific variants. Cancer Res; 77(11); 2789–99. ©2017 AACR.

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Genetic testing for cancer susceptibility is a cornerstone of precision cancer prevention and care. Major communication hurdles remain for the differently specialized professionals involved in the identification, counselling, and clinical management of at-risk individuals. This may be ascribed to gaps in the genetic/genomic literacy of health care providers and to an ambiguous lexicon used for variant description. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) international consortium endorses controlled terminology and a framework for interpretation and reporting of germline variants in cancer susceptibility genes (PMID: 30962250). However, for most ENIGMA affiliates a language other than English is used for written and verbal communication of genetic test results, potentially confounding local application of the published framework. The ENIGMA Clinical Working Group thus launched a Vocabulary Translation Project (VTP) to translate the ENIGMA recommendations into the various languages spoken by the membership. The VTP involved 65 ENIGMA members from 22 countries organized into 19 language-specific teams, covering Catalan, Chinese, Czech, Danish, Dutch, Finnish, French, Galician, German, Greek, Italian, Japanese, Malay, Norwegian, Polish, Portuguese, Spanish (Castilian), Swedish, and Tagalog. Excerpts from the original publication were selected for translation based on a majority consensus and included a glossary of terms and recommendations for interpreting and reporting germline sequence variants in (breast) cancer susceptibility genes. Using a two-step process, each team conducted the relevant translation followed by independent back-translation to English. The VTP proved useful to reappraise the reference text. It disclosed transnational issues, which prompted revision of the original source to emphasize that risk estimates and actionability were based on breast cancer as an exemplar. It also highlighted country-specific differences with regards to breast cancer risk assessment (e.g. different absolute/relative breast cancer risk cut points) and management. As a secondary outcome, via electronic survey of the participating teams we documented the perceived high value of the translation effort and its expected positive impact on more consistent clinical management of carrier individuals. The identified target audience encompasses medical geneticists, physicians of other specialties participating in multidisciplinary teams, genetic counselors, primary care physicians, as well as non-health care professionals, e.g. journalists and science communicators. The outreach program includes dissemination of the translations via local, regional, and especially national networks and their use for education and training purposes. Because French, Portuguese, and Spanish are widely used as official, co-official, or secondary languages, the reach of the VTP potentially extends to a greater number of countries and territories, mostly in Central and South America, Caribbean, and Africa. By moving a step forward towards terminological coherence across disciplines and borders, we will facilitate more precise delivery and clinical application of genetic test results for breast cancer predisposition. Our translated recommendations will improve interdisciplinary cross-talk and carriers’ awareness of the risks and implications associated with their status, contributing to more informed decision-making. We used breast cancer as a blueprint. Application of the model to other cancer types will require calibration on the cancer-specific absolute and relative risks. Citation Format: Arcangela De Nicolo, Diana M. Eccles, Sarah Louise Ariansen, Michela Biancolella, Miguel de la Hoya, Orland Diez, Hans Ehrencrona, Florentia Fostira, Tiara Hassan, Issei Imoto, Artur Kowalik, Fabienne Lesueur, Arjen R. Mensenkamp, Heli Nevanlinna, Joanne Ngeow, Edenir I. Palmero, Inge Søkilde Pedersen, Frances Que, Jana Soukupová, Yen Tan, Ana Vega, Amanda B. Spurdle, Paolo Radice. Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-02-15.

Background. Carcinoma in situ (CIS) of the breast is a non-obligatory pre-malignant breast lesion and a highly suspected precursor of invasive cancer. Although the selection criteria for referral to genetic testing are well established for patients diagnosed with invasive breast cancer, these are not clear for patients diagnosed with CIS. Our goal is to assess the prevalence of predisposing pathogenic variants in key genes, as well as to describe factors in patients with CIS that might be associated with genetic predisposition. Methods. A total of 267 patients solely diagnosed with CIS (mean age 43.65 years, range 18-74) through years 2010-2022, were referred for genetic testing and were analyzed, following their informed consent, implementing a 42-gene panel. Of these, 81.5%, 12.4% and 5.8% were ductal, lobular and mixed CIS, respectively. Patients having a synchronous invasive breast cancer diagnosis were not included in the study. Of patients with known grade, 52.28%, 28.9% and 18.8% were grade 3, 2 and 1, respectively. Strong family history for breast cancer (>2 close family relatives) was positive in 28% (75/267) of patients, while 67.8% of CIS were hormone receptor positive. Results. A total of 12.7% (34/267) of patients carried pathogenic variants in seven clinically actionable genes, i.e. CHEK2 (10), BRCA2 (9), BRCA1 (5), ATM (5), PALB2 (2), MSH6 (2) and TP53 (1). Mean age at diagnosis of carriers was 42.1 years (range 29-61 years, p=0.26), 60% of patients had a grade 3 CIS diagnosis (OR 1.1, 95% 0.589-2.1, p=0.73), 96% had a hormone positive diagnosis (OR 1.4, 95% 0.78-2.50, p=0.24), while 73.5% (25/34) reported as having at least two close family relatives with breast cancer (OR 2.9, 95% 1.6-5.1, p=0.0001). The vast majority of carriers had a ductal CIS (DCIS) diagnosis, i.e. 91.2% (31/34) although this did not reach statistical significance (OR 1.087, 95% 0.64-1.82, p=0.72), probably due to small numbers. Notably, carriers were more likely to have a diagnosis with comedo characteristics, although this parameter has not been monitored closely for the whole cohort. Conclusion. Herein, an important fraction of patients with breast CIS carried pathogenic variants in clinically actionable genes, with the most frequent being CHEK2, BRCA2, BRCA1 and ATM. Notably, the prevalence is comparable to that of patients with invasive breast cancer. Strong family history for breast cancer was strongly associated with the identification of predisposing variants. Other factors, such as high grade, hormone positivity, age at diagnosis and others might also be associated with predisposition to CIS, but larger, prospective, studies are needed to confirm these. This is one of the few studies evaluating the inherited predisposition associated with both high and moderate penetrant breast cancer genes, highlighting that individuals with CIS diagnosis and strong family history for breast cancer should be offered the option to genetic testing via multigene panel. Citation Format: Florentia Fostira, Dimitrios Nasikas, Paraskevi Apostolou, Vassiliki Dellatola, Anna Fokianou, Panagiota Kontogianni, Romina Alevizou, Sofia Filippidou, Dimitrios Maniatis, Lazaros Papadopoulos, Emmanouil Pavlakis, Panagiota Ntasiou, Sofia Karageorgopoulou, Grigorios Xepapadakis. Prevalence of breast cancer predisposing variants in patients with breast carcinoma in situ [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-02-10.

Background: Individuals carrying pathogenic, cancer-predisposing, variants face an increased lifetime risk for various diagnoses, depending on the gene and maybe, variant. Traditionally, germline genetic testing is ordered for a cancer patient who fulfills certain eligibility criteria; mainly based on early age at diagnosis and family history for cancer. Although the test results provide useful information for the clinical management of the patients themselves, this information is pivotal for the primary disease prevention of their blood relatives. Targeted testing for the causative variant (so-called cascade testing), already identified in an affected individual, provide valuable information, while minimizing testing cost. Asymptomatic carriers who are at elevated risk may be offered risk-reducing measurements or early-stage diagnoses, resulting in improved outcomes or even no cancer diagnosis. Methods: In a single breast oncology clinic, with a turnaround of ~200 new breast cancer patients annually, and throughout years 2014-2019, a total of ninety-five cancer patients fulfilled the NCCN criteria, agreed and consented to genetic testing. Of these, eighty-nine were breast cancer patients, of which two were males, while six were ovarian cancer patients. A comprehensive cancer-gene panel was implemented, which targets a total of 94 genes. Results: Overall, 25.2% (24/95) of the tested patients carried a loss-of-function variant in a breast/ovarian cancer susceptibility gene (BRCA1, BRCA2, PALB2, TP53, ATM, RAD51C and PMS2), while secondary findings were identified in 5.2% (5/95) of the patients. All carriers in the first group were counselled and were informed to notify their blood relatives. Of these, thirty-nine family relatives from fourteen different families (range 1-6 per family), actually pursued cascade testing based on their relatives’ genetic test result, contributing in a rate of 56%. Interestingly, all the aforementioned were first-degree relatives and no second-degree relative came forward. Of these, twenty individuals (51.2%; 20/39) tested positive, five males and fifteen females. Conclusion: Although cascade testing for high penetrant pathogenic variants can provide primary prevention options for asymptomatic carriers, as well as assurance in non-carriers, patients with a positive genetic test result are reluctant to inform their family relatives. The rate of notification is inversely proportional to the degree of relation, with individuals in our study being restricted only to their first degree relatives. Changes in law and health policies will maximize the benefits of targeted testing. Considering the clinical actionability and the potential for preventing tumor diagnoses through cascade testing, raising awareness and educating our patients seems inevitable. Citation Format: Florentia Fostira, Vasiliki Rapti, Paraskevi Apostolou, Angeliki Delimitsou, Myrto Papamentzelopoulou, Drakoulis Yannoukakos, Ioannis Misitzis, Georgios Koumakis, Stamatina Demiri, Irene Konstantopoulou, Dimitrios Tryfonopoulos. A remarkably high yield of identified hereditary breast cancer cases in a single oncology center, followed by inversely proportional rate of cascade testing in family relatives [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-33.

Male breast cancer is a rare cancer entity, representing just 1% of all cancers diagnosed in men. It shares clinical and biological characteristics with post-menopausal female breast cancer, although recent reports showed that they are not identical. Approximately 10% of unselected male breast cancer cases carry BRCA1 & BRCA2 mutations, with the most frequently mutated gene being BRCA2. Multiple-gene sequencing has already entered clinical practice and leads in detection of potential pathogenic variants in other cancer predisposition genes, in as many as 15% female breast cancer patients without BRCA1 & BRCA2 mutations. We evaluated the performance of a customized panel for cancer risk assessment in a representative sample of male breast cancer patients. Ninety eight male individuals diagnosed with breast cancer (mean age 59y, range 38y-81y) and referred for genetic counseling between 2010 and 2015 were invited to donate a research blood sample. Patients were eligible irrespectively of family history and age at diagnosis. Genomic DNAs were used to prepare libraries for the capture of the entire coding regions of twenty six known -or suspected- breast cancer genes. These were: BRCA1, BRCA2, TP53, STK11, CDH1, PTEN, PALB2, ATM, CHEK2, NBN, BRIP1, BARD1, RAD51C, RAD51D, RAD50, BLM, SLX4, MRE11A, FAM175A, MLH1, MSH2, EPCAM, MHS6, PMS2, MUTYH, NF1. Loss-of-function mutations have been found in three genes; BRCA2, BRCA1 and PMS2. Not surprisingly, the most frequently mutated gene was BRCA2 accounting for 5% of the cases, while BRCA1 and PMS2 mutations have been detected in single cases, accounting each for 1%. Interestingly, no mutations were identified in any of other genes that have been reported in female breast-ovarian cancer families (PALB2, TP53, ATM, CDH1, RAD51C, RAD51D, etc). Compared to BRCA1 & BRCA2 testing, gene panel prompted consideration of change in care (early colon cancer screening) in a single family (1%). This is the first evaluation of the performance a multiple-gene panel in male patients with breast cancer. Our findings confirm that genetic counseling and BRCA1 & BRCA2 should be the primary concern for these patients and their families. Mutations in other genes been reported in female breast-ovarian cancer families were not identified in this cohort. If larger studies confirm our results, then multiple-gene sequencing panels may be of limited benefit for male breast cancer patients and their families. Citation Format: Fostira F, Saloustros E, Konstantopoulou I, Tryfonopoulos D, Barbounis V, Mauroudis D, Georgoulias V, Fountzilas G, Yannoukakos D. Multiple-gene panel sequencing prompts no change in care compared to BRCA1 and BRCA2 testing in male breast cancer patients and their families. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-05.

Background: Guidelines recommend germline mutation testing of breast cancer predisposition genes in triple negative (TN) breast cancer cases with a family history of breast or ovarian cancer or when diagnosed under age 60. However, the prevalence of mutations in these genes among TN cases unselected for family history of breast or ovarian cancer is not known. Methods: To assess the frequency of mutations in 16 predisposition genes in TN cases we screened a large cohort of TN patients (n=1824) unselected for family history of breast or ovarian cancer from 12 centers and 824 study matched unaffected controls for mutations using a panel-based sequencing approach. Results: Deleterious mutations were identified in 15% of TN patients: 8.5% had BRCA1, 2.7% had BRCA2, and 3.6% had mutations in 12 other genes. Mutations in non-BRCA1/2 genes encoding proteins implicated in homologous recombination repair of DNA double strand breaks were detected at the same frequency as in breast cancer families. TN cases with mutations had high-grade tumors and were diagnosed at an earlier age than non-mutated cases. However, 10% of TN cases diagnosed at ≥60 years and 5% with no family history of cancer were also found to carry mutations. Inactivating mutations in non-BRCA1/2 predisposition genes were associated with moderate to high risks of TN breast cancer. Conclusions: National Comprehensive Cancer Network (NCCN) guidelines support clinical genetic testing of breast cancer predisposition genes in 95% of TN breast cancer patients carrying mutations in susceptibility genes. In contrast, National Institute of Health and Care Excellence (NICE) guidelines in the U.K. do not support genetic testing of a substantial proportion of TN patients with predisposing alleles. Frequency tables for inherited mutations in known predisposition genes based on age of diagnosis and family history of cancer will allow for selection of TN patients most likely to carry mutations in the predisposition genes. Citation Format: Fergus J Couch, Steven N Hart, Priyanka Sharma, Amanda Ewart Toland, Penelope Miron, Janet E Olson, Andrew Godwin, Vernon S Pankratz, Curtis Olswold, Seth Slettedahl, Lucia Guidugli, Matthias W Beckmann, Brigitte Rack, Arif B Ekici, Irene Konstantopoulou, Florentia Fostira, George Fountzilas, Liisa M Pelttari, Song Yao, Judy Garber, Angela Cox, Hiltrud Brauch, Christine Ambrosone, Heli Nevanlinna, Drakoulis Yannoukakos, Susan L Slager, Celine M Vachon, Diana M Eccles, Peter A Fasching. Triple-negative breast cancer: Frequency of inherited mutations in breast cancer susceptibility genes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-03.

Background – aim: In carriers of BRCA1/2 pathogenic mutations (mut), it is expected that the germline mut is present in all tissues, particularly in normal; the somatic mut status in normal tissues from these patients is usually not addressed. We investigated the mut status in normal and tumor tissues in a real-life cohort of BRCA1/2 carriers who underwent prophylactic surgery. Methods: All 53 women had known BRCA1/2 germline mut that had been assessed independently; 42 had previous cancer manifestation (PCM); all had prophylactic mastectomy; 22 had prophylactic hystero-salpingo-oophorectomy. By using a 60-gene NGS panel, we examined the mut status of 231 samples, 39 peripheral blood and 192 paraffin tissues (FFPE: 46 tumors, out of which 43 breast; 97 normal breast [NB]; 49 normal ovary and salpinx [NGYN]). Germline mut status was interrogated in tissues with the above panel, Sanger sequencing and a multiplex PCR protocol for large exonic deletions, along with extensive FFPE DNA quality control (QC) to exclude false negatives. Results: Eight patients carried germline BRCA2 and 45 BRCA1 mut (29 in the BRCT-domain; 31 substitutions/indels). We identified somatic mut in 85% of the tumors and in 64% of the normal samples; mut were found significantly more often (p=0.003) and in higher numbers (p Conclusions: In BRCA1/2 carriers, somatic mut in BRCA genes and TP53 are present in normal breast and GYN tissues, more frequently in the latter, and seem associated with the mutated gene and with the type of mut in the germline. The mut status of normal breast tissue does not seem to be affected by neoadjuvant chemotherapy for breast cancer. The observed BRCA1 germline mut loss, particularly in normal tissues, may be approached as a negative selection for the inherited mut; similarly to the described germline mut reversion after chemotherapy, tissues may react to deleterious effects of haploinsufficiency, which needs functional validation. Citation Format: Kotoula V, Demiri E, Fostira F, Vrettou E, Papadopoulou K, Tikas I, Papazisis K, Zaramboukas T, Asimaki-Vlachopoulou A, Miliaras S, Fountzilas E, Ananiadis A, Chrisafi S, Poulios C, Natsiopoulos I, Tsiftsoglou A, Fountzilas G. Germline and somatic mutation status in tissues from BRCA1/2 carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-04.

Non-mucinous epithelial ovarian cancer diagnosis is a stand-alone criterion for genetic testing referral, irrespectively of family history or age at diagnosis. Identification of at least BRCA1 & BRCA2 mutations are fundamental for clinical decision making of ovarian cancer (OC) patients. The purpose of our study was to evaluate the prevalence of damaging variants that can lead to defects of DNA repair by homologous recombination (HR) in an unselected cohort of epithelial non-mucinous OC patients. Identification of such variants can indicate ideal candidates for Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor therapies.We therefore comprehensively analyzed genomic DNA from 578 epithelial non-mucinous OC patients for mutations in 94 genes that are involved in DNA repair, implementing a commercially available gene-panel. Additionally, 121 tumors were collected from OC patients that had negative germline testing and were assessed for somatic BRCA1 & BRCA2 mutations. Tumor cell content and necrosis were evaluated prior to tumor DNA extraction and were above 50% and below 20%, respectively.Overall, 25.4% (147/578) of the patients carried germline loss-of-function (LoF) variants, distributed in 18 genes namely, BRCA1, BRCA2, RAD51C, ATM, FANCL, CHEK2, FANCM, BRIP1, TP53, NBN, FANCA, RECQL4, MLH1, MSH2, MSH6, PMS2, ERCC2 and SLX4. After BRCA1 & BRCA2, which accounted for 72.1% of the total LoF variants, RAD51C LoF variants were the most frequent, accounting for 4.8% of the total LoF variants. Interestingly, the vast majority of LoF variants (136/147; 92.5%) involved homologous recombination/Fanconi anemia genes, detected in 23.5% of the total OC patients tested. Notably, 4.5% of the pathogenic variants were detected in genes involved in Mismatch Repair (MMR) pathway. Subsequently, tumor analysis resulted in the identification of damaging BRCA1 & BRCA2 variants in 11.5% of the ovarian tumors tested. Variant allele frequencies varied from 9%-47%. Altogether, approximately one in three of OC patients in our cohort could be good candidates for therapies targeting defective mechanisms of DNA repair, including HR and MMR.Our study highlights the high prevalence of LoF variants in HR genes, when combining germline and tumor testing in unselected, non-mucinous epithelial OC patients. These genetic defects can predominantly lead to HR deficiency, the ultimate biomarker for therapeutic intervention by PARP inhibition leading to tumor-cell death. Citation Format: Florentia Fostira, Despoina Kalfakakou, Myrto S. Papamentzelopoulou, Aggeliki Delimitsou, Paraskevi Apostolou, Andromachi Vagena, Christos Papadimitriou, Gerasimos Aravantinos, Georgios Fountzilas, Drakoulis Yannoukakos, Irene Kostantopoulou. Combination of germline and tumor testing yields a high rate of loss-of-function variants in non-mucinous epithelial ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4170.

Background - aim: Germline mutation carriers in BRCA1 and related genes frequently develop triple-negative breast carcinomas (TNBC) that presumably preserve the inherited mutation. Herein, we examined the clinicopathologic features and prognostic impact of paired germline and tumor genotypes in this disease. Methods: We compared baseline germline and paraffin tumor genotype data (next generation [NGS] and Sanger sequencing) from 194 patients with operable TNBC who were treated with anthracyclines-taxanes based chemotherapy within adjuvant trials by our Group. Because tissue NGS did not target BRCA-related genes, we additionally interrogated the presence of germline mutations in tumor and non-cancerous breast tissues, in carriers with available tissue material (n=33). We validated all NGS germline mutations with Sanger sequencing and ensured matched germline/tumor identity with microsatellite testing. Results: We identified 50 (26%) germline mutation carriers, 39 in BRCA1, 4 in BRCA2 and the rest in RAD51C, BARD1, RAD50, NBN, MRE11A and BRIP1. BRCA1 carriers were younger as compared to non-BRCA1 carriers (median 42y.o. vs. 56y.o, respectively; p=0.020), premenopausal (p=0.014), and had lower nodal status (pA), and 2 BRCA2 mutations. In one such tumor, the germline mutation was replaced by a somatic BRCA1 (p.Q1811X [c.5431C>T]). Tumors with mut-LOH were more often TP53 mutant as compared to those with preserved germline mutations, which had mutations in other genes as well (p=0.034). Overall though, TP53 mutations were not associated with germline status. Germline mutations and tumor TP53 mutations were not individually associated with patient disease-free survival (DFS) but interacted with each other: in non-carriers, TP53 status did not affect outcome; in carriers, those with mutant TP53 had unfavorable DFS compared to those with wild-type TP53 tumors (interaction p, multivariate analysis: 0.053). Conclusions: Germline BRCA-related TNBC may be distinguished into two groups with potentially different biological characteristics, those with preserved and lost inherited mutation. The interaction between germline status and tumor TP53 mutations may have treatment implications and seems worthy pursuing in larger studies. Combined germline/tumor genotype testing may be needed for TNBC patient classification, especially in the context of clinical trials. Citation Format: Vassiliki Kotoula, Florentia Fostira Fostira, Kyriaki Papadopoulou, Paraskevi Apostolou, Eleftheria Tsolaki, Georgios Lazaridis, Kyriaki Manoussou, Flora Zagouri Zagouri, Dimitrios Pectasides, Ioannis Vlachos, Ioannis Tikas, Sotiris Lakis, Irene Konstantopoulou, George Pentheroudakis, Helen Gogas, Pavlos Papakostas, Christos Christodoulou, Drakoulis Yannoukakos, George Fountzilas. The fate of germline BRCA related mutations in breast tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2017-1755

Germ-line mutations in the BRCA1 and BRCA2 genes confer a high lifetime risk of developing breast and other cancers. However, remarkable differences exist regarding disease manifestation in mutation carriers, suggesting the existence of other genetic modifier factors. Given that both BRCA1 and BRCA2 are involved in the repair of double-strand breaks (DSBs) mainly by Homologous Recombination, SNPs in genes involved in DNA repair are good candidates to be tested as phenotypic modifiers. The Base Excision Repair (BER) pathway could be particularly interesting given the relation of synthetic lethality that exists between one of the components of this pathway, PARP1, and both BRCA1 and BRCA2. To test our hypothesis we have performed a comprehensive analysis of the 18 genes involved in BER, in a series of 2000 BRCA1 and BRCA2 mutation carriers from the Spanish Consortium for the Study of Hereditary Breast Cancer, the Istituto Nazionale Tumori and the National Centre for Scientific Research “Demokritos”. For this purpose we have used a tagging SNP approach in which we have evaluated the common genetic variation in the 18 genes using the VeraCode GoldenGate 144 SNP Genotyping Kit. Preliminary results have showed statistically significant associations with cancer risk in BRCA1 and/or BRCA2 mutation carriers for 46 SNPs (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 90. doi:1538-7445.AM2012-90