Hereditary Breast Cancer Genes Research Articles

Clinical testing of BRCA1 and BRCA2: a worldwide snapshot of technological practices

Clinical testing of BRCA1 and BRCA2 began over 20 years ago. With the expiration and overturning of the BRCA patents, limitations on which laboratories could offer commercial testing were lifted. These legal changes occurred approximately the same time as the widespread adoption of massively parallel sequencing (MPS) technologies. Little is known about how these changes impacted laboratory practices for detecting genetic alterations in hereditary breast and ovarian cancer genes. Therefore, we sought to examine current laboratory genetic testing practices for BRCA1/BRCA2. We employed an online survey of 65 questions covering four areas: laboratory characteristics, details on technological methods, variant classification, and client-support information. Eight United States (US) laboratories and 78 non-US laboratories completed the survey. Most laboratories (93%; 80/86) used MPS platforms to identify variants. Laboratories differed widely on: (1) technologies used for large rearrangement detection; (2) criteria for minimum read depths; (3) non-coding regions sequenced; (4) variant classification criteria and approaches; (5) testing volume ranging from 2 to 2.5 × 105 tests annually; and (6) deposition of variants into public databases. These data may be useful for national and international agencies to set recommendations for quality standards for BRCA1/BRCA2 clinical testing. These standards could also be applied to testing of other disease genes.

Abstract PD1-10: Multi-gene panel testing results in patients with multiple breast cancer primaries

Abstract Background: Every woman diagnosed with breast cancer has a risk to develop another breast primary throughout their lifetime. This risk is significantly elevated in individuals with a BRCA1 or BRCA2 mutation. For these individuals, the risk of a second primary breast cancer is 15% within 5 years of their initial cancer and the lifetime risk could be as high as 44%. Currently, the NCCN guidelines recommend testing of the BRCA1 and BRCA2 genes for individuals with multiple breast primaries, if their first diagnosis was under the age of 50. With the advent of Next Generation Sequencing and multiple gene panels, testing for hereditary breast cancer genes outside of BRCA1 and BRCA2 has become more prevalent. Although there are currently no guidelines that dictate when multiple gene panel testing should be ordered, recent studies have shown that there may be some associations between non-BRCA breast cancer genes such as ATM, CDH1, CHEK2, PALB2, PTEN, and TP53, in individuals with multiple primary breast cancers. Our study aimed to look at our institution's cohort of individuals with multiple primary breast cancers that underwent panel testing, to determine the rates of pathogenic mutations in non-BRCA genes. Methods: Sixty-five patients that had multiple breast cancer primaries and underwent a multi-gene breast cancer panel test were identified using MD Anderson's Clinical Cancer Genetics Database. The genetic testing results for each of these patients was reviewed and analyzed. Results: Out of the sixty-five patients found to have multiple breast cancer primaries and had a multiple gene panel test, 23 (29%) tested positive for a pathogenic mutation in a breast cancer predisposition gene: 6 (26%) BRCA1, 3 (13%) BRCA2, 4 (17%) ATM, 3 (13%) CHEK2, 1 PALB2 (4%), 1 PTEN (4%), 1 TP53 (4%). Overall, 15% of this cohort tested positive for a mutation in a non-BRCA breast cancer predisposition gene. The average age of first breast cancer diagnosis in these positive patients was 42 (range 26-75), which was similar to the average age in patients that tested negative or were found to have a variant of uncertain significance (VUS), 42 (range26-65). The average age of the first breast cancer diagnosis in patients positive for non-BRCA breast cancer genes was 46 (range 28-75). Conclusion: In our cohort, 29% of patients with multiple breast primaries who underwent panel testing were found to have a germline mutation in a breast cancer predisposition gene. Interestingly, 15% of our cohort were found to have a mutation in a non-BRCA breast cancer gene. There was no difference between age of onset in those that tested positive versus those that tested negative or were found to have a VUS. The high positive rate for all individuals with multiple breast cancers, regardless of age, for both BRCA1/2 and non-BRCA genes suggests that panel testing for patients with multiple breast cancer primaries should be considered. This finding should be validated in larger cohorts in order to help clarify whether all patients meeting guidelines for BRCA1 and BRCA2 genetic testing due to multiple breast cancer primaries would benefit from a multiple gene panel test. Citation Format: Ross JL, Woodson AH, Gutierrez Barrera AM, Litton JK, Arun B. Multi-gene panel testing results in patients with multiple breast cancer primaries [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 PD1-10.

Abstract A37: Complex landscape of germline variants in hereditary and early-onset breast cancer ascertained through whole exome sequencing

Abstract When considering family history, hereditary breast cancer (HBC) syndromes correspond to nearly 5-10% of all breast cancer (BC) cases. However, pathogenic variants in known moderate- and high-risk BC genes explain only ~30% of familial BC cases. Recent advances in sequencing technology resulted in an increasing number of BC genes being revealed in previously negative families by using extended gene panels and whole-exome sequencing (WES). WES offers the opportunity to concomitantly investigate several rare risk genes as well as to identify new BC-predisposing genes. Thus, in this study we used WES to disclose variants contributing to BC increased risk in patients that were negative for mutations in three major BC genes (BRCA1/2 and TP53) and met stringent clinical criteria indicating a genetic predisposition to BC. We selected 17 women (two of them sisters) who fulfilled one or more of the following criteria: early onset BC (<36 years); bilateral BC; breast and other primary related tumor (ovary, fallopian tube, or primary peritoneal tumors). Firstly, we used WES data to search for rare variants in 27 well-established and emerging HBC predisposing genes. This analysis identified two patients harboring truncating mutations, one in ATM (p.Tyr2334Glnfs*4 – pathogenic in ClinVar database) and one in BARD1 (Tyr739Leufs*2 – not described, probably pathogenic according to ACMG guidelines). Additionally, we identified nine variants of unknown clinical significance in these genes, occurring in seven patients. For the remaining 15 patients with no putative pathogenic mutations in HBC genes, we first applied quality filters to exclude false-positive results (included variants: variant base coverage ≥5X, variant frequency ≥25%, QD>2, FS<6). Remaining variants were excluded if present in population databases with a minor allele frequency (MAF) > 0.01 or present in five BRCA1 mutated patients sequenced in the same platforms. Next, using a functional-based variant prioritization, we selected candidate variants according to the predicted impact in the protein function, the affected gene, and segregation with the phenotype (in the family-based study of the two sisters). Candidate variants included all loss-of-function variants as well as missense and in-frame indels occurring in a specific list of 651 genes of interest (DNA repair and cancer-related genes) and if predicted to be damaging by at least 3/6 prediction software. The resulting 244 selected variants were submitted for technical validation by targeted NGS and, of these, 220 were validated (89%). Using the same custom panel, we evaluated 25 control samples of healthy Brazilian women for filtering common polymorphisms in our population, resulting in a final 139 variants in 129 genes (89 LOF and 50 missense variants). For two families---one with WES of two affected sisters and target NGS of one affected aunt; one with WES of the affected daughter and target NGS of the affected mother---we were able to perform cosegregation analysis in other family members and 11 out of 23 variants were found to be segregating with the disease. These 11 distinct genes as well as 82 genes harboring LOF variants were evaluated in an independent cohort of 34 WES of high-risk BC patients. Several rare, possibly damaging variants were identified in this cohort, providing additional evidence of the potential role in BC predisposition of some new genes. Of those, we highlight ATRX, FANCC, TET2, ERCC1, PTPRD, and ROS1 genes, which are involved in DNA repair and other tumorigenic pathways. Overall, our results provide a set of putative BC-predisposing genes and underpin WES as a useful tool for assessing the complex landscape of HBC predisposition. The discovery and validation of new HBC genes in different populations will continue to provide insights into disease mechanisms, eventually leading to the development of more effective therapies and improved management of affected families. Citation Format: Giovana T. Torrezan, Fernanda GSR Almeida, Marcia CP Figueiredo, Bruna DF Barros, Claudia A. Paula, Renan Valieris, Jorge ES Souza, Elisa N. Ferreira, Felipe CC Silva, Amanda F. Nobrega, Maria Isabel Achatz, Edenir I. Palmero, Rodrigo F. Ramalho, Sandro J. Souza, Dirce M. Carraro. Complex landscape of germline variants in hereditary and early-onset breast cancer ascertained through whole exome sequencing [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A37.

Abstract 1450: Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes

Abstract Genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming increasingly widespread in the era of precision medicine. The implementation of next-generation sequencing (NGS) has resulted in an explosion of genetic data. While the majority of patients receive definitive results, germline unclassified variants with unknown function are regularly detected in thousands of patients. In particular, variants of unknown significance (VUS) in the HBOC susceptibility genes BRCA1 and BRCA2 pose a quandary to medical providers and patients because these genes are clinically actionable. A large percentage of VUS in BRCA1/2 are predicted to affect splicing. Previous efforts have focused on interrogating splicing VUS using low-throughput and/or imprecise techniques. Therefore, we developed and validated a method for using RT-PCR NGS to accurately and efficiently characterize germline splicing defects. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium recommends capillary electrophoresis and Sanger sequencing of subcloned transcripts as the gold standard to identify the BRCA1/2 transcripts that are present in patient samples. We followed the recommendations of the ENIGMA consortium and were able to largely replicate their results when we analyzed the cDNA of lymphoblastoid cell lines (LCLs) generated by the kConFab consortium from carriers of BRCA1 or BRCA2 variants known to be associated with splicing defects. In addition to LCLs, we analyzed RNA extracted from normal blood controls and normal breast tissue for the entire BRCA1 gene. We compared our capillary electrophoresis and Sanger sequencing results to our newly developed RT-PCR NGS assay. For this assay, we used RT-PCR NGS coupled to our custom in-house bioinformatics analysis to identify splicing events including exon skipping, novel intron insertion, and alternative 5’ donor and 3’ acceptor splicing sites. Capillary electrophoresis allowed us to roughly visualize the various transcripts present in our samples, though sequencing was needed to confidently identify the exact splicing event. Similar to Sanger sequencing, our RT-PCR NGS assay detected all major splicing events. Interestingly, we found that RT-PCR NGS allowed us to visualize more minor splicing events present in the samples due to our high coverage. We sequenced tens of thousands of reads with RT-PCR NGS, as opposed to Sanger sequencing, which was limited to low-throughput sequencing of single colonies containing subcloned RT-PCR products. In conclusion, RT-PCR NGS is a reliable high-throughput alternative to the gold-standard splicing assays. Our assay will greatly improve the interpretation of splicing VUS detected by clinical genetic tests for BRCA1/2. Citation Format: Suzette Farber-Katz, Vickie Hsuan, Jayne Hoo, Sitao Wu, Huy Vuong, Dong Xu, Hsiao-Mei Lu, Phillip Gray, Aaron Elliott, Rachid Karam. Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes [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 1450. doi:10.1158/1538-7445.AM2017-1450

Breast cancer phenotype in patients with hereditary gene mutations other than BRCA1 and BRCA2.

e13121 Background: Use of multi-gene panel (MGP) testing for hereditary breast cancer (BC) has increased significantly. Unlike in case of TNBC diagnosed before age 60 and ordering BRCA testing; for MGP testing, healthcare providers depend on personal and/or family history of cancer as tumor phenotype and on patient characteristics for non-BRCA hereditary BC genes which are not well described. Therefore, we planned to evaluate tumor phenotype of BCs associated with hereditary gene mutations other than BRCA 1 and BRCA2. Methods: Consecutive patients with invasive BC who are in a prospective cohort study and who underwent MGP testing based on published criteria as clinically indicated were included in the study. All patients’ breast pathology slides were reviewed by breast pathologists at our institution. For statistical analysis: Patients’ demographic and clinical characteristics were summarized using descriptive statistics such as frequency distribution, mean (± s.d.) and median (range). SAS version 9.4 and S-Plus version 8.04 were used to carry out the computations for all analyses. Institutional IRB approval was obtained. Results: Between 2013 and 2017, 1607 patients underwent MGP testing and 347 were found to be positive for a pathogenic variant. For the study purposes we included only patients with invasive breast cancer (N = 146). Median age was 45.1 yrs (Range: 25-78). Genetics testing results were as follows: BRCA1: 38 (26%), BRCA2: 37 (25%), CHEK2 15 (10%), PALB2: 12 (8%) , ATM: 12 (8%), TP53: 9 (6%), CDH1 : 5 (3%), PTEN: 3 (2%), BRIP1: 3 (2%), and 12 patients (8%) positive for other genes (CDKN2A, MUTYH, PMS2, APC, BARD1, MLH1, NBN, RAD51C, and SDHD). Tumor phenotype by gene mutation in shown in table 1. Conclusions: Several hereditary BC genes are associated with ER positive BC which could have implications for chemoprevention, while others associated with ER neg/TNBC that could have therapeutic implications. These findings and implications need to be further studied in larger cohorts. [Table: see text]

A multi-gene panel study in hereditary breast and ovarian cancer in Colombia.

Germline mutations in BRCA1 and BRCA2 account for approximately 50% of inherited breast and ovarian cancers. Three founder mutations in BRCA1/2 have been reported in Colombia, but the pattern of mutations in other cancer susceptibility genes is unknown. This study describes the frequency and type of germline mutations in hereditary breast and/or ovarian cancer genes in a referral cancer center in Colombia. Eighty-five women referred to the oncogenetics unit of the Instituto de Cancerologia Las Americas in Medellin (Colombia), meeting testing criteria for hereditary breast and ovarian cancer syndrome (NCCN 2015), who had germline testing with a commercial 25-gene hereditary cancer panel, were included in the analysis. Nineteen patients (22.4%) carried a deleterious germline mutation in a cancer susceptibility gene: BRCA1 (7), BRCA2 (8), PALB2 (1), ATM (1), MSH2 (1) and PMS2 (1). The frequency of mutations in BRCA1/2 was 17.6%. One BRCA2 mutation (c.9246dupG) was recurrent in five non-related individuals and is not previously reported in the country. Seventeen mutation-carriers had a diagnosis of breast cancer (median age of diagnosis of 36 years) and two of ovarian cancer. All BRCA1 mutation-carriers with breast cancer had triple negative tumors (median age of diagnosis of 31 years). Variants of unknown significance were reported in 35% of test results. This is the first report of a multi-gene study for hereditary breast and/or ovarian cancer in a Latin American country. We found a high frequency and a wide spectrum of germline mutations in cancer susceptibility genes in Colombian patients, some of which were not previously reported in the country. We observed a very low frequency of known Colombian founder BRCA1/2 mutations (1.2%) and we found mutations in other genes such as PALB2, ATM, MSH2 and PMS2. Our results highlight the importance of performing multi-gene panel testing, including comprehensive BRCA1/2 analysis (full gene sequencing and large rearrangement analysis), in high-risk breast and/or ovarian cancer patients in Colombia.

Ancestry-based differences in hereditary cancer genetic testing.

e13107 Background: Ancestry-based disparities in genetic testing for the hereditary breast and ovarian cancer (HBOC) genes BRCA1/2 have been well documented, but it is unclear whether this extends to other cancer-risk genes. Given reduced costs and broader access to multi-gene panels, we evaluated ancestry-based differences in the utilization and outcomes of HBOC and pan-cancer panel testing. Methods: Individuals who had genetic testing for BRCA1/2only (2006-2016) or with a multi-gene pan-cancer panel (2013-2016) were assessed. Clinical information was obtained from provider-completed test request forms. The most commonly reported ancestries [European (EU), Latin American/Caribbean (LA/C), African (AF), Asian (AS)] were evaluated. Individuals of Ashkenazi Jewish ancestry were not included in the EU group. Results: The relative utilization of HBOC testing in 2013-2016 increased in AF and LA/C individuals (vs 2006-2013) and was similar to panel testing in all ancestries (Table). The positive mutation rate for panel testing was 6.7%; AF (6.4%), LA/C (6.6%), AS (7.1%), EU (7.1%). The positive rate for HBOC testing from 2006-2013 was 5.7%. Of significance, the HBOC positive rate from 2013-2016 dropped to 4.1% and ancestry-specific positive rates were much lower relative to panel testing (Table). Gene-specific mutation prevalence differed by ancestry, but mutations were identified in a wide range of genes for all ancestries. Possible founder mutations were identified in BRCA1/2 and other genes. Conclusions: In this cohort, broader access to genetic testing has reduced disparities in recent years and panel testing shows improved clinical utility relative to HBOC testing in all ancestries. [Table: see text]

Abstract P5-10-04: Spectrum of hereditary breast and ovarian cancer gene variants in an African American cohort

Abstract Background: Few reports describe the spectrum of mutations in breast and ovarian cancer predisposition genes found specifically in African Americans. Methods: 560 women who self-identified as African American (AA) from the University of Pennsylvania and Wayne State University were included in this IRB-approved, case-control study. Cases (n=218 with a personal history of breast and/or ovarian cancer) and controls (n=342 without breast or ovarian cancer) underwent germline genetic testing using the Color Genomics 19-gene breast and ovarian cancer risk panel. The subset of AA patients diagnosed with breast cancer ≤40 (n=185) was compared to an institutional cohort of white patients with breast cancer diagnosed ≤40 (n=189). Results: Of 218 AA cases, 70 had pathogenic or likely pathogenic (P/LP) mutations (BRCA1: n=36; BRCA2: n=24; TP53: n=3; RAD51D: n=2; ATM: n=2; CHEK2: n=2 and MSH6: n=1). Forty-two of 218 patients (19%) had at least one variant of uncertain significance (VUS). Of 342 AA controls, 5 women had P mutations in 5 distinct genes: BRCA2, ATM, BRIP1, PALB2 and PMS2. 55 control patients (16%) had at least one VUS. Many of the 75 P/LP mutations (cases, 70 mutations; controls, 5 mutations) in the full AA cohort were unique variants. In the 135 patients who had BRCA1/BRCA2 sequencing prior to testing under this protocol, the Color Genomics platform identified all 56 pathogenic mutations. Among AA patients diagnosed with breast cancer ≤40 (n=185), the incidence of TP53 and ATM pathogenic mutations was similar to the white, early-onset breast cancer cohort (n=189): TP53, 1% in both cohorts; ATM = 1% in AA patients and 2% in whites. However, no patients in the AA, early-onset cohort had germline CHEK2 mutations, compared to 4% of white, early-onset breast cancer patients (p=0.007). Conclusions: Taken together, the results of this study demonstrate the importance of considering germline mutation testing in the AA population. Examination of mutations and disease phenotypes within the AA population may facilitate understanding of the clinical risk associated with variants of uncertain significance. Further comparative data between the AA and white cohorts will be presented. Citation Format: Shah PD, Digiovanni L, Maxwell KN, Bradbury AR, Van Den Akker J, Kim S, Gil E, Simon MS, Nathanson KL, Domchek SM. Spectrum of hereditary breast and ovarian cancer gene variants in an African American cohort [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-10-04.

Abstract P5-09-03: Associations between breast cancer subtypes and mutations in cancer predisposition genes identified by clinical genetic testing of breast cancer patients

Abstract Clinical genetic testing of individuals with a personal or family history of breast and ovarian cancer using panels for BRCA1/2 and other candidate cancer predisposition genes has become routine clinical practice. Several of the genes on hereditary cancer testing panels have been strongly associated with specific subtypes of breast cancer. In particular, individuals with germline mutations in BRCA1 predominantly develop estrogen receptor (ER)-negative and triple negative (TN) (estrogen receptor negative, progesterone receptor negative, HER2 negative) breast tumors. In contrast, CHEK2 and ATM mutations have been associated with ER-positive breast cancer. In this study, associations between mutations in panel genes and breast cancer subtypes were evaluated. A cohort of 60,000 breast cancer patients tested for germline cancer predisposing mutations using hereditary cancer gene panels was utilized. Information on personal and family cancer history, age of diagnosis, tumor pathology, and ethnicity of patients was obtained from test requisition forms or by follow up with ordering health care providers. Mutations in each gene were combined into four histological subtypes (triple negative; HER2 positive; ER-positive,HER2-positive; and ER-positive,HER2 negative). Associations for each subtype were estimated by case-control analyses comparing the frequencies of pathogenic mutations in each subtype with frequencies from non-TCGA controls from the Exome Aggregation Consortium (ExAC) database. In addition, case-case analyses were conducted to assess enrichment of gene mutations in specific breast cancer subtypes. Among the observed associations between genes and breast cancer subtypes, mutations in CHEK2 and ATM were highly enriched in luminal breast cancers and BARD1 was specifically associated with TN breast cancer. Refining the spectrum of pathological correlates with mutations in hereditary breast cancer genes will aid gene specific cancer risk management, and may accelerate the development of novel gene-specific therapeutic interventions. Citation Format: Couch FJ, Lilyquist J, Na J, Hu C, Polley EC, Shimelis H, Akinhanmi M, McFarland R, LaDuca H, Goldgar DE, Dolinsky JS. Associations between breast cancer subtypes and mutations in cancer predisposition genes identified by clinical genetic testing of breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-09-03.

Fork Protection and Therapy Resistance in Hereditary Breast Cancer.

The BRCA-Fanconi anemia (FA) pathway preserves the genome and suppresses cancer and is a main determinant of chemotherapeutic efficacy. The hereditary breast cancer genes BRCA1 and BRCA2 function in DNA double-strand break repair mediating distinct steps of homologous recombination (HR). More recently, independent of DNA repair, functions in the replication stress response have come to light, providing insight as to how the BRCA-FA pathway also balances genome preservation with proliferation. The BRCA-FA proteins associate with the replisome and contribute to the efficiency and recovery of replication following perturbations that slow or arrest DNA replication. Although the full repertoire of functions in the replication stress response remains to be elucidated, the function of BRCA1 and BRCA2 in protecting stalled replication forks contributes along with HR to the sensitivity of BRCA-associated tumors to chemotherapy. Moreover, chemoresistance evolves from restoration of either HR and/or fork protection. Although mechanisms underlying the restoration of HR have been characterized, it remains less clear how restoration of fork protection is achieved. Here, we outline mechanisms of “rewired” fork protection and chemotherapy resistance in BRCA cancer. We propose that mechanisms are linked to permissive replication that limits fork remodeling and therefore opportunities for fork degradation. Combating this chemoresistance mechanism will require drugs that inactivate replication bypass mechanisms.

FOXC1 identifies basal-like breast cancer in a hereditary breast cancer cohort.

Breast cancers arising in the setting of the hereditary breast cancer genes BRCA1 and BRCA2 are most commonly classified as basal-like breast cancer (BLBC) or luminal breast cancer, respectively. BLBC is an aggressive subtype of breast cancer associated with liver and lung metastases and poorer prognosis than other subtypes and for which chemotherapy is the only systemic therapy. Multiple immunohistochemical markers are used to identify the basal-like subtype, including the absence of estrogen receptor alpha, progesterone receptor, and human epidermal growth factor receptor 2. Forkhead box C1 (FOXC1) has been identified as a specific marker expressed in BLBC in general breast cancer cohorts. We examined an institutional cohort of breast cancer patients with germline BRCA1 (n=46) and BRCA2 (n=35) mutations and found that FOXC1 expression on immunohistochemical staining is associated with BRCA1 vs BRCA2 mutations [30/46 vs. 6/35]. In BRCA1 mutant tumors, FOXC1 was expressed in 28/31 BLBC tumors and 2/13 non-BLBC tumors, In BRCA2 mutant tumors, FOXC1 was expressed in 5/5 BLBC tumors and 1/30 non-BLBC tumors. In cell culture models of BRCA1-mutant breast cancer, FOXC1 is associated with increased proliferation and may serve as a marker for sensitivity to PARP-inhibitor therapy with olaparib.

Variations in the Genome: The Mutation Detection 2015 Meeting on Detection, Genome Sequencing, and Interpretation.

The content of the 13th Mutation Detection meeting (Leiden, April 2015) is summarized in this report. Topics discussed at the meeting included current challenges of clinical NGS, advances in bioinformatics, data quality control, single cell analysis and RNA sequencing, among others. Social, ethical and regulatory challenges of genomic data handling and data sharing were the focus of an expert panel debate. The 14th International Symposium on Variants in the Genome will take place in Santiago de Compostela, June 5-8, 2017. http://isv.variome.org.

Abstract 2597: Breast and ovarian cancer risks associated with cancer predisposition gene mutations identified by multigene panel testing

Abstract Multigene panel testing (MGPT) for hereditary cancer is increasing in popularity in the USA. Many panels include genes identified as hereditary breast and/or ovarian cancer (HBOC) genes despite limited data regarding the precise cancer risks associated with mutations in these genes. Here we report on results from BreastNext and OvaNext panel testing of 20 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11A, MSH2, MSH6, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, TP53) in a cohort of 15,083 individuals. The majority of individuals were from high-risk breast and/or ovarian (Br/Ov) cancer families, with 92.4% of all probands meeting National Comprehensive Cancer Network HBOC testing criteria. Pathogenic mutations were identified in 9.4% of the overall cohort. To estimate gene-specific breast and ovarian cancer risks, case-control analyses were performed comparing the frequencies of pathogenic mutations from Caucasian breast or ovarian cancer cases from BreastNext and OvaNext with frequencies from Caucasian, non-Finnish, non-TCGA controls from the Exome Aggregation Consortium (ExAC) database. Mutations in the well studied ATM and CHEK2 genes were associated with moderate risks (OR>2) of breast cancer and mutations in PALB2 were associated with high-risks (OR>5) of breast cancer, consistent with previous reports. In addition, the study suggested that pathogenic mutations in MSH6, RAD51D, CDH1, and NF1 are associated with moderate to high risks of breast cancer. In contrast, RAD51C, RAD51D, and BRIP1 mutations were associated with high risks of ovarian cancer, PALB2 mutations were associated with moderate risks, but ATM and CHEK2 mutations were not associated with increased ovarian cancer risk. In addition, modeling of missense mutations in the predisposition genes using in silico prediction algorithms suggested that missense mutations in CDH1, CHEK2, MSH2, and MSH6 are associated with moderate risks of breast cancer and missense mutations in RAD51C increase risks of ovarian cancer. This large breast and ovarian cancer case-control analysis provides useful data for many predisposition genes previously lacking risk estimates, and should prove useful for clinical risk management of patients after clinical panel testing. Citation Format: Fergus J. Couch, David E. Goldgar, Steven N. Hart, Emily Hallberg, Raymond Moore, Huong Meeks, Robert Huether, Holly LaDuca, Elizabeth Chao, Jill Dolinsky. Breast and ovarian cancer risks associated with cancer predisposition gene mutations identified by multigene panel testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2597.

PALB2 mutation carriers: Are clinicians acting on the molecular diagnosis?

e13121Background: PALB2 is a frequently mutated hereditary breast and ovarian cancer gene. Recent studies have demonstrated up to a 58% lifetime risk for breast cancer with increased risks for pancreatic and other cancers. However, the precise risks and recommendations for management in PALB2 mutation carriers (PALB2+) are not well established. The National Comprehensive Cancer Network guidelines include breast MRI surveillance for female PALB2+, but comment on prophylactic surgeries is vague and other cancer screening is not mentioned. This study aimed to provide preliminary data on how clinicians manage cancer risks in PALB2+. Methods: 156 patients with a PALB2 mutation were identified from a clinical diagnostic laboratory from Aug 2012 to Sept 2015. Clinicians with ≥ 2 PALB2+ were invited to participate by completing a survey on the impact the molecular diagnosis had on management for their patient. Results: Of 19 females and 1 male, 70% (n = 14) had a history of breast cancer only and 15% (n = 3) had ...

Risks of triple negative breast cancer associated with cancer predisposition gene mutations.

1513Background: Multigene panel testing for hereditary cancer includes genes identified as hereditary breast and/or ovarian cancer (HBOC) genes despite limited data regarding the precise cancer risks associated with mutations in these genes. Here we report on risks of breast cancer and separately risks of triple negative breast cancer associated with mutations in 20 predisposition genes. Methods: A cohort of 10,091 individuals with breast cancer, including 2,400 with triple negative breast cancer, were tested for mutations with the BreastNext and OvaNext clinical genetic testing panels. A further 2,100 individuals with triple negative breast cancer were tested for the same genes by custom capture-based sequencing. Case-control analyses were performed comparing the frequencies of pathogenic mutations from Caucasian breast cancer cases with frequencies from Caucasian study matched controls, and Caucasian, non-Finnish, non-TCGA controls from the ExAC database. Results: Pathogenic mutations were identified in...

How Far Do We Go With Genetic Evaluation? Gene, Panel, and Tumor Testing.

The traditional model by which an individual was identified as harboring a hereditary susceptibility to cancer was to test for a mutation in a single gene or a finite number of genes associated with a particular syndrome (e.g., BRCA1 and BRCA2 for hereditary breast and ovarian cancer or mismatch repair genes for Lynch syndrome). The decision regarding which gene or genes to test for was based on a review of the patient's personal medical history and their family history. With advances in next-generation DNA sequencing technology, offering simultaneous testing for multiple genes associated with a hereditary susceptibility to cancer is now possible. These panels typically include high-penetrance genes, but they also often include moderate- and low-penetrance genes. A number of the genes included in these panels have not been fully characterized either in terms of their cancer risks or their management options. Another way some patients are unexpectedly identified as carrying a germline mutation in a cancer susceptibility gene is at the time they undergo molecular profiling of their tumor, which typically has been carried out to guide treatment choices for their cancer. This article first focuses on the issues that need to be considered when deciding between recommending more targeted testing of a single or a small number of genes associated with a particular syndrome (single/limited gene testing) versus performing a multigene panel. This article also reviews the issues regarding germline risk that occur within the setting of ordering molecular profiling of tumors.

Prioritizing Variants in Complete Hereditary Breast and Ovarian Cancer Genes in Patients Lacking Known BRCA Mutations.

BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including noncoding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize noncoding variants of uncertain significance in regulatory, coding, and intronic regions based on changes in binding sites in these genes. Besides mRNA splicing, IT provides a common framework to evaluate potential affinity changes in transcription factor (TFBSs), splicing regulatory (SRBSs), and RNA-binding protein (RBBSs) binding sites following mutation. We prioritized variants affecting the strengths of 10 splice sites (four natural, six cryptic), 148 SRBS, 36 TFBS, and 31 RBBS. Three variants were also prioritized based on their predicted effects on mRNA secondary (2°) structure and 17 for pseudoexon activation. Additionally, four frameshift, two in-frame deletions, and five stop-gain mutations were identified. When combined with pedigree information, complete gene sequence analysis can focus attention on a limited set of variants in a wide spectrum of functional mutation types for downstream functional and co-segregation analysis.

Abstract P6-06-03: Novel hereditary breast cancer gene mutations: Should there be greater concern regarding ovarian cancer risk?

Abstract INTRODUCTION: As the use of multi-gene breast cancer panel testing increases the phenotype of the included genes continues to evolve. PALB2 and NBN have a well characterized association with breast cancer and are included on many breast cancer gene panels. Germline PALB2 and NBN mutations have been identified in a small percentage of ovarian carcinoma cases with one study reporting a non-significant twofold increase in carriers of the PALB2 mutations amongst ovarian cancer patients (P= 0.4). Similar to BRCA1 and BRCA2 both genes are members of the Fanconi Anemia pathway therefore a potential increased risk for both breast and ovarian cancer could be anticipated. However at present overall the current literature on the association with ovarian cancer is sparse. Here we present the cases of three hereditary breast and ovarian cancer families found to carry pathogenic mutations within the PALB2 and NBN genes. In all three families the proband was diagnosed with ovarian or fallopian tube cancer and carries a pathogenic PALB2 or NBN mutation. Our PALB2 family carries the well-known French Canadian founder mutation, c.2323C>T, and includes the proband who was diagnosed with a stage II-C, grade 3, fallopian tube carcinoma at age 58, her heterozygote sister with ovary cancer at 68 and her mother with ovary cancer at 43 who was not able to be tested. This PALB2 proband has 5 sisters, 7 brothers and 41 nieces and nephews with only one sister diagnosed with breast cancer at 69 who also is PALB2 positive and a maternal grandmother with breast cancer at 48 who was unable to be tested. The NBN Slavic founder mutation, c.657_661del, was discovered in a 66 year old woman with stage IV, high-grade serous ovarian cancer having a mother with breast cancer at 91 and a maternal aunt with ovary cancer at 59 who have not yet been tested. Lastly, our patient from Laos with a diagnosis of a stage II-C endometrioid adenocarcinoma of the ovary diagnosed at 38 years old was found to carry the deleterious NBN mutation, c.1550dupA. She is not aware of any cancer family history and reports a large family including five brothers, four sisters and multiple aunts and uncles on both side of the family. CONCLUSION: These case studies suggest a link between PALB2 and NBN, two known breast cancer susceptibility genes, and hereditary ovarian cancer risk. These observations suggest that further data are needed to accurately evaluate ovarian cancer risk within novel hereditary breast cancer genes now commonly tested as part of multiplex panel analysis. Citation Format: Nassikas N, Wilbur JS, Laprise JK, Legare RD. Novel hereditary breast cancer gene mutations: Should there be greater concern regarding ovarian cancer risk?. [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 P6-06-03.

How Far Do We Go With Genetic Evaluation? Gene, Panel, and Tumor Testing

The traditional model by which an individual was identified as harboring a hereditary susceptibility to cancer was to test for a mutation in a single gene or a finite number of genes associated with a particular syndrome (e.g., BRCA1 and BRCA2 for hereditary breast and ovarian cancer or mismatch repair genes for Lynch syndrome). The decision regarding which gene or genes to test for was based on a review of the patient’s personal medical history and their family history. With advances in next-generation DNA sequencing technology, offering simultaneous testing for multiple genes associated with a hereditary susceptibility to cancer is now possible. These panels typically include high-penetrance genes, but they also often include moderate- and low-penetrance genes. A number of the genes included in these panels have not been fully characterized either in terms of their cancer risks or their management options. Another way some patients are unexpectedly identified as carrying a germline mutation in a cancer ...

A Systematic Comparison of Traditional and Multigene Panel Testing for Hereditary Breast and Ovarian Cancer Genes in More Than 1000 Patients

Gene panels for hereditary breast and ovarian cancer risk assessment are gaining acceptance, even though the clinical utility of these panels is not yet fully defined. Technical questions remain, however, about the performance and clinical interpretation of gene panels in comparison with traditional tests. We tested 1105 individuals using a 29-gene next-generation sequencing panel and observed 100% analytical concordance with traditional and reference data on >750 comparable variants. These 750 variants included technically challenging classes of sequence and copy number variation that together represent a significant fraction (13.4%) of the pathogenic variants observed. For BRCA1 and BRCA2, we also compared variant interpretations in traditional reports to those produced using only non-proprietary resources and following criteria based on recent (2015) guidelines. We observed 99.8% net report concordance, albeit with a slightly higher variant of uncertain significance rate. In 4.5% of BRCA-negative cases, we uncovered pathogenic variants in other genes, which appear clinically relevant. Previously unseen variants requiring interpretation accumulated rapidly, even after 1000 individuals had been tested. We conclude that next-generation sequencing panel testing can provide results highly comparable to traditional testing and can uncover potentially actionable findings that may be otherwise missed. Challenges remain for the broad adoption of panel tests, some of which will be addressed by the accumulation of large public databases of annotated clinical variants.