BRCA1 Variants Of Uncertain Significance Research Articles

Abstract B013: A quest for deeper understanding of BRCA2 Variants of Uncertain Significance (VUS)

Abstract Deleterious mutations in homology-directed repair pathway genes impact both future cancer risk and therapeutic intervention options for cancer diagnosis and prognosis. Homology-directed repair pathway is a high-fidelity DNA repair pathway for resolving double-strand breaks (DSBs) throughout the human genome. BRCA2, as a homology-directed repair factor, is essential to ensure genome integrity through an error-free process. Deleterious BRCA2 mutations increase the lifetime risk of breast, ovarian, melanoma, prostate, and pancreatic cancers. Deleterious BRCA2 mutations cover only 25% of the total BRCA2 mutations identified in the population by genetic testing whereas 60% are mutations with unknown connection to the disease called Variants of Uncertain Significance (VUS). The majority of BRCA2 VUS are missense mutations with one point mutation which could potentially alter BRCA2 cellular localization and/or nuclear functions. Thus, evaluating BRCA2 cellular localization and function in BRCA2 missense carriers and tumors is urgently needed to provide clinical guidance regarding cancer risk and treatment options. We have identified a paradoxical relationship between BRCA2 nuclear import and treatment. Deleterious missense mutations in the DNA binding domain of BRCA2 direct the protein to the cytosol which increase sensitivity to PARP inhibitors (PARPi) and platinum drugs. Therefore, although BRCA2 mislocalization might be a cause of cancer, keeping it out of the nucleus allows for much better treatment outcomes. Here, we present how BRCA2 cancer-driver missense mutations lead to cytosolic mislocalization and how BRCA2 localization can be used as a diagnostic tool to stratify patients. Our robust multi-step molecular approach will guide future clinical decision rules surrounding BRCA2 VUS. By providing reliable and accurate functional assays to correctly evaluate the pathogenicity of BRCA2 VUS, our goal is to improve personalized medicine by providing actionable information to patients and their physicians. Citation Format: Cole Niedmann, Judit Jimenez Sainz. A quest for deeper understanding of BRCA2 Variants of Uncertain Significance (VUS) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B013.

Association Between Germline BRCA1/2 Gene Variants and Clinicopathological Features of Ovarian Cancer.

To investigate the relationship between BRCA1/2 gene mutation and clinicopathological features in ovarian cancer patients, so as to develop precise individualized treatment plan for patients. Patients diagnosed with ovarian cancer between January 2018 and July 2023 who underwent BRCA1/2 genetic testing were retrospectively analyzed. The clinicopathological characteristics (age, body mass index (BMI), family history of ovarian cancer, pregnancy history, menopause status, tumor size, histopathology, Federation of Gynecology and Obstetrics (FIGO) staging, and ascites) of non-carriers and BRCA1/2 variant carriers were compared. Logistic regression analysis was used to explore the relationship between BRCA1/2 variants and clinicopathological characteristics of ovarian cancer. A total of 284 ovarian cancer patients were collected, and the subjects were divided into two groups, 197 non-carriers and 87 BRCA1/2 variants carriers. The proportion of serous ovarian carcinoma in BRCA1/2 variant carriers is higher than that in non-BRCA variant carriers (78.2% vs 60.9%, p=0.015). There were 51 patients with BRCA pathogenic or likely pathogenic variant, 22 patients with BRCA likely benign variant, and 14 patients with BRCA variants of uncertain significance (VUS). The proportion of serous ovarian carcinoma in patients with BRCA pathogenic/likely pathogenic variant is higher than that in patients with BRCA likely benign variant and BRCA VUS (94.1% vs 50.0% and 64.3%. p<0.001). There were no statistically significant differences in BMI, family history of ovarian cancer, pregnancy history, menopause status, maximum diameter of the tumor lesion, FIGO stage, and ascites among patients with different grades of variants. Multivariate logistic regression analysis showed that serous ovarian carcinoma was related to BRCA mutation (Serous carcinoma vs non-serous carcinoma: OR 2.145, 95% CI: 1.044-4.407) (p=0.038). Patients with BRCA1 variant develop ovarian cancer at a younger age than those with the BRCA2 variant. The proportion of FIGO stage III-IV in patients with BRCA pathogenic + likely pathogenic variant was significantly higher than those in patients with other variants. Germline BRCA1/2 variants were most frequently identified in serous ovarian carcinoma patients.

DNA repair function scores for 2172 variants in the BRCA1 amino-terminus.

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1.

Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells.

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

BRCA1-specific machine learning model predicts variant pathogenicity with high accuracy.

Identification of novel BRCA1 variants outpaces their clinical annotation which highlights the importance of developing accurate computational methods for risk assessment. Therefore our aim was to develop a BRCA1-specific machine learning model to predict the pathogenicity of all types of BRCA1 variants and to apply this model and our previous BRCA2-specific model to assess BRCA variants of uncertain significance (VUS) among Qatari patients with breast cancer. We developed an XGBoost model that utilizes variant information such as position frequency and consequence as well as prediction scores from numerous in silico tools. We trained and tested the model with BRCA1 variants that were reviewed and classified by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. In addition we tested the model's performance on an independent set of missense variants of uncertain significance with experimentally determined functional scores. The model performed excellently in predicting the pathogenicity of ENIGMA-classified variants (accuracy: 99.9%) and in predicting the functional consequence of the independent set of missense variants (accuracy: 93.4%). Moreover it predicted 2 115 potentially pathogenic variants among the 31 058 unreviewed BRCA1 variants in the BRCA exchange database. Using two BRCA-specific models we did not identify any pathogenic BRCA1 variants among those found in patients in Qatar but predicted four potentially pathogenic BRCA2 variants, which could be prioritized for functional validation.

Refinement of the assignment to the ACMG/AMP BS3 and PS3 criteria of eight BRCA1 variants of uncertain significance by integrating available functional data with protein interaction assays.

The clinical screening of cancer predisposition genes has led to the identification of a large number of variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is limited by the amount of necessary data, which are difficult to obtain for rare variants. The guidelines for variant interpretation of the American College of Medical Genetics and Genomics along with the Association for Molecular Pathology (ACMG/AMP) state that "well-established" functional studies provide strong support of a pathogenic or benign impact (criteria PS3 and BS3, respectively) and can be used as evidence type to reach a final classification. Moreover, the Clinical Genome Resource Sequence Variant Interpretation Working Group developed rule specifications to refine the PS3/BS3 criteria. Recently, Lira PC etal. developed the "Hi Set" approach that generated PS3/BS3 codes for over two-thousands BRCA1 VUS. While highly successful, this approach did not discriminate a group of variants with conflicting evidences. Here, we aimed to implement the outcomes of the "Hi-set" approach applying Green Fluorescent Protein (GFP)-reassembly assays, assessing the effect of variants in the RING and BRCT domains of BRCA1 on the binding of these domains with the UbcH5a or ABRAXAS proteins, respectively. The analyses of 26 clinically classified variants, including 13 tested in our previous study, showed 100% sensitivity and specificity in identifying pathogenic and benign variants for both the RING/UbcH5a and the BRCTs/ABRAXAS interactions. We derived the strength of evidences generated by the GFP-reassembly assays corresponding to moderate for both PS3 and BS3 criteria assessment. The GFP-reassembly assays were applied to the functional characterization of 8 discordant variants from the study by Lyra etal. The outcomes of these analyses, combined with those reported in the "Hi Set" study, allowed the assignment of ACMG/AMP criteria in favor or against pathogenicity for all 8 examined variants. The above findings were validated with a semi-quantitative Mammalian Two-Hybrid approach, and totally concordant results were observed. Our data contributes in shedding light on the functional significance of BRCA1 VUS and on their clinical interpretation within the ACMG/AMP framework.

Racial and ethnic variation in BRCA1 and BRCA2 genetic test results among individuals referred for genetic counseling at a large urban comprehensive cancer center.

The prevalence of pathogenic variants in BRCA1 and BRCA2 in populations other than Ashkenazi Jewish (AJ) is not well defined. We describe the racial and ethnic-specific prevalence of BRCA1/2 pathogenic variants and variants of uncertain significance (VUS) among individuals referred for genetic testing in a large urban comprehensive cancer center over a 20-year period. The population included 3,537 unrelated individuals who underwent genetic testing from January 1999 to October 2019 at the Karmanos Cancer Institute. We estimated the prevalence of pathogenic variants and VUS and evaluated associations with race and ethnicity for African American (AA), Arab, AJ and Hispanic individuals compared to Non-Hispanic Whites (NHW). We used multivariable models to adjust for other predictors of pathogenic variants. We also reported the most common pathogenic variants by racial and ethnic group. The racial and ethnic breakdown of our population was: NHW (68.9%), AA (20.3%), AJ (2.5%), Arab (2.2%), Hispanic (1.0%), Asian Pacific Islander, Native American/Alaskan Native (4.7%), and < 1% unknown. The overall prevalence of pathogenic variants in BRCA1/2 was 8.9% and the prevalence of VUS was 5.6%. Compared to NHW, there were no racial or ethnic differences in the rate of pathogenic variants. However, AA individuals were more likely to have VUS in BRCA1 (adjusted OR 2.43, 95% CI 1.38-4.28) and AJ weremore likely to have VUS in BRCA2 (adjusted OR 3.50, 95% CI 1.61-6.58). These results suggest the continued need for genetic testing and variant reclassification for individuals of all racial and ethnic groups.

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair.

Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic; however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele, whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

Classification of BRCA2 Variants of Uncertain Significance (VUS) Using an ACMG/AMP Model Incorporating a Homology-Directed Repair (HDR) Functional Assay.

The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer-testing laboratory. The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2. Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS. Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.

Identification of deleterious variants of uncertain significance in BRCA2 BRC4 repeat through molecular dynamics simulations.

Large quantity of variants of uncertain significance (VUS) has been identified in cancer predisposition genes, but classification of VUS remains a big challenge. We proposed that the impact of VUS on protein structure stability can be used to identify these with deleterious effects by using molecular dynamics simulation (MDS)-based approach and developed a MDS-based method for missense VUS classification. In the current study, we applied the system to classify the missense VUS in BRCA2. BRCA2 plays an important role in maintaining genome stability by repairing double-strand DNA damage through homologous recombination. BRCA2 BRC repeats bring RAD51 from cytoplasm to the break sites in nucleus to initiate the repairing process. Missense variants in BRCA2 BRC repeats can interfere the interaction between BRCA2 and RAD51, impair double-strand break repair, cause genome instability and increase cancer risk. We characterized the missense VUS in BRCA2 BRC4 repeat, the primary site of BRCA2 interacting with RAD51. Based on the well-determined BRC4 structure, we applied MDS to measure the impact of BRC4 missense VUS on the stability of BRC4 structure by testing the equilibrium state, flexibility, compactness, hydrogen bonds and surface accessibility. Of the 46 missense VUS analyzed, we were able to differentiate them into 24 Deleterious and 22 Tolerated variants. Comparison between the MDS-based and other 24 existing computational methods for variant classification showed that the MDS-based approach is highly sensitive and specific for classifying missense VUS in cancer predisposition genes.

The functional impact of BRCA1 BRCT domain variants using multiplexed DNA double-strand break repair assays

Pathogenic variants in BRCA1 are associated with a greatly increased risk of hereditary breast and ovarian cancer (HBOC). With the increased availability and affordability of genetic testing, many individuals have been identified with BRCA1 variants of uncertain significance (VUSs), which are individually detected in the population too infrequently to ascertain a clinical risk. Functional assays can be used to experimentally assess the effects of these variants. In this study, we used multiplexed DNA repair assays of variants in the BRCA1 carboxyl terminus to functionally characterize 2,271 variants for homology-directed repair function (HDR) and 1,427 variants for cisplatin resistance (CR). We found a high level of consistent results (Pearson’s r = 0.74) in the two multiplexed functional assays with non-functional variants located within regions of the BRCA1 protein necessary for its tumor suppression activity. In addition, functional categorizations of variants tested in the multiplex HDR and CR assays correlated with known clinical significance and with other functional assays for BRCA1 (Pearson’s r = 0.53 to 0.71). The results of the multiplex HDR and CR assays are useful resources for characterizing large numbers of BRCA1 VUSs.

Abstract PO-139: BRCA sequence variants in breast cancer patients with African ancestry

Abstract Introduction Breast cancer is the most common cause of cancer related deaths in women worldwide; however, significant disparities are evident in the epidemiology of breast cancer subtypes and clinical outcomes of different racial/ethnic groups. African Americans are 40% more likely to die from breast cancer—a disparity that is partially explained by genetic predisposition to early onset breast cancer and triple negative breast cancer (TNBC), an aggressive subtype linked to West African ancestry and associated with BRCA-1 germline mutations. Pathogenic BRCA mutations significantly alter breast cancer treatment, informing alternate screening, chemoprevention, prophylactic resections and use of PARB inhibitors; however, AAs have higher prevalence of BRCA variants of uncertain significance (VUS) secondary to underrepresentation of African ancestry in the reference genome. The aim of this study is therefore to further characterize BRCA mutations and BRCA VUS in breast cancer patients with African ancestry. Methods RNA Sequencing platforms were used to determine somatic mutation profiles from primary tumor samples of over 70 breast cancer patients from Ethiopia and Ghana, by identifying single nucleotide polymorphisms (SNPs) in BRCA 1 and 2. Each sequence variant was evaluated for consequence on the gene, previously reported clinical significance, and allele frequency by self-reported race in comparison to global allele frequencies. Results BRCA1: 10 BRCA1 SNPs were identified: Missense: Rs1799966, rs16942, rs16941, rs799917, rs4986850, rs1799950; synonymous: rs1060915, rs16940, rs1799949; variant of 3'UTR: rs3092995. Rs1799966, rs16942, rs16941 and rs1799950 cause deleterious missense mutations, and have been reported as uncertain clinical significance. No SNPs were previously reported pathogenic, 6 had uncertain significance, and 4 were classified as either benign or likely benign. Allele frequency of Rs799917 was high among Ethiopian, Ghanaian, and 1KG African subgroups. BRCA2: 13 BRCA2 SNPs were identified: Missense: rs766173, rs144848, rs1799944, rs169547; synonymous: rs1801439, rs1801499, rs1801406, rs543304, rs206075, rs1799955, rs9590940; downstream/missense: rs1801426; variant of 5'Prime UTR: rs1799943. Rs766173 caused a deleterious missense mutation, with conflicting interpretations of pathogenicity. No SNPs were previously reported pathogenic, 4 had uncertain significance, and 8 were either benign or likely benign. No SNPs had high allele frequency in African ancestry. Conclusions The BRCA-1 SNP Rs799917 has a high allele frequency rate among Ethiopian, Ghanaian, and 1KG African ancestry, suggesting this mutation is potentially unique to African ancestry. Further investigation with RNA seq or whole genome sequencing in larger cohorts is warranted. Citation Format: Solange Bayard, Rachel Martini, Yalei Chen, Brittany Jenkins, Isra Elhussin, Esther Cheng, Syed Hoda, Paula Ginter, Jeffrey Hanover, Rozina Zeidan, Joseph Oppong, Kofi Gyan, Clayton Yates, Rick Kittles, Engida Abebe, Ishmael Kyei, Frances Aitpillah, Michael Adinky, Lisa Newman, Melissa Davis. BRCA sequence variants in breast cancer patients with African ancestry [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-139.

Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance.

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes.

Reliability of Tumor Testing Compared to Germline Testing for Detecting BRCA1 and BRCA2 Mutations in Patients with Epithelial Ovarian Cancer

Background: BRCA 1/2 mutation status has become one of the most important parameters for treatment decision in patients with epithelial ovarian cancer (EOC). The aim of this study was to compare tumor DNA with blood DNA sequencing to evaluate the reliability of BRCA tumor testing results. Methods: Patients who were treated for EOC between 2003 and 2019 at the Medical University of Vienna and underwent both germline (gBRCA) and tumor (tBRCA) testing for BRCA mutations were identified. We calculated the concordance rate and further analyzed discordant cases. Results: Out of 140 patients with EOC, gBRCA mutation was found in 47 (33.6%) and tBRCA mutation in 53 (37.9%) patients. Tumor testing identified an additional 9/140 (6.4%) patients with somatic BRCA mutation and negative germline testing. The comparison of germline testing with tumor testing revealed a concordance rate of 93.5% and a negative predictive value of tumor testing of 96.0%. After BRCA variants of uncertain significance were included in the analysis, concordance rate decreased to 90.9%. Conclusion: Tumor testing identified the majority of pathogenic germline BRCA mutations but missed three (2.1%) patients. In contrast, nine (6.4%) patients harboring a somatic BRCA mutation would have been missed by gBRCA testing only.

Prevalence and Spectrum of Germline BRCA1 and BRCA2 Variants of Uncertain Significance in Breast/Ovarian Cancer: Mysterious Signals From the Genome.

About 10–20% of breast/ovarian (BC/OC) cancer patients undergoing germline BRCA1/2 genetic testing have been shown to harbor Variants of Uncertain Significance (VUSs). Since little is known about the prevalence of germline BRCA1/2 VUS in Southern Italy, our study aimed at describing the spectrum of these variants detected in BC/OC patients in order to improve the identification of potentially high-risk BRCA variants helpful in patient clinical management. Eight hundred and seventy-four BC or OC patients, enrolled from October 2016 to December 2020 at the “Sicilian Regional Center for the Prevention, Diagnosis and Treatment of Rare and Heredo-Familial Tumors” of University Hospital Policlinico “P. Giaccone” of Palermo, were genetically tested for germline BRCA1/2 variants through Next-Generation Sequencing analysis. The mutational screening showed that 639 (73.1%) out of 874 patients were BRCA-w.t., whereas 67 (7.7%) were carriers of germline BRCA1/2 VUSs, and 168 (19.2%) harbored germline BRCA1/2 pathogenic/likely pathogenic variants. Our analysis revealed the presence of 59 different VUSs detected in 67 patients, 46 of which were affected by BC and 21 by OC. Twenty-one (35.6%) out of 59 variants were located on BRCA1 gene, whereas 38 (64.4%) on BRCA2. We detected six alterations in BRCA1 and two in BRCA2 with unclear interpretation of clinical significance. Familial anamnesis of a patient harboring the BRCA1-c.3367G>T suggests for this variant a potential of pathogenicity, therefore it should be carefully investigated. Understanding clinical significance of germline BRCA1/2 VUS could improve, in future, the identification of potentially high-risk variants useful for clinical management of BC or OC patients and family members.

Genetic and clinical characterization of multigene hereditary breast and ovarian cancer (HBOC) panels in Málaga (Spain).

e22530 Background: Next Generation Sequencing (NGS) technologies have transformed hereditary breast and ovarian cancer (HBOC) testing process. Several multigene panels (MP) include from 10 to 100 candidate cancer susceptibility genes, but there is a debate about what genes should and should not be tested because of lack of actionability. Few studies have been reported about MP in Europe or Spanish cancer families and no studies in Andalusian population (southern Spain). Methods: We investigated a panel of 17 known genes of high/moderate-risk for HBOC in 938 clinically suspicious HBOC Andalusian families (SEOM 2015 criteria), tested from 2017 to 2019. Multigene panel including BRCA,1 BRCA2, CHEK2, PALB2, BRIP1, ATM, MLH1, MSH2, MSH6, PMS2, CDH1, NF1, PTEN, p53, STK11, RAD51C and RAD51D was performed. Results: We identified 130 patients who carried a high- or moderate-risk pathogenic variants: 61 in BRCA2 (47%), 30 in BRCA 1 (23%), 10 in CHEK2 (8%), 7 in ATM (5%), 7 in PALB2 (5%), 4 in RAD51 (3%), 4 in BRIP1 (3%), 4 in MSH6 (3%), 2 in MLH1 (1,5%) and 1 in MSH2. We detected 220 patients carry variants of uncertain significance (VUS), with a total of 248 VUS (some patients carried more than one VUS): 46 (19%) in ATM, 38 (15%) in BRCA 2, 28 (11%) in MSH6, 19 (8%) in PMS2, 17 (7%) in BRIP1, 16 (6%) in NF1, 14 (6%) in MSH2 and 12 (5%) in CDH1 and PALB2. The most frequent criteria in the entire cohort was “High-grade epithelial non-mucinous ovarian cancer”, reported in 243 cases (26%)”, whereas “Breast cancer (BC) diagnostic under 35” was the most frequent criteria between positives (48 cases (40%)). One case carried two pathogenic variants: BRCA2 and MUTYH. Conclusions: This is the first study reporting the mutational profile of MP gene testing in Andalusia. 70% of mutations were due to BRCA1 and 2 followed by far by CHEK2, ATM and PALB2. We also identified a large amount of VUS in BRCA2, ATM and MSH6. MP improve the diagnostic in andalusian HBOC patients.

Imprecise Medicine: BRCA2 Variants of Uncertain Significance (VUS), the Challenges and Benefits to Integrate a Functional Assay Workflow with Clinical Decision Rules.

Pathological mutations in homology-directed repair (HDR) genes impact both future cancer risk and therapeutic options for patients. HDR is a high-fidelity DNA repair pathway for resolving DNA double-strand breaks throughout the genome. BRCA2 is an essential protein that mediates the loading of RAD51 onto resected DNA breaks, a key step in HDR. Germline mutations in BRCA2 are associated with an increased risk for breast, ovarian, prostate, and pancreatic cancer. Clinical findings of germline or somatic BRCA2 mutations in tumors suggest treatment with platinum agents or PARP inhibitors. However, when genetic analysis reveals a variant of uncertain significance (VUS) in the BRCA2 gene, precision medicine-based decisions become complex. VUS are genetic changes with unknown pathological impact. Current statistics indicate that between 10–20% of BRCA sequencing results are VUS, and of these, more than 50% are missense mutations. Functional assays to determine the pathological outcome of VUS are urgently needed to provide clinical guidance regarding cancer risk and treatment options. In this review, we provide a brief overview of BRCA2 functions in HDR, describe how BRCA2 VUS are currently assessed in the clinic, and how genetic and biochemical functional assays could be integrated into the clinical decision process. We suggest a multi-step workflow composed of robust and accurate functional assays to correctly evaluate the potential pathogenic or benign nature of BRCA2 VUS. Success in this precision medicine endeavor will offer actionable information to patients and their physicians.

Racial disparities in genetic testing of breast cancer patients.

10591 Background: TNBC is disproportionately prevalent in African American (AA) populations and in women with BRCA-1 germline mutations. BRCA mutation carriers are candidates for targeted therapy with PARP-inhibitors, and testing results may influence risk-reducing surgery choice. Methods: We evaluated genetic testing patterns and outcomes for TNBC patients treated in the prospectively maintained databases of academic cancer programs in two metropolitan cities in the Northeast (New York City, NYC) and Midwest (Detroit, Det), 1998-2018. Median follow up was 3.73 years. Testing patterns were also analyzed by time, comparing pts diagnosed before versus after the mid-2013 Supreme Court ruling that expanded testing availability by banning gene patenting. Results: Of 810 pts, 600 were from NYC and 200 from Det; 202 were AA and 488 WA. Pts undergoing genetic testing were younger (median age 50 vs 62; p &lt; 0.0001). Compared to WA, AA pts were less likely to undergo genetic testing overall (23.8% vs 42.0%; p &lt; 0.0001) and within site (NYC: 25.6% vs 42.8%, p = 0.008; Det: 22.3% vs 38.6%, p = 0.025). No significant differences were seen in frequency of pathogenic BRCA mutations (AA-14.6%; WA-29.3%) or VUSs (AA-6.3%; WA- 4.9%); p = 0.20. Genetic testing disparities were reduced among pts diagnosed after mid-2013 (AA-31.4% vs WA-44.0%; p = 0.01) compared to pre-mid-2013 (AA-18.3% vs WA-40.7%; p &lt; 0.0001). No differences were seen in local or distant recurrence free survival between patients with BRCA, BRCA variants of uncertain significance, non-BRCA mutations, and patients without genetic mutations (local recurrence p = 0.827; distant recurrence p = 0.574). This outcome equivalence was consistent when stratified by WA vs AA identity. Conclusions: Genetic testing has increased for TNBC pts following the mid-2013 Supreme Court ban on gene patenting, but race-associated disparities persist. Pts undergoing genetic testing are more likely to undergo risk-reducing mastectomy, but testing results do not affect survival outcomes, regardless of race. Addressing genetic testing disparities will become increasingly important as mutation-associated targeted therapies are identified through advances in precision medicine.

A Population-Based Analysis of BRCA1/2 Genes and Associated Breast and Ovarian Cancer Risk in Korean Patients: A Multicenter Cohort Study.

Simple SummaryAlthough it has been suggested that cancer risk and genetic variation vary by population, there is still a lack of research on non-European populations. In this study, we applied Korean patients as a model to find out the way to conduct BRCA1/2-related clinical studies in non-European populations who do not have as much clinical data as Europeans. The BRCA1/2 variants were classified following the 2015 ACMG standards/guidelines and using a multifactorial probability-based approach. To estimate the additional sample numbers needed to resolve BRCA1/2 unclassified status, we applied a simulation analysis considering population-specific clinical characteristics. In addition, we estimated the risks of breast or ovarian cancer for BRCA1/2 carriers by mutation regions. Data from this study reveal that BRCA1/2 variants in the non-European population are highly specific; therefore, population-specific study is essential for clinical application of treatment or prevention for breast or ovarian cancer.In this study, we performed a comprehensive analysis of BRCA1/2 variants and associated cancer risk in Korean patients considering two aspects: variants of uncertain significance (VUS) and pathogenic or likely pathogenic variants (PLPVs) in BRCA1 and BRCA2. This study included 5433 Korean participants who were tested for BRCA1/2 genes. The BRCA1/2 variants were classified following the standards/guidelines for interpretation of genetic variants and using a multifactorial probability-based approach. In Korea, 15.8% of participants had BRCA1 or BRCA2 PLPVs. To estimate the additional sample numbers needed to resolve unclassified status, we applied a simulation analysis. The simulation study for VUS showed that the smaller the number of samples, the more the posterior probability was affected by the prior probability; in addition, more samples for BRCA2 VUS than those of BRCA1 VUS were required to resolve the unclassified status, and the presence of clinical information associated with their VUS was an important factor. The cumulative lifetime breast cancer risk was 59.1% (95% CI: 44.1–73.6%) for BRCA1 and 58.3% (95% CI: 43.2–73.0%) for BRCA2 carriers. The cumulative lifetime ovarian cancer risk was estimated to be 36.9% (95% CI: 23.4–53.9%) for BRCA1 and 14.9% (95% CI: 7.4–28.5%) for BRCA2 carriers.