BRCA1 Variants Of Unknown Significance Research Articles

BRCA1 VUS: A functional analysis to differentiate pathogenic from benign variants identified in clinical diagnostic panels for breast cancer.

Genetic testing for susceptibility genes through next‑generation sequencing (NGS) has become a widely used technique. Using this, a number of genetic variants have been identified, several of which are variants of unknown significance (VUS). These VUS can either be pathogenic or benign. However, since their biological effect remains unclear, functional assays are required to classify their functional nature. As the use of NGS becomes more mainstream as a diagnostic tool in clinical practice, the number of VUS is expected to increase. This necessitates their biological and functional classification. In the present study, a VUS was identified in the BRCA1 gene (NM_007294.3:c.1067A>G) in two women at risk for breast cancer, for which no functional data has been reported. Therefore, peripheral lymphocytes were isolated from the two women and also from two women without the VUS. DNA from all samples were sequenced by NGS of a breast cancer clinical panel. Since the BRCA1 gene is involved in DNA repair and apoptosis, the functional assays chromosomal aberrations, cytokinesis‑blocked micronucleus, comet, γH2AX, caspase and TUNEL assays were then conducted on these lymphocytes after a genotoxic challenge by ionizing radiation or doxorubicin to assess the functional role of this VUS. The micronucleus and TUNEL assays revealed a lower degree of DNA induced‑damage in the VUS group compared with those without the VUS. The other assays showed no significant differences between the groups. These results suggested that this BRCA1 VUS is likely benign, since the VUS carriers were apparently protected from deleterious chromosomal rearrangements, subsequent genomic instability and activation of apoptosis.

ATR/ATM-Mediated Phosphorylation of BRCA1 T1394 Promotes Homologous Recombinational Repair and G2-M Checkpoint Maintenance.

BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSB) and DNA damage-induced cell-cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response (DDR). Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single-strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G2-M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DDR and may have implications in the development or prognosis of BRCA1-associated cancers. SIGNIFICANCE: This study identifies a BRCA1 phosphorylation event critical for its DNA repair function and reveals the functional defects of several BRCA1 variants of unknown significance.

Predicting Ovarian/Breast Cancer Pathogenic Risks of Human BRCA1 Gene Variants of Unknown Significance.

High-throughput sequencing is gaining popularity in clinical diagnoses, but more and more novel gene variants with unknown clinical significance are being found, giving difficulties to interpretations of people's genetic data, precise disease diagnoses, and the making of therapeutic strategies and decisions. In order to solve these issues, it is of critical importance to figure out ways to analyze and interpret such variants. In this work, BRCA1 gene variants with unknown clinical significance were identified from clinical sequencing data, and then, we developed machine learning models so as to predict the pathogenicity for variants with unknown clinical significance. Through performance benchmarking, we found that the optimized random forest model scored 0.85 in area under receiver operating characteristic curve, which outperformed other models. Finally, we applied the best random forest model to predict the pathogenicity of 6321 BRCA1 variants from both sequencing data and ClinVar database. As a result, we obtained the predictive pathogenic risks of BRCA1 variants of unknown significance.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Structure-based functional analysis of BRCA1 RING domain variants: Concordance of computational mutagenesis, experimental assay, and clinical data

A significant impediment to the improvement of clinical outcomes in treating breast and ovarian cancers rests with the lack of available interpretations for BRCA1 variants of unknown significance. Two research groups recently implemented large-scale functional assays for quantifying effects of single missense mutations on homology-directed DNA repair activity of BRCA1 variants, which is critical for tumor suppression and strongly correlates with cancer risk, and their results are significantly concordant with each other as well as with known pathogenic and benign variant clinical data. In this work, we implemented an established computational mutagenesis procedure to characterize structural impacts of single residue replacements to the BRCA1 RING domain. The computational data showed similarly strong concordance with known clinical data as well as with experimental data from both functional assays. Predictions made by models trained on our computational data offer a complementary and orthogonal approach for classifying all remaining unexplored BRCA1 RING domain variants.

High-throughput functional evaluation of BRCA2 variants of unknown significance

Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance and they thus remain unactionable. To assess the pathogenicity of variants of unknown significance (VUSs) within BRCA2, here we develop a method, the MANO-B method, for high-throughput functional evaluation utilizing BRCA2-deficient cells and poly (ADP-ribose) polymerase (PARP) inhibitors. The estimated sensitivity and specificity of this assay compared to those of the International Agency for Research on Cancer classification system is 95% and 95% (95% confidence intervals: 77–100% and 82–99%), respectively. We classify the functional impact of 186 BRCA2 VUSs with our computational pipeline, resulting in the classification of 126 variants as normal/likely normal, 23 as intermediate, and 37 as abnormal/likely abnormal. We further describe a simplified, on-demand annotation system that could be used as a companion diagnostic for PARP inhibitors in patients with unknown BRCA2 VUSs.

Points to consider: is there evidence to support BRCA1/2 and other inherited breast cancer genetic testing for all breast cancer patients? A statement of the American College of Medical Genetics and Genomics (ACMG)

Points to consider: is there evidence to support BRCA1/2 and other inherited breast cancer genetic testing for all breast cancer patients? A statement of the American College of Medical Genetics and Genomics (ACMG)

Abstract P4-09-05: Somatic genomic profiles of breast tumors differ by BRCA1 and BRCA2 variants of unknown significance status

Abstract Background: Among women with invasive breast cancer, the clinical significance of BRCA1 and BRCA2 variants of unknown significance (VUS) remain uncertain. A closer analysis of the somatic mutational profiles of women with invasive breast cancer and BRCA1 or 2 VUS could provide a better understanding of the pathogenesis of the disease and potentially allow for the development of targeted treatment regimens. We hypothesized that breast cancers with BRCA1/2 VUS will have different somatic genomic profiling patterns and tumor mutational burden (TMB) compared with BRCA1/2 wildtype (WT). Methods: We compared the tumor molecular profiling patterns for breast cancer with somatic BRCA1/2 VUS to BRCA1/2 WT using a multiplatform profiling service (Caris Life Sciences) consisting of next generation sequencing (NGS), protein expression (immunohistochemistry [IHC]) and gene amplification (fluorescence or chromogenic in situ hybridization [FISH or CISH]). We also determined the differences in TMB between VUS and WT. We defined ER/PR/Her2 status based on IHC and/or CISH results. DNA or mutation results with >1% frequency were analyzed using Chi Square test; p<0.05 was considered statistically significant. Results: Among 4,172 breast cancer specimens available in the Caris database acquired from more 800 participating institutions between 2013 and 2018, 125 (3%) and 341 (5.5%) had VUS in BRCA1 and BRCA2 respectively. Women with BRCA1 VUS were more likely to have tumors that are hormone receptor (HR) negative, Her2/neu positive (6.1%) compared to triple negative, triple positive or HR positive, Her2 negative (3.6%, 3.6% and 2.3% respectively) (p-value=0.0096) however BRCA2 VUS were more evenly distributed among tumor subtypes. Tumors with either BRCA1 or BRCA2 VUS were more likely to have a high TMB compared to WT (mean TMB 10.3 mut/Mb vs. 7.5 mut/Mb, p=7.2 × 10−7). Compared to WT tumors, tumors with BRCA1 or BRCA2 VUS were more likely to have pathogenic mutations in RB1 (6.4% vs 3.9%) and TP53 (62.5% vs 56%) and less likely to have GATA3 mutations (5.9% vs 8.8%) and PIK3CA amplifications (0.3% vs 35.3 %%). Furthermore, BRCA1/2 VUS cancers were 3 times more likely to have FGFR2 amplification (P=0.0065). There was no difference in AR or PD-L1 expression between VUS and WT, however breast cancer associated with BRCA1/2 VUS had a significantly less WT1 amplification compared to WT (0.3% vs 72.9%, p value <0.001). Conclusion: Our results suggest that breast cancer associated with BRCA 1 or 2 VUS may be phenotypically and genetically different than WT. Our findings suggest the potential for different responses to immune and targeted therapies, which should be investigated in future prospective studies. Citation Format: Hadeel Assad, Isabel Hart, Kristen Purrington, Joanne Xiu, Paula R Pohlmann, Sandra Swain, Gregory Vidal, Michael Simon. Somatic genomic profiles of breast tumors differ by BRCA1 and BRCA2 variants of unknown significance status [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 P4-09-05.

Reclassification of BRCA1 and BRCA2 variants found in ovarian epithelial, fallopian tube, and primary peritoneal cancers.

ObjectiveWe investigated the proportions of and reclassified BRCA1/2 variants of unknown significance (VUS) in Korean patients with epithelial ovarian, tubal, and primary peritoneal cancers.MethodsData from 805 patients who underwent genetic testing for BRCA1/2 from January 1, 2006 to August 31, 2018 were included. The VUS in BRCA1/2 were reclassified using the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology standards and guidelines.ResultsA BRCA1 pathogenic variant was found in 17.0% (137/805) of the patients, and BRCA1 VUS were found in 15.9% (128/805) of the patients. Further, 8.7% (69/805) of the patients possessed a BRCA2 pathogenic variant and 18.4% (148/805) of the patients possessed BRCA2 VUS. Fifty-three specific BRCA1 VUS were found and 20 were further reclassified as benign (n=11), likely benign (n=5), likely pathogenic (n=3), and pathogenic (n=1). The remaining 33 remained classified as VUS. For BRCA2, 55 specific VUS were detected; among these, 14 were reclassified as benign or likely benign, and 2 were reclassified as likely pathogenic. Among the 805 patients, 195 were found to have only VUS and no pathogenic variants (PV), and 41.5% (81/195) were reclassified as benign or likely benign, and 10.3% (20/195) as pathogenic or likely pathogenic variants.ConclusionsApproximately 33.3% (36/108) of the specific BRCA1/2 variants analyzed in this study that were initially classified as VUS over a 13-year period were reclassified. Among these, 5.6% (6/108) were reclassified as pathogenic or likely pathogenic variants.

Highlights of This Issue

Three-dimensional organoid systems have emerged as potentially powerful tools to understand cancer biology, and they show promise as personalized models that can be used to guide therapeutic decisions. However, their clinical use is limited by the lack of in-depth characterization of their morphological and genetic features. In this study, Romero-Calvo and colleagues describe comprehensive histopathological, genomic and transcriptomic comparisons between patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids and their corresponding primary tumors. The organoid models recapitulate patient-specific structural and molecular features, making them potentially valuable in understanding the etiology and pathogenesis of the patient's tumor. Further, patient-specific drug sensitivities are also reproduced by the organoids, suggesting their utility as personalized models for drug testing. These findings potentiate patient-derived organoids as a promising system for precision oncology.Although the two major breast and ovarian cancer predisposing genes, BRCA1 and BRCA2, were identified over 20 years ago, new variants are still identified daily. Half of them do not show a premature stop signal, but remain classified as variants of unknown significance (VUS), which does not guide the management of the index case and her relatives. Their classification is a major challenge for the diagnosis of breast and ovarian cancer predisposition. More than 1,000 different VUS of each gene are to be classified, each constituting an independent classification effort. The functional complexity of the BRCA1 and BRCA2 macroproteins is such that the silico VUS classification tools are insufficient. This project aims to strengthen the knowledge of the impact of large sets of BRCA1 VUS on cancer risk. The current study identifies structural and functional defects in BRCA1 VUS, to improve their classification and support clinical interpretation and therapeutic selection. BRCA1/2 VUS are emblematic of the interpretation difficulties faced by geneticists with the explosion of next generation sequencing.C-X-C chemokine receptor type 7 (CXCR7), a seven-transmembrane G-protein-coupled chemokine receptor, is an androgen receptor (AR)-repressed gene in prostate cancer cells. Derepression of CXCR7 expression, after androgen deprivation therapy, is one of the survival mechanisms for castration-resistant prostate cancer (CRPC). This study suggests that CXCR7 plays more important roles than well-studied CXCR4 in CRPC development and progression. Both CXCR4 and CXCR7 are druggable targets. However, targeting CXCR7, in combination with anti-androgen, inhibits CRCP growth and potentially prevents incurable bone metastasis.Transforming growth factor ß1 (TGFß1) causes cell cycle arrest by suppressing transcriptional events and inhibiting activation of Cdc45-MCM-GINS (CMG) helicases. Notably, the retinoblastoma (Rb) tumor suppressor plays a pivotal role in both these mechanisms. However, in Rb-deficient cells, TGFß1 remains potent at eliciting growth arrest due to a previously unknown ability of TGFß1 to inhibit assembly of MCM hexamers. Overexpression of the Cdt1 oncoprotein promotes MCM assembly and abrogates this process, inducing G1-S transit in the presence of TGFß1. These results demonstrate additional redundancy in the mechanisms underlying TGFß1 growth arrest and highlight how cancer cells can overcome normal growth-regulatory signals.

BRCA1/2 mutations are not a common cause of malignant melanoma in the Polish population.

The association of BRCA1/2 mutations with melanoma is not completely determined; the interpretation of variants of unknown significance is also problematic. To evaluate these issues we explored the molecular basis of melanoma risk by performing whole-exome sequencing on a cohort of 96 unrelated Polish early-onset melanoma patients and targeted sequencing of BRCA1/2 genes on additional 30 melanoma patients with familial aggregation of breast and other cancers. Sequencing was performed on peripheral blood. We evaluated MutationTaster, Polyphen2, SIFT, PROVEAN algorithms, analyzed segregation with cancer disease (in both families with identified BRCA2 variants) and in one family performed LOH (based on 2 primary tumors). We found neither pathogenic mutations nor variants of unknown significance within BRCA1. We identified two BRCA2 variants of unknown significance: c.9334G>A and c.4534 C>T. Disease allele frequency was evaluated by genotyping of 1230 consecutive melanoma cases, 5000 breast cancer patients, 3500 prostate cancers and 9900 controls. Both variants were found to be absent among unselected cancer patients and healthy controls. The MutationTaster, Polyphen2 and SIFT algorithms indicate that c.9334G>A is a damaging variant. Due to lack of tumour tissue LOH analysis could not be performed for this variant. The variant segregated with the disease. The c.4534 C>T variant did not segregate with disease, there was no LOH of the variant. The c.9334G>A variant, classified as a rare variant of unknown significance, on current evidence may predisposes to cancers of the breast, prostate and melanoma. Functional studies to describe how the DNA change affects the protein function and a large multi-center study to evaluate its penetrance are required.

PO-023 Chloroquine sensitises oncogenic NOTCH1 driven human T cell leukaemia to γ-secretase inhibition

IntroductionFamilial breast cancer (BC) cases account for 5%–10% of all BC cases, and are essentially correlated with the prevalence of pathogenic BRCA1 and BRCA2 mutations in cancer patients. Recent advances in next generation sequencing (NGS) have enabled panel gene testing, or simultaneous testing for mutations in multiple genes for a clinical condition. With more widespread genetic testing, an increased detection of alleles with moderate penetrance without established clinical guidelines and of variants of unknown significance (VUS) as either benign or disease-causing will occur. Functional analyses on VUS may identify pathogenicity, and clearly categorise their mutational status. We carried-out a proof-of concept in vitro functional analysis in peripheral blood lymphocytes of VUS-harbouring individuals and healthy controls assessing the cellular response to γ-radiation.Material and methodsFour samples were collected, one BC patient with a pathogenic ATM mutation; one BRCA2 VUS detected without diagnosed disease, and two controls (A, B, no variant detected). All samples were previously sequenced by NGS to confirm the presence of pathogenic mutations or VUS detected in a genetic testing panel. Several methodologies were selected to evaluate the cellular response to genetic lesions induced by γ-radiation (2Gy): chromosomal aberrations (CA), micronuclei (MN) and comet assay.Results and discussionsComet assay results revealed a higher DNA damage in the patient with a pathogenic ATM mutation (14% DNA in tail) after irradiation, which might be related with the non-optimal function of the ATM protein. The BRCA2 VUS sample also showed a higher%DNA in tail (6.5%), contrasting with the damage quantified for control samples (2.8% and 0.9% DNA in tail for control A and B, respectively). These results revealed that the presence of such variants might be correlated with their biological function, being crucial to categorise the mutational status. Regarding CA and MN, these assays were not sensitive enough to discriminate pathogenic from control samples.ConclusionResults obtained for comet so far suggest a significant difference between variant carriers and controls, which may indicate an increased susceptibility to ionising radiation. However, more sensitive assays (e.g. H2AX) will be necessary allowing more informative results. As this study is a proof-of concept approach, in the near future the sample size will be incremented.

Next Generation Sequencing Reveals High Prevalence of BRCA1 and BRCA2 Variants of Unknown Significance in Early-Onset Breast Cancer in African American Women.

Variants of unknown significance (VUSs) have been identified in BRCA1 and BRCA2 and account for the majority of all identified sequence alterations. Notably, VUSs occur disproportionately in people of African descent hampering breast cancer (BCa) management and prevention efforts in the population. Our study sought to identify and characterize mutations associated with increased risk of BCa at young age. In our study, the spectrum of mutations in BRCA1 and BRCA2 was enumerated in a cohort of 31 African American women of early age at onset breast cancer, with a family history of breast or cancer in general and/or with triple negative breast cancer. To improve the characterization of the BRCA1 and BRCA2 variants, bioinformatics tools were utilized to predict the potential function of each of the variants. Using next generation sequencing methods and in silico analysis of variants, a total of 197 BRCA1 and 266 BRCA2 variants comprising 77 unique variants were identified in 31 patients. Of the 77 unique variants, one (1.3%) was a pathogenic frameshift mutation (rs80359304; BRCA2 Met591Ile), 13 (16.9%) were possibly pathogenic, 34 (44.2%) were benign, and 29 (37.7%) were VUSs. Genetic epidemiological approaches were used to determine the association with variant, haplotype, and phenotypes, such as age at diagnosis, family history of cancer and family history of breast cancer. There were 5 BRCA1 SNPs associated with age at diagnosis; rs1799966 (P=.045; Log Additive model), rs16942 (P=.033; Log Additive model), rs1799949 (P=.058; Log Additive model), rs373413425 (P=.040 and .023; Dominant and Log Additive models, respectively) and rs3765640 (P=.033 Log Additive model). Additionally, a haplotype composed of all 5 SNPs was found to be significantly associated with younger age at diagnosis using linear regression modeling (P=.023). Specifically, the haplotype containing all the variant alleles was associated with older age at diagnosis (OR= 5.03 95% CI=.91-9.14). Knowing a patient's BRCA mutation status is important for prevention and treatment decision-making. Improving the characterization of mutations will lead to better management, treatment, and BCa prevention efforts in African Americans who are disproportionately affected with aggressive BCa and may inform future precision medicine genomic-based clinical studies.

Next-generation sequencing of BRCA1/2 in breast cancer patients: potential effects on clinical decision-making using rapid, high-accuracy genetic results.

PurposeWe evaluated the clinical role of rapid next-generation sequencing (NGS) for identifying BRCA1/2 mutations compared to traditional Sanger sequencing.MethodsTwenty-four paired samples from 12 patients were analyzed in this prospective study to compare the performance of NGS to the Sanger method. Both NGS and Sanger sequencing were performed in 2 different laboratories using blood samples from patients with breast cancer. We then analyzed the accuracy of NGS in terms of variant calling and determining concordance rates of BRCA1/2 mutation detection.ResultsThe overall concordance rate of BRCA1/2 mutation identification was 100%. Variants of unknown significance (VUS) were reported in two cases of BRCA1 and 3 cases of BRCA2 after Sanger sequencing, whereas NGS reported only 1 case of BRCA1 VUS, likely due to differences in reference databases used for mutation identification. The median turnaround time of Sanger sequencing was 22 days (range, 14–26 days), while the median time of NGS was only 6 days (range, 3–21 days).ConclusionNGS yielded comparably accurate results to Sanger sequencing and in a much shorter time with respect to BRCA1/2 mutation identification. The shorter turnaround time and higher accuracy of NGS may help clinicians make more timely and informed decisions regarding surgery or neoadjuvant chemotherapy in patients with breast cancer.

Functional isogenic modeling of BRCA1 alleles reveals distinct carrier phenotypes.

Clinical genetic testing of BRCA1 and BRCA2 is commonly performed to identify specific individuals at risk for breast and ovarian cancers who may benefit from prophylactic therapeutic interventions. Unfortunately, it is evident that deleterious BRCA1 alleles demonstrate variable penetrance and that many BRCA1 variants of unknown significance (VUS) exist. In order to further refine hereditary risks that may be associated with specific BRCA1 alleles, we performed gene targeting to establish an isogenic panel of immortalized human breast epithelial cells harboring eight clinically relevant BRCA1 alleles. Interestingly, BRCA1 mutations and VUS had distinct, quantifiable phenotypes relative to isogenic parental BRCA1 wild type cells and controls. Heterozygous cells with known deleterious BRCA1 mutations (185delAG, C61G and R71G) demonstrated consistent phenotypes in radiation sensitivity and genomic instability assays, but showed variability in other assays. Heterozygous BRCA1 VUS cells also demonstrated assay variability, with some VUS demonstrating phenotypes more consistent with deleterious alleles. Taken together, our data suggest that BRCA1 deleterious mutations and VUS can differ in their range of tested phenotypes, suggesting they might impart varying degrees of risk. These results demonstrate that functional isogenic modeling of BRCA1 alleles could aid in classifying BRCA1 mutations and VUS, and determining BRCA allele cancer risk.

Proper genomic profiling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes

IntroductionBRCA1-mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1-mutated breast carcinomas could lead to a better understanding of BRCA1-associated oncogenic events and could prove valuable in clinical testing for BRCA1-involvement in patients. MethodsFor this purpose, genomic and gene expression profiles of basal-like BRCA1-mutated breast tumors (n = 27) were compared with basal-like familial BRCAX (non-BRCA1/2/CHEK2*1100delC) tumors (n = 14) in a familial cohort of 120 breast carcinomas. ResultsGenome wide copy number profiles of the BRCA1-mutated breast carcinomas in our data appeared heterogeneous. Gene expression analyses identified varying amounts of tumor infiltrating lymphocytes (TILs) as a major cause for this heterogeneity. Indeed, selecting tumors with relative low amounts of TILs, resulted in the identification of three known but also five previously unrecognized BRCA1-associated copy number aberrations. Moreover, these aberrations occurred with high frequencies in the BRCA1-mutated tumor samples. Using these regions it was possible to discriminate BRCA1-mutated from BRCAX breast carcinomas, and they were validated in two independent cohorts. To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas with estimated TIL percentages of 40% and higher. Genomic profiles of sorted and unsorted fractions were compared by shallow whole genome sequencing and confirm our findings. ConclusionThis study shows that genomic profiling of in particular basal-like, and thus BRCA1-mutated, breast carcinomas is severely affected by the presence of high numbers of TILs. Previous reports on genomic profiling of BRCA1-mutated breast carcinomas have largely neglected this. Therefore, our findings have direct consequences on the interpretation of published genomic data. Also, these findings could prove valuable in light of currently used genomic tools for assessing BRCA1-involvement in breast cancer patients and pathogenicity assessment of BRCA1 variants of unknown significance. The BRCA1-associated genomic aberrations identified in this study provide possible leads to a better understanding of BRCA1-associated oncogenesis.

Estimating risks for variants of unknown significance according to their predicted pathogenicity classes with application to BRCA1

Sequence-based testing of disease-susceptibility genes has identified many variants of unknown significance (VUSs) whose pathogenicity is unknown at the time of their measurement. Female breast cancer cases aged 20-49 years at diagnosis and who have VUSs in BRCA1 and no mutations in BRCA2 have previously been identified through the population-based Los Angeles County Cancer Surveillance Program. These nominal BRCA1 VUSs have been classified as "low," "medium," and "high" risk by four classification methods: Align-GVGD, Polyphen, Grantham matrix scores, and sequence conservation in mammalian species. Average hazard ratios (HRs) for classes of variants, i.e., the age-specific incidences of cancer for carriers of such variants divided by the population incidences, were estimated from the cancer family histories of first- and second-degree relatives of the index cases using modified segregation analysis. The study sample comprised 270 index cases and 4,543 of their relatives. There was weak evidence that the risk of breast cancer increases with the degree of sequence conservation (P = 0.03) and that missense variants at highly conserved sites are associated with a 5.6-fold (95 % confidence interval 1.4-22.2; P = 0.05) increased incidence of breast cancer. An upper bound of 2.3 is given for the average breast cancer HRs corresponding to variants classified as "low risk" by any of the four VUS classification methods. In summary, we have given a method to estimate cancer risks for groups of VUSs by combining existing classification methods with traditional penetrance analyses. This analysis suggests that classification methods for BRCA1 variants based on sequence conservation might be useful in a clinical setting. We have shown in principle that our method can be used to classify VUSs into clinically useful risk categories, but our specific findings should not be put into clinical practice unless confirmed by larger studies.

Follow-up of carriers of BRCA1 and BRCA2 variants of unknown significance: Variant reclassification and surgical decisions

PurposeApproximately 5–10% of patients who undergo genetic testing of BRCA1 and BRCA2 receive a variant of unknown significance (VUS) result. The ambiguous nature of a VUS may increase difficulty in patient understanding and decision making regarding risk reduction and surveillance options, including cancer risk-reducing surgeries. VUS reclassification to benign or deleterious may occur in time; however, clinical decisions may need to be made expeditiously, and some patients may pursue irreversible treatments before VUS reclassification. MethodsWe reviewed the surgical decisions of 107 women postdisclosure of a BRCA VUS result counseled at our institute between 1998 and 2009. ConclusionAmong women receiving a BRCA VUS result at our center, 11 of 107 (10.3%) pursued cancer risk-reducing mastectomy and 22 of 107 (20.6%) pursued cancer risk-reducing bilateral salpingo-oophorectomy. Reclassification of VUS occurred up to 9 years after testing, and 5 of 22 (22.7%) women followed up for 8 or more years continue to have a VUS result. We discuss considerations for providers of genetic services to discuss with patients who receive a VUS result.

Assessment of rare BRCA1 and BRCA2 variants of unknown significance using hierarchical modeling

Current evidence suggests that the genetic risk of breast cancer may be caused primarily by rare variants. However, while classification of protein-truncating mutations as deleterious is relatively straightforward, distinguishing as deleterious or neutral the large number of rare missense variants is a difficult on-going task. In this article, we present one approach to this problem, hierarchical statistical modeling of data observed in a case-control study of contralateral breast cancer (CBC) in which all the participants were genotyped for variants in BRCA1 and BRCA2. Hierarchical modeling permits leverage of information from observed correlations of characteristics of groups of variants with case-control status to infer with greater precision the risks of individual rare variants. A total of 181 distinct rare missense variants were identified among the 705 cases with CBC and the 1,398 controls with unilateral breast cancer. The model identified three bioinformatic hierarchical covariates, align-GV, align-GD, and SIFT scores, each of which was modestly associated with risk. Collectively, the 11 variants that were classified as adverse on the basis of all the three bioinformatic predictors demonstrated a stronger risk signal. This group included five of six missense variants that were classified as deleterious at the outset by conventional criteria. The remaining six variants can be considered as plausibly deleterious, and deserving of further investigation (BRCA1 R866C; BRCA2 G1529R, D2665G, W2626C, E2663V, and R3052W). Hierarchical modeling is a strategy that has promise for interpreting the evidence from future association studies that involve sequencing of known or suspected cancer genes.

BRCA1 c.5181_5183delGTT (p.Val1688del) is a true pathogenic mutation that affects both protein stability and function: implications for genetic counseling.

Abstract Abstract #1106 Germ line mutations in the BRCA1 tumor suppressor gene are associated with a significantly increased lifetime risk of breast and/or ovarian cancers. Since genetic testing has become a routinely step in the clinical practice, the discrimination between pathogenic mutations and benign variants appears a mandatory task as it can facilitate risk estimation and surveillance, as well as implementation of cancer prevention and treatment strategies. Most of the deleterious BRCA1 mutations identified so far are frameshift or nonsense ones that result in a premature translational truncation of the protein; only a small percentage is represented by missense mutations residing in the N-terminal RING finger or in the C-terminal BRCT domains. Interestingly, a growing number of variants of unknown significance (VUSs) are being described, which lack ad hoc functional assays thus remaining unclassified with respect to their pathogenic or neutral effect.
 Recently, disease-causality was predicted, based upon a multifactorial likelihood approach, for a BRCA1 VUS, c.5181_5183delGTT (p.Val1688del), recurrent in Italian breast/ovarian cancer families (Malacrida et al., 2008). The same VUS was also identified in one of the families recruited at the Dana-Farber Cancer Institute: pedigree analysis revealed early onset breast cancer in both the proband and her mother; family history confirmed Italian descent.
 To gain insights into the specific risk associated with the BRCA1 c.5181_5183delGTT (p.Val1688del) VUS, we investigated its impact on BRCA1 function. The sequence variant causes a single amino acid in frame deletion that falls within the first of the two tandem BRCA1 BRCTs. A series of approaches, including protein stability assays and co-immunoprecipitation experiments, as well as structural modeling based on sequence alignment, were employed to characterize both the full-length mutant protein (ΔValBRCA1) and its C-terminal fragment.
 We observed that ΔValBRCA1 displayed decreased stability when compared to the wild type counterpart. Moreover, when tested, interactions with known BRCA1 partners, BRIP1 and CtIP, were completely abrogated. In addition, ΔValBRCA1 failed to interact with Rap80 and was deficient in DNA damage response and repair by homologous recombination. These results demonstrate that BRCA1 c.5181_5183delGTT (p.Val1688del) is a true pathogenic sequence alteration and provide the first evidence of its effect on the stability and function of the encoded protein. We believe that our findings will impact the genetic counseling of those patients who harbor this newly defined mutation and their family members thus allowing them to benefit of appropriate risk assessment, prevention and therapeutic strategies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1106.