BRCA1 Variants Of Uncertain Significance Research Articles

Contralateral prophylactic mastectomy rate and predictive factors among patients with breast cancer who underwent multigene panel testing for hereditary cancer.

Although multigene panel testing is increasingly common in patients with cancer, the relationship between its use among breast cancer patients with non‐BRCA mutations or variants of uncertain significance (VUS) and disease management decisions has not been well described. This study evaluated the rate and predictive factors of CPM patients who underwent multigene panel testing. Three hundred and fourteen patients with breast cancer who underwent multigene panel testing between 2014 and 2017 were included in the analysis. Of the 314 patients, 70 elected CPM. Election of CPM by gene status was as follows: BRCA carriers (42.3%), non‐BRCA carriers (30.1%), and VUS (10.6%). CPM election rates did not differ between non‐BRCA carriers and BRCA carriers (P = 0.6205). Among non‐BRCA carriers, negative hormone receptor status was associated with CPM (P = 0.0115). For those with a VUS, hormone receptor status was not associated with CPM (P = 0.1879). Although the rate of CPM between BRCA carriers and non‐BRCA carriers was not significantly different, the predictors of CPM were different in each group. Our analyses shed the light on the increasing use of CPM among patients who are non‐BRCA carriers as well those with a VUS. Our study elucidates the differing predictive factors of CPM election among BRCA carriers, non‐BRCA carries, and those with a VUS. Our findings reveal the need for providers to be cognizant that non‐BRCA genes and VUS drive women to elect CPM despite the lack of data for contralateral breast cancer risk associated with these genes.

Characteristics of African American women at high-risk for ovarian cancer in the southeast: Results from a Gynecologic Cancer Risk Assessment Clinic

ObjectivesDescribe patient characteristics in African American (AA) women seen for gynecologic cancer related genetic counseling at a large southeastern comprehensive cancer center. MethodsWe reviewed an IRB approved, prospective observational cohort of patients from a Gynecologic Cancer Risk Assessment Clinic. Data evaluated included personal cancer history, family history, frequency of genetic testing, frequency/type of genetic mutations, and frequency of surgical intervention. Standard statistical statistics were utilized. Results1227 patients were evaluated from 2003 to 2015, of which 95 (7.7%) were AA. Sixteen patients had a personal history of ovarian cancer. 21 women (22%) underwent genetic counseling only; subsequent genetic testing was not recommended based on absence of risk factors. Of the seventy-four AA patients in whom genetic testing was recommended, sixty-six (69.5%) completed testing. Of women tested, 37 (56%) had abnormal results. Eight and 14 patients had pathogenic variants in BRCA1 and BRCA2, respectively. Two were found to have pathogenic PALB2 variants; one had a pathogenic ATM variant and one constitutional MLH1 epimutation case was identified. Eleven had BRCA variants of uncertain significance. Of the patients with abnormal testing, six of 22 women with pathogenic BRCA variants underwent risk-reducing salpingo-oophorectomy (RRSO). ConclusionsOur study demonstrates that in a region where AAs represent 27% of the population, the proportion of AA patients referred to a Gynecologic Cancer Risk Assessment Clinic remains low. Pathogenic variant and variant of uncertain significance rates were high in patients tested, likely representing a selection bias of high-risk patients. Endeavors should continue to identify minorities at risk for ovarian cancer and institute measures to provide thorough genetic counseling and testing.

Abstract GS4-06: Cancer risks and response to targeted therapy associated with BRCA2 variants of uncertain significance

Abstract Background: Germline genetic testing of individuals with a diagnosis of triple negative breast cancer, young age at diagnosis, or a family history of breast and/or ovarian cancer has led to the identification of many unique BRCA2 missense variants of uncertain significance (VUS). VUS in BRCA2 are predominantly missense mutations that have unclear relevance to breast, ovarian, and other cancers. Thus, patients found to have a germline BRCA2 VUS do not know if the variant is associated with high risks of these cancers similar to truncating mutations, intermediate risks more similar to CHEK2 mutations, or low risks of no clinical significance. Furthermore, it is unclear if germline BRCA2 VUS, or somatic VUS identified by tumor sequencing, are associated with hypersensitivity to selected DNA damaging and cross-linking agents. Methods: We have used a homology directed repair (HDR) cell-based assay to characterize missense variants in the DNA binding domain (DBD) of BRCA2. The method has been validated using known pathogenic and known non-pathogenic BRCA2 missense variants and has 100% sensitivity (95% confidence interval (CI): 75.3%–100%) and 100% specificity (95% CI: 81.5%–100%) for pathogenic BRCA2 variants. A classifier of variant pathogenicity based on the mean and variances of the distributions of the HDR results of the known pathogenic and neutral variants has been established. We have also developed PARP inhibitor and cisplatin drug response assays for BRCA2 missense variants. Results: Assessment of 207 BRCA2 missense variants, identified in public databases such as BRCA exchange and ClinVar, by the HDR assay identified 71 deleterious variants with >99% probability of pathogenicity, 116 neutral variants with >99% probability of neutrality, and 20 with hypomorphic activity and potentially intermediate risk. A combination of the functional data and sequence-based predictors of protein activity in a Bayesian prediction model resulted in classification of the deleterious variants as pathogenic cancer predisposing variants and the neutral variants as non-pathogenic with low clinical significance. The influence of the deleterious/pathogenic variants on PARPi and cisplatin response was also assessed. Conclusion: The HDR assay is effective for characterization of BRCA2 VUS. The combination of functional data and in silico prediction models provides a robust tool for clinical annotation of BRCA2 VUS. HDR function of BRCA2 missense variants is strongly correlated with response to targeted therapy. Citation Format: Couch FJ, Shimelis H, Hart SN, Moore RM, Thomas A, Lipton GB, Iversen E. Cancer risks and response to targeted therapy associated with BRCA2 variants of uncertain significance [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 GS4-06.

Assessment of the Clinical Relevance of BRCA2 Missense Variants by Functional and Computational Approaches

Many variants of uncertain significance (VUS) have been identified in BRCA2 through clinical genetic testing. VUS pose a significant clinical challenge because the contribution of these variants to cancer risk has not been determined. We conducted a comprehensive assessment of VUS in the BRCA2 C-terminal DNA binding domain (DBD) by using a validated functional assay of BRCA2 homologous recombination (HR) DNA-repair activity and defined a classifier of variant pathogenicity. Among 139 variants evaluated, 54 had ?99% probability of pathogenicity, and 73 had ?95% probability of neutrality. Functional assay results were compared with predictions of variant pathogenicity from the Align-GVGD protein-sequence-based prediction algorithm, which has been used for variant classification. Relative to the HR assay, Align-GVGD significantly (p < 0.05) over-predicted pathogenic variants. We subsequently combined functional and Align-GVGD prediction results in a Bayesian hierarchical model (VarCall) to estimate the overall probability of pathogenicity for each VUS. In addition, to predict the effects of all other BRCA2 DBD variants and to prioritize variants for functional studies, we used the endoPhenotype-Optimized Sequence Ensemble (ePOSE) algorithm to train classifiers for BRCA2 variants by using data from the HR functional assay. Together, the results show that systematic functional assays in combination with in silico predictors of pathogenicity provide robust tools for clinical annotation of BRCA2 VUS.

Germline BRCA mutations in Asian patients with pancreatic adenocarcinoma: a prospective study evaluating risk category for genetic testing.

Introduction Germline BRCA mutations may have therapeutic implications as surrogate markers of DNA-damage repair status in pancreatic ductal adenocarcinoma (PDAC). We performed a prospective study to evaluate the efficiency of risk criteria based on personal or family history of breast and ovarian cancer for determining germline BRCA mutations in PDAC patients with Asian ethnicity. Methods Between November 2015 and May 2016, we screened consecutive PDAC patients with locally advanced unresectable or metastatic disease who were referred for systemic chemotherapy. Analyses for germline BRCA mutations were performed if patients had one or more first-degree or second-degree relatives with breast or ovarian cancers or had a personal medical history of these diseases. DNA was extracted from whole blood, and all coding exons and their flanking intron regions of BRCA1 and BRCA2 were sequenced. Results A total of 175 patients were screened for personal and family history and 10 (5.7%) met the inclusion criteria for genetic sequencing. Pathogenic germline BRCA2 mutation [c.7480C>T (p.Arg2494*)] was identified in one male patient, resulting in a frequency of 10% for the risk-stratified patients and 0.6% for the unselected PDAC population. Two patients had germline BRCA2 variants of uncertain significance [c.1744A>C (p.Thr582Pro) and c.68-7T>A]. Conclusion Personal or family history of breast or ovarian cancers is a feasible, cost-effective risk categorization for screening germline BRCA mutations in Asian PDAC patients as 10% of this population had the pathogenic mutation herein. Future validation from a large, prospective cohort is needed.

Clinical Decision-Making in Patients with Variant of Uncertain Significance in BRCA1 or BRCA2 Genes.

How diagnosis with a variant of uncertain significance (VUS) in a BRCA gene impacts clinical decision-making is not well known. We queried for all patients attending Mayo Clinic Rochester from 2004 to 2016 who tested positive for BRCA1 or BRCA2 VUS and reviewed patient management choices. Groups were compared by using Wilcoxon rank-sum and Chi-square tests. We identified 97 patients (95 females, 2 males) with BRCA VUS. For patients without cancer history (n=20), 80% had a mother or sister with breast cancer, and median Tyrer-Cuzick (IBIS) lifetime breast cancer risk score was 27% (range 16-62%). Management included bilateral prophylactic mastectomy (BPM) in 39%, where choice for BPM was significantly associated with IBIS score (median 32 vs. 24%, p=0.02) and first-degree family history of breast cancer (100 vs. 64%, p=0.03) but not Gail score or total number of family members with cancer. For patients with breast cancer who had known VUS statusprior to surgery (n=9), the rate of contralateral prophylactic mastectomy (CPM) was 22% compared with 25% without known VUS and 83% with known BRCA pathogenic mutation. In 21 of 97 (22%) patients, the BRCA VUS has been reclassified (95% benign, 5% deleterious). BRCA VUS carriers with cancer elected surgical choices similar to average-risk breast cancer patients. However, VUS carriers without cancer had high rates of BPM, associated with first-degree family history and IBIS score. Over time, a significant proportion of BRCA VUS were reclassified, illustrating the importance of appropriate counseling regarding VUS.

29 - Persistence of Preleukemic Clonal Hematopoiesis after Chemotherapy is Associated with Poor Prognosis in Patients with High Risk MDS and AML

Describe patient characteristics in African American (AA) women seen for gynecologic cancer related genetic counseling at a large southeastern comprehensive cancer center.We reviewed an IRB approved, prospective observational cohort of patients from a Gynecologic Cancer Risk Assessment Clinic. Data evaluated included personal cancer history, family history, frequency of genetic testing, frequency/type of genetic mutations, and frequency of surgical intervention. Standard statistical statistics were utilized.1227 patients were evaluated from 2003 to 2015, of which 95 (7.7%) were AA. Sixteen patients had a personal history of ovarian cancer. 21 women (22%) underwent genetic counseling only; subsequent genetic testing was not recommended based on absence of risk factors. Of the seventy-four AA patients in whom genetic testing was recommended, sixty-six (69.5%) completed testing. Of women tested, 37 (56%) had abnormal results. Eight and 14 patients had pathogenic variants in BRCA1 and BRCA2, respectively. Two were found to have pathogenic PALB2 variants; one had a pathogenic ATM variant and one constitutional MLH1 epimutation case was identified. Eleven had BRCA variants of uncertain significance. Of the patients with abnormal testing, six of 22 women with pathogenic BRCA variants underwent risk-reducing salpingo-oophorectomy (RRSO).Our study demonstrates that in a region where AAs represent 27% of the population, the proportion of AA patients referred to a Gynecologic Cancer Risk Assessment Clinic remains low. Pathogenic variant and variant of uncertain significance rates were high in patients tested, likely representing a selection bias of high-risk patients. Endeavors should continue to identify minorities at risk for ovarian cancer and institute measures to provide thorough genetic counseling and testing.

Abstract OI-1: OI-1 Decoding breast cancer predisposition genes

Abstract Women with one or more first-degree female relatives with a history of breast cancer have a two-fold increased risk of developing breast cancer. This risk increases with the number of affected family members, particularly for those diagnosed at a young age or with bilateral disease. Segregation analyses of breast cancer families led to the identification of inactivating mutations in the BRCA1 and BRCA2 breast cancer susceptibility genes. Pathogenic variants resulting in truncation of these proteins, along with a small number of pathogenic missense variants, have been associated with 55% to 65% lifetime risk of breast cancer in the general population and up to 85% lifetime risks among women with a strong family history of the disease. In contrast, the influence on cancer risk of many rare variants of uncertain significance (VUS) is not known. Mutations in several genes in addition to BRCA1 and BRCA2 have been implicated in predisposition to breast cancer through family segregation studies. These include moderate risk mutations (relative risk between 2.0 and 5.0) in CHEK2 and ATM, high-risk mutations (relative risk greater than 5.0) in PALB2, and high-risk mutations in CDH1, PTEN, STK11, and TP53 that are associated with hereditary diffuse gastric cancer, Cowden, Peutz-Jeghers, and Li-Fraumeni syndromes. However, risks of breast and other cancers associated with mutations in these and other genes that have been suggested to predispose to cancer have not been established or have very broad confidence intervals. Despite the absence of accurate risk estimates clinical testing with gene panels for hereditary cancer gene mutations has become an integral component of medical management for high-risk families and individuals with triple negative breast cancer. To estimate breast cancer risks several large studies of breast cancer patients from the general population and from high-risk families are underway. These studies are expected to identify the subset of panel genes that are associated with increased risk of breast cancer or breast cancer subtypes and to establish more accurate risks of breast and other cancers. In addition, classification of the clinical relevance of VUS in BRCA1, BRCA2, and other predisposition genes remains a significant problem, with patients unable to benefit from enhanced risk assessment, prevention, and clinical management. Thousands of VUS in BRCA1 and BRCA2 have already been identified through clinical testing and many more in BRCA1, BRCA2, and other predisposition genes are being identified through panel-based genetic testing and tumor sequencing studies. Efforts to classify VUS as neutral or pathogenic have focused on large case-control studies and quantitative multifactorial models based on family history of cancer and pathology. However, more recent studies have established the utility of validated functional assays for VUS assessment. Hundreds of BRCA1 and BRCA2 VUS have been reclassified by commercial testing laboratories or by the ENIGMA consortium that serves as the expert review panel for variants in these genes for the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and BRCA Exchange (http://brcaexchange.org) publically accessible websites. Common genetic variants associated with breast cancer risk have also been identified through genome-wide association studies (GWAS). The magnitude of these associations have ranged from 1.03 to 1.29 in women of European ancestry. While the majority of the 179 known breast cancer variants are associated with estrogen receptor (ER) positive breast cancer, 21 of these show specific associations with ER negative or triple negative (ER negative, PR negative, HER2 negative) breast cancer. The variants in risk loci account for approximately 19% of the familial relative risk of breast cancer. Several studies have recently established that polygenic risk scores derived from these risk variants improve personalized risk assessment by identifying women at increased or reduced risk of breast cancer in the general population and by modifying risks of breast cancer among BRCA1 and BRCA2 mutation carriers. In summary, germline genetic studies have substantially changed the landscape of breast cancer, identifying mutations associated with moderate and high risks of breast cancer in up to 5% of population-based breast cancer cases and 35% of high-risk families and providing personalization of underlying breast cancer risk for all women. Citation Format: Couch FJ. OI-1 Decoding breast cancer predisposition genes [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 OI-1.

In silico, in vitro and case-control analyses as an effective combination for analyzing BRCA1 and BRCA2 unclassified variants in a population-based sample

Ascertaining the clinical consequences of BRCA1 and BRCA2 variants of uncertain significance (VUS) is currently indispensable for providing effective genetic counseling and preventive actions for families with hereditary breast and ovarian cancer (HBOC). To this end, we conducted a combination of in silico prediction and cDNA splicing analyses of 13 BRCA1 and 10 BRCA2 VUS identified in our cohort as well as a case-control analysis in a population-based sample of 10 recurrent VUS. We observed consistent results between the in silico predictions and sequencing analyses for all analyzed VUS. An abnormal cDNA pattern was observed for variants c.212+1G>A and c.5278-1G>A in BRCA1 and c.516+2T>A and c.8168A>G in BRCA2 according to in silico splicing prediction. A case-control study of VUS confirmed the polymorphisms of the c.67+62A>G, c.7008-62A>G and c.8851G>A BRCA2 variants previously published. c.4068G>A in the BRCA2 gene can also be considered a polymorphism due to its occurrence at a frequency greater than 1% in our population. Our study shows that employing population-based analysis and a combination of several in silico methods yields highly accurate information, resulting in a reliable tool for selecting variants for cDNA sequencing analysis in routine cancer genetic counseling units.

Comparative analysis of BRCA1 and BRCA2 variants of uncertain significance in patients with breast cancer: a multifactorial probability-based model versus ACMG standards and guidelines for interpreting sequence variants.

To investigate variants of uncertain significance (VUS) in BRCA1 and BRCA2, we assessed the multifactorial posterior probability of VUS in BRCA1 and BRCA2 and compared these analyses with interpretations according to the recently released American College of Medical Genetics and Genomics (ACMG) standards and guidelines. The analysis involved 715 Korean patients with breast cancer. The multifactorial probability of a VUS was analyzed using the prior probability and combined likelihoods of personal and family history, the pathologic profile of the breast cancer, and co-occurrence with pathogenic variants. Results were compared with those obtained according to the ACMG standards/guidelines. Sixteen VUS from 51 BRCA1 VUS carriers and 28 VUS from 62 BRCA2 VUS carriers were analyzed. There was a slight agreement between the two analyses, with a kappa value of 0.14 (95% confidence interval (CI) = -0.34 to 0.62) for the BRCA1 VUS and a kappa value of 0.17 (95% CI = -0.10 to 0.49) for the BRCA2 VUS. We propose that genetic counseling should be based on the concordant results between these two analyses. When discrepancies are found, those variants are still considered VUS and careful counseling should be provided.Genet Med 18 12, 1250-1257.

Improving performance of multigene panels for genomic analysis of cancer predisposition

Screening multiple genes for inherited cancer predisposition expands opportunities for cancer prevention; however, reports of variants of uncertain significance (VUS) may limit clinical usefulness. We used an expert-driven approach, exploiting all available information, to evaluate multigene panels for inherited cancer predisposition in a clinical series that included multiple cancer types and complex family histories. For 1,462 sequential patients referred for testing by BROCA or ColoSeq multigene panels, genomic DNA was sequenced and variants were interpreted by multiple experts using International Agency for Research on Cancer guidelines and incorporating evolutionary conservation, known and predicted variant consequences, and personal and family cancer history. Diagnostic yield was evaluated for various presenting conditions and family-history profiles. Of 1,462 patients, 12% carried damaging mutations in established cancer genes. Diagnostic yield varied by clinical presentation. Actionable results were identified for 13% of breast and colorectal cancer patients and for 4% of cancer-free subjects, based on their family histories of cancer. Incidental findings explaining cancer in neither the patient nor the family were present in 1.7% of subjects. Less than 1% of patients carried VUS in BRCA1 or BRCA2. For all genes combined, initial reports contained VUS for 10.5% of patients, which declined to 7.5% of patients after reclassification based on additional information. Individualized interpretation of gene panels is a complex medical activity. Interpretation by multiple experts in the context of personal and family histories maximizes actionable results and minimizes reports of VUS.Genet Med 18 10, 974-981.

Investigating the effect of 28 BRCA1 and BRCA2 mutations on their related transcribed mRNA.

Germline inactivating mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome (HBOCS). Genetic testing of these genes identifies a significant proportion of variants of uncertain significance (VUS). Elucidation of the clinical impact of these variants is an important challenge in genetic diagnostics and counseling. In this study, we assess the RNA effect of 28 BRCA1 and BRCA2 VUS identified in our set of HBOCS families with the aim of gaining insight into their clinical relevance. mRNA was extracted from VUS carriers and controls lymphocytes cultured for 5-6 days and treated with puromycin. RNA was reverse transcribed to perform transcriptional analysis for the study of splicing aberrations. In silico prediction tools were used to select those variants most likely to affect the RNA splicing process. Six out of the 28 variants analyzed showed an aberrant splicing pattern and could therefore be classified as probably pathogenic mutations. Reclassification of VUS improves the genetic counseling and clinical surveillance of carriers of these mutations and highlights the importance of RNA studies in routine diagnostic laboratories.

Understanding of BRCA VUS genetic results by breast cancer specialists.

BackgroundMainstreaming genetic medicine, increased media coverage and clinical trials for BRCA mutation carriers are leading oncologists into more patient discussions about BRCA genetic testing. BRCA variants of uncertain significance (VUS) occur in 10–20 % of tests. VUS detection introduces additional uncertainty for patient and potentially clinician. We aimed to explore the ability of breast cancer specialists (BCS) in the UK to correctly respond to a VUS report.MethodsA survey sent to 800 UK BCS collected demographics data, VUS general knowledge and interpretation and communication based on two genetics reports. A separate survey of UK clinical geneticists collected demographics data, laboratory reporting practice and methods used to clarify VUS pathogenicity including classification systems.ResultsOf the 155 BCS (22.5 %) who completed the survey, 12 % reported no genetics training. Ninety five percent referred patients for BRCA genetic tests, 71 % felt unsure about the clinical implications of the test reports presented here. A VUS report from a patient with a positive family history was interpreted and theoretically communicated correctly by 94 % but when presented with a different VUS report with no management guidance and negative family history, 39 % did not know how to communicate this result to the patient. Geneticists reported multiple VUS classification systems; the most commonly used was word-based in 32 %.ConclusionsA consistent and standardised format to report particularly VUS results across all diagnostic laboratories plus additional training of UK BCS will be necessary for effective mainstreaming of BRCA testing to the oncology clinic.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1934-1) contains supplementary material, which is available to authorized users.

Abstract 4685: Characterization of variants of uncertain significance (VUS) in breast and ovarian cancer predisposition genes

Abstract Panel-based germline genetic testing of established breast and ovarian cancer predisposition genes is widely used in high-risk clinics to inform on risks of breast and ovarian cancer. While most protein truncating mutations identified in predisposition genes are associated with moderate to high risks of these cancers, variants of uncertain significance (VUS), in the form of missense and intronic variants, have an undefined relevance to cancer. Many hundreds of VUS in BRCA1 and BRCA2 identified by clinical genetic testing over the last 20 years still have uncertain clinical relevance. In addition, VUS in other known predisposition genes are being identified by current panel-testing in up to 10% of individuals without BRCA1 or BRCA2 mutations. These VUS cause problems in cancer risk estimation and clinical management of patients undergoing clinical genetic testing. In an effort to evaluate the clinical significance of VUS in cancer predisposition genes we have developed functional assays that characterize the impact of the variants on the functions of the proteins encoded by the predisposition genes. In particular, a homology directed repair reporter assay providing a quantitative measure of homologous recombination repair of DNA double strand breaks has been developed for characterization of VUS in homologous recombination associated genes. When applied to 13 known pathogenic and 30 known neutral variants from the DNA binding domain of BRCA2, this assay exhibits 100% specificity and 100% sensitivity. Based on these results a model estimating the probability of pathogenicity of each BRCA2 VUS was developed. The HDR assay and probability model has now been applied to 230 BRCA2 VUS identified through genetic testing. Here we identify 80 BRCA2 variants that are deficient in HDR activity. We further combined these results with prior probabilities of pathogenicity derived from the sequence based Align GVGD in silico prediction model to estimate the posterior probability of pathogenicity for each VUS. Our results suggest that in the absence of family based segregation information, results from the HDR functional assay and in silico prediction models can be used to predict the pathogenicity of BRCA2 VUS. The assay has also been used to characterize VUS in the PALB2 and RAD51C predisposition genes, leading to identification of a subset of missense variants that are deficient in HDR repair activity and may be associated with increased risk of breast and ovarian cancer. These results will prove useful for improved risk assessment of carriers of these BRCA2, PALB2 and RAD51C variants and their family members and for validation of other approaches to mutation classification. Citation Format: Fergus J. Couch, Hermela Shimelis, Andreas Schroeder, Chunling Hu, Emily Hallberg, Gary Lipton, Edwin Iversen, Noralane M. Lindor. Characterization of variants of uncertain significance (VUS) in breast and ovarian cancer predisposition genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4685. doi:10.1158/1538-7445.AM2015-4685

The influence of BRCA variants of uncertain significance in cancer risk management decision-making.

1555 Background: Among patients undergoing genetic testing for BRCA1/2 mutations, 7% will harbor a variant of uncertain significance (VUS), a finding with more emotional than clinical weight. The o...

Abstract P4-12-08: Utilizing next generation sequencing technologies for hereditary breast cancer risk assessments in a private oncology practice

Abstract Background: The recent introduction of clinical Next Generation Sequencing (NGS) technologies has transformed the ability of health care providers to provide personalized hereditary breast cancer risk assessments to patients and their family members. However, along with the plethora of data derived from NGS technologies come challenges regarding how to interpret this data in a clinically meaning way to guide medical management decisions. Methods: A retrospective analysis was performed on a population of 242 patients with a personal or family history of breast and/or ovarian cancer and completed NGS genetic testing between March 2012 and May 2014 at Ambry Genetics. Data points included age, gender, primary diagnosis, type of NGS genetic test and result. Patients with prior genetic testing were not excluded from the analysis. Results: Of the 242 patients who completed NGS genetic testing 199 (82%) had a personal history of cancer and 43 (18%) had a family history of cancer. Ten (5%) clinically significant deleterious mutations were found in the affected population; 3 MUTYH mutations were consider not clinically significant and excluded from the analysis. Four (40%) were CHEK2 mutations, 2(20%) BRCA1 mutations, 1(10%) NBN mutation, 1(10%) ATM mutation, 1(10%) MSH6 mutation and 1(10%) PTEN mutation. One patient with a history of ovarian cancer had two deleterious mutations in the BRCA1 and NBN genes, and three affected patients had a deleterious mutation in addition to a variant of uncertain significance (VUS); a patient with synchronous breast and ovarian cancers had a MSH6 mutation and a BRCA2 VUS, a patient with breast cancer had a CHEK2 mutation and a CHEK2 VUS, and a patient with breast cancer had a CHEK2 mutation and a MSH6 VUS. A total of 37 (19%) VUS were found in the affected population, including one patient with a history of breast cancer and a VUS in both ATM and MRE11A. A single (2%) clinically significant BRCA1 deleterious mutation and 6(14%) VUS were detected in the unaffected population. Conclusions: NGS technologies allow for health care providers to complete more comprehensive hereditary breast cancer risk assessments for patients and their families. However, the high VUS rate (14-19%) and the possibility for multiple mutations and/or variants in a single patient create unique challenges when interpreting NGS genetic test results. Furthermore, the paucity of prospective studies and consensus guidelines limits the ability of the health care provider to interpret NGS genetic test results in a clinically meaningful way to guide patient management. This is highlighted by the case example above regarding the patient with synchronous breast and ovarian cancers who was found to have a MSH6 mutation and a BRCA2 VUS. Given that the patient does not meet Amsterdam criteria, the MSH6 mutations would have been missed had it not been for the NGS technology utilized for this patient. However, challenges still exist about how to interpret this data for both the patient and her family in a clinically meaningful way given that the family does not look like a classic Lynch Syndrome family. Citation Format: Barbara Hamlington, Scot Sedlacek, Lucy Langer, Brittney Goetsch, Katie Lemas, Sami Diab. Utilizing next generation sequencing technologies for hereditary breast cancer risk assessments in a private oncology practice [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-08.

453P - Phase 1 Study of the Parp Inhibitor E7449 As a Single Agent in Patients with Advanced Solid Tumors or B-Cell Lymphoma

ABSTRACT Aim: E7449 is an orally bioavailable, brain penetrable, potent small-molecule inhibitor of poly (ADP-ribose) polymerase (PARP) 1 and PARP 2 with an IC50 of 1.0 and 1.2 nmol/L respectively. E7449 is a poor P-gp substrate. Preclinically, E7449 potentiates the antitumour activity of chemotherapy and radiotherapy and has activity as a single-agent in BRCA-deficient and other tumours. A Phase 1 study of E7449 as a single agent is underway to determine the MTD, safety, PK, PD, preliminary activity and perform exploratory biomarker analysis. Methods: Patients (pts) with advanced solid tumours or B-cell lymphoma, ECOG PS 0-1, > 18 years (yrs) and adequate organ function are eligible. E7449 is administered orally, once daily (QD), continuously. Blood samples for PK, PD and biomarkers are collected at multiple time points. Results: 28 pts have been treated at 6 dose levels: 50, 100, 200, 400, 800 and 600 mg QD. Tumour types most frequently included were pancreatic (n = 5), ovarian (n = 5) and lung (n = 4). Five DLTs have been observed, four at 800 mg: Gr3 fatigue (n = 1) and Gr2 fatigue requiring more than 7days (d) dose interruption (n = 3) and one at 600 mg, drug interruption for more than 7d and Gr3 allergic reaction. The MTD was achieved at 600 mg QD. Frequently occurring related adverse events (AEs) (any grade) are fatigue: 48% (Gr3: 12%), photosensitive rash (48%), diarrhoea and nausea: 32% each, chromaturia: 28%, depression: 20%, and decreased appetite: 20% (Gr3: 4%). No significant haematological toxicity and no Gr4 related or fatal AEs have been reported. One PR in a high grade serous ovarian (HGSO) cancer pt (BRCA unknown) and 6 SD for >23 weeks have been observed (one in a pancreatic cancer pt with BRCA2 variant of uncertain significance, one in a HGSO cancer pt BRCA mutant). E7449 PK exhibits rapid absorption, tmax ∼1.5 hrs and elimination t1/2 ∼9 hrs, with dose proportional exposures. Sustained PARP inhibition (PARPi) of ∼90% is observed in PMBCs at 600 mg QD. Predictive biomarker analysis is ongoing. Conclusions: In this study, E7449 was generally well tolerated at the MTD of 600 mg QD, with preliminary evidence of antitumour activity and sustained PARPi in PMBCs. Further evaluation will continue in Phase 1b/2 combination studies. Disclosure: B.D.L. Heras: I am Director of Clinical Development, Eisai Inc; B. Ink: I am Director of Project Management, Eisai Ltd; M. Matijevic: Associate Director, Eisai Inc.; D. Sarker: I have a consultant/advisory relationship to disclose at Eisai Inc.All other authors have declared no conflicts of interest.

Pathogenicity Evaluation of BRCA1 and BRCA2 Unclassified Variants Identified in Portuguese Breast/Ovarian Cancer Families

Hereditary breast/ovarian cancer syndrome is caused by germline deleterious mutations in BRCA1 and BRCA2. A major problem of genetic testing and counseling is the finding of variants of uncertain significance (VUS). We sought to ascertain the pathogenicity of 25 BRCA1 and BRCA2 VUS identified in Portuguese families during genetic testing. We performed cosegregation analysis of VUS with cancer in families, evaluated their frequency in unaffected controls, and looked for loss of heterozygosity in tumors. In addition, three different bioinformatic algorithms were used (Interactive Biosoftware, ESEfinder, and PolyPhen). Finally, six VUS located in exon-intron boundaries were analyzed by RT-PCR. We found that seven variants segregated with the disease, six variants co-occurred with a pathogenic mutation in the same gene, and four variants co-occurred with a deleterious mutation in the other BRCA gene. By RT-PCR, we observed that four variants (BRCA1 c.4484G>T, BRCA2 c.682-2A>C, BRCA2 c.8488-1G>A, and BRCA2 c.8954-5A>G) disrupted splicing. After the combined analysis, we were able to classify 4 splicing variants as pathogenic mutations, 16 variants as neutral, and 3 variants as polymorphisms; only 2 variants remained classified as VUS. This work highlights the contribution of DNA, RNA, and in silico data to assess the pathogenicity of BRCA1/2 VUS, which, in turn, allows more accurate genetic counseling and clinical management of the families carrying them.

A High-Throughput Functional Complementation Assay for Classification ofBRCA1Missense Variants

Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains. BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance.

Functional analysis of BRCA1 missense variants of uncertain significance in Japanese breast cancer families

Germline mutations in the tumor suppressor genes BRCA1 and BRCA2 are responsible for a large proportion of familial breast cancer cases, and therefore, BRCA1 and BRCA2 genetic testing has become increasingly common in clinical practice. However, variants of uncertain significance (VUS) have been detected in 16.3% of Japanese patients suspected of having hereditary breast and ovarian cancers. The clinical importance of VUS is unknown, and their incidence has led to issues in risk counseling, assessment and treatment of cancer patients. In the present study, we performed functional analyses of two VUS in BRCA1, A1752G and Y1853C that were detected in two independent breast cancer patients who were suspected of having hereditary breast cancer. Segregation analysis revealed that Y1853C, but not A1752G, was cosegregated in affected family members. Conservation, transcription and structure analyses also supported the pathogenic potential of Y1853C. Detailed segregation and in silico and in vitro analyses will enhance our understanding of VUS and improve the management of cancer patients and their families.