Functional characterization of splice variants in hereditary breast and ovarian cancer susceptibility genes.

Simple SummaryGenotyping of BRCA genes is required for treatment with PARP inhibitors. Purposing to address treatment needs and familial cancer risk, the aim of our study was to introduce the most appropriate testing workflow in epithelial ovarian cancer patients (EOC) using germline and tumor genotyping of BRCA and other hereditary breast and/or ovarian cancer (HBOC) susceptibility genes. In consecutive patients with advanced non-mucinous EOC, who responded to platinum-based chemotherapy, germline and tumor genotyping were performed using Illumina’s next generation sequencing (NGS) panels. Sensitivity of tumor genotyping for detection of germline pathogenic/likely pathogenic variants (PV/LPV) was 96.2% for BRCA and 93.3% for HBOC genes. With germline genotyping-only strategy, 58.8% of HBOC PV/LPV and 68.4% of BRCA PV/LPV were detected. By tumor genotyping-only, 96.1% of HBOC PV/LPV and 97.4% of BRCA PV/LPV were detected. Tumor genotyping first followed by germline genotyping detects nearly all germline and somatic PV/LPV in the shortest time.Detection of germline and somatic pathogenic/likely pathogenic variants (PV/LPV) in BRCA genes is at the moment a prerequisite for use of PARP inhibitors in different treatment settings of different tumors. The aim of our study was to determine the most appropriate testing workflow in epithelial ovarian cancer (EOC) patients using germline and tumor genotyping of BRCA and other hereditary breast and/or ovarian cancer (HBOC) susceptibility genes. Consecutive patients with advanced non-mucinous EOC, who responded to platinum-based chemotherapy, were included in the study. DNA extracted from blood and FFPE tumor tissue were genotyped using NGS panels TruSightCancer/Hereditary and TruSight Tumor 170. Among 170 EOC patients, 21.8% had BRCA germline or somatic PV/LPV, and additionally 6.4% had PV/LPV in other HBOC genes. Sensitivity of tumor genotyping for detection of germline PV/LPV was 96.2% for BRCA genes and 93.3% for HBOC genes. With germline genotyping-only strategy, 58.8% of HBOC PV/LPV and 68.4% of BRCA PV/LPV were detected. By tumor genotyping-only strategy, 96.1% of HBOC PV/LPV and 97.4% of BRCA PV/LPV were detected. Genotyping of tumor first, followed by germline genotyping seems to be a reasonable approach for detection of PV/LPV in breast and/or ovarian cancer susceptibility genes in non-mucinous EOC patients.

BRCA1 and BRCA2 are the most well-known breast and ovarian cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. Recently, RAD51C, a new Fanconi Anemia gene, essential for homologous recombination repair, has been reported to be a rare hereditary breast and ovarian cancer susceptibility gene. Indeed, several pathogenic mutations have been identified in BRCA1/BRCA2-negative hereditary breast and ovarian cancer families. Here, we present the results of the screening of RAD51C mutations in a large series of 516 BRCA1/BRCA2-negative Spanish patients from breast and/or ovarian cancer families, and the evaluation of these results in the context of all RAD51C carriers. RAD51C mutation screening was performed by DNA analysis for all index cases. All the genetic variants identified were analyzed in silico for splicing and protein predictions. cDNA analysis was performed for three selected variants. All previous RAD51C mutation studies on breast and/or ovarian cancer were reviewed. We identified three inactivating RAD51C mutations. Two mutations were found in breast and ovarian cancer families and one mutation in a site-specific breast cancer family. Based on the mean age of ovarian cancer diagnosis in RAD51C carriers, we would recommend prophylactic bilateral salpingo-ophorectomy in premenopausal RAD51C mutation carriers. Our results support that RAD51C is a rare breast and ovarian cancer susceptibility gene and may contribute to a small fraction of families including breast and ovarian cancer cases and families with only breast cancer. Thus, RAD51C testing should be offered to hereditary breast and/or ovarian cancer families without selecting for specific cancer origin.

Background: Strategies exist to decrease cancer incidence among individuals with HBOC syndromes. These include: 1) surgeries such as risk-reducing mastectomy (RRM) and risk reducing bilateral salpingo-oophorectomy (RRBSO) and 2) risk-reducing medications with selective estrogen receptor modulators (SERM) or aromatase inhibitors (AI). RRM decreases the incidence of breast cancer by >90%. RRBSO decreases ovarian cancer risk by >80%, may decrease breast cancer risk, and decreases mortality in individuals with BRCA1 and BRCA2 mutations. Use of 5 years of SERM or AI reduces risk of future breast cancer by 40-50% in individuals at high risk, but data regarding efficacy in individuals with HBOC syndromes are limited. To date, few studies have explored cancer risk reducing treatment decisions among individuals with hereditary cancer susceptibility without personal history of malignancy. Methods: Individuals evaluated in the Breast and Ovarian Cancer Risk Evaluation Clinic (BOCREC) at the University of Michigan Rogel Cancer Center with pathogenic germline variants (PGVs) conferring increased cancer risk without personal history of breast, ovarian, pancreatic, or prostate cancer from 1/1/2012 to 1/1/2022 were eligible for analysis. Basic demographic information (age, sex, and race/ethnicity), pursuit of cancer risk reduction measures, and subsequent cancer diagnoses were recorded. A chi-squared test was used to compare rates of RRM among carriers of high penetrance versus moderate penetrance breast cancer susceptibility genes. Results: 402 individuals were included in this analysis. The majority were female (80%) and white (91%; Not listed 5%; Asian 3%; Black 1%). The most common PGVs identified were in BRCA1 (32%, n=134) and BRCA2 (32%, n=133), followed by CHEK2 (16%, n=66), ATM (7%, n=30), PALB2 (4%, n=18), BRIP1 (3%, n=14), RAD51C/RAD51D (2%, n=8), Lynch Syndrome associated PGVs (1.2%, n=5), CDH1 (0.5%, n=2), TP53 (0.5%, n=2), CDKN2A (0.2%, n=1), and PTEN (0.2%, n=1). Of the 295 individuals eligible for RRM (female, PGV in breast cancer susceptibility gene), 108 (37%) underwent RRM. Of those who had a RRM, the average time to surgery following PGV diagnosis was 21 months (range 0.5-97 months). RRM was most common in the 40-49 age group (31%, n=35), closely followed by the 30-39 age group (30%, n=34). Only 13% of individuals had a RRM below age 30 (n=15) and 9% of RRMs occurred in individuals aged 60 or older (n=10). Likelihood of undergoing RRM was significantly greater for individuals with a highly penetrant breast cancer PGV (42% for BRCA1, BRCA2, PALB2, PTEN, TP53, CDH1) versus a moderately penetrant breast cancer PGV (21% for CHEK2, ATM, RAD51C/D) (p = 0.002). Among 116 individuals eligible for RRBSO (female, PGV in ovarian cancer susceptibility gene) 86% underwent RRBSO within the timeframe recommended. Of the 178 patients eligible for breast cancer risk-reducing medication (breast cancer PGV, female, age ≥ 35, no RRM), 80% (n=142) had a documented discussion regarding risks and benefits of SERM or AI at time of PGV diagnosis and 7 patients (3.9%, BRCA1 n=2, BRCA2 n=2, CHEK2 n=3) initiated therapy. Of the 402 patients analyzed, 41 (11%) were subsequently diagnosed with cancer during the follow up period, 51% (n=21) of which were breast cancer. Of those diagnosed with breast cancer, 57% were carcinoma in situ and 24% were stage I. Conclusions: In this cohort, nearly 40% of unaffected females with hereditary breast cancer risk opted to undergo RRM. Approximately 1 in 5 individuals with moderate penetrance breast cancer PGVs opted for RRM despite insufficient evidence of benefit. Compliance with RRBSO among those with hereditary ovarian cancer risk was high. Uptake of risk-reducing medication (SERM or AI) was exceptionally low despite its potential to significantly decrease breast cancer risk. Additional research regarding efficacy of risk-reducing medication in those with hereditary breast cancer predisposition is needed. Citation Format: Abby Hadley, Grayson Buning, Nicholas C. Henderson, Kara J. Milliron, Bailey B. Hulswit, Lauren E. Hipp, Versha A. Pleasant, James M. Rae, Melissa L. Pilewskie, Daniel F. Hayes, N. Lynn Henry, Sofia D. Merajver, Erin F. Cobain. A real-world analysis of cancer prevention measures among individuals with hereditary breast and ovarian cancer (HBOC) syndromes and no personal history of malignancy [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-15.

BackgroundRoutine genetic testing for germline pathogenic variants (GPVs) in cancer susceptibility genes (CSGs) in individuals with suspected hereditary cancer risk, and subsequent cascade testing in close relatives, has led to a significantly increased number of individuals identified to be at increased lifetime cancer risk in the UK. These individuals have differing clinical needs over their lifetime to implement cancer surveillance and discuss risk-reducing interventions, to advise on risk to close relatives and counsel on reproductive options. Regional clinical genetics services across the UK have implemented a range of clinical pathways to manage this patient cohort, with differences in practice across centres. Our aim was to provide consensus guidelines on best practice for recontact and follow-up for individuals with GPVs in BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51C, RAD51D and mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) to ensure appropriate lifelong clinical management.MethodsThe UK Cancer Genetics Group convened a virtual consensus meeting involving key stakeholders. Consensus for best practice guidance was achieved through premeeting surveys, structured discussions and in-meeting polling.ResultsWe identified variability in the extent of active recontact and follow-up for individuals with a GPV in CSGs across the UK. The availability of resources was a key determinant of the level of follow-up currently provided. We achieved consensus on best practice guidelines on key time points for recontact for those with a GPV in a CSG, processes for referral to specialist services and follow-up, the preferred method for recontact and the content of information given during recontact.ConclusionThe consensus meeting broadly supported recontact and follow-up for most individuals with a GPV in a CSG. The consensus achieved by the multidisciplinary and expert group of healthcare professionals gives clear guidance on the long-term management of this patient cohort at increased lifetime risk of cancer and highlights the additional resources that would be required to effectively deliver this.

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)

1010P - Frequency of mutations in 21 hereditary breast and ovarian cancer susceptibility genes among 882 high-risk individuals

BRCA1 and BRCA2 in hereditary breast cancer

Introduction: BRCA1, as well as BRCA2, were identified as the hereditary breast and ovarian cancer susceptibility genes that can account for almost all of inherited cases of breast cancers. BRCA1 is a multifunctional nuclear protein with a RING domain at the N-terminus. Heterochromatin is chromatin containing inactive genes. Pericentromeric heterochromatin is composed of highly repetitive tandem DNA repeats whose transcription is constitutively silenced. In mice, the repeats are classified as major and minor satellite repeats. Heterochromatin protein 1 (HP1) plays a critical role in maintaining heterochromatin structure. Methods: To study the biological function of the tumor suppressor BRCA1, we deleted BRCA1 in nestin positive neuronal stem cells in the brain and showed that the animals displayed severe neuro-developmental deficits. In the course of this work we discovered a novel molecular defect in BRCA1 deficient cells, i.e., a failure to maintain integrity of the heterochromatic structure. Results: In the absence of BRCA1, the number of heterochromatic centers is severely reduced in mouse neuronal cells. HP1 proteins are destabilized, and more importantly, the transcription of satellite DNA is derepressed in mouse neural progenitor cells, mouse fibroblasts, and human cancer cells, including a BRCA1 deficient breast cancer cell line. The defect in silencing invariably appears after the loss of the BRCA1 allele in our paradigm prior to exhibiting any neoplastic phenotype. What is the mechanism of BRCA1 in regulating the heterochromatin integrity? ChIP analysis showed that BRCA1 physically associates with both major satellite DNA and minor satellite DNA, whereas the association is diminished in the BRCA1 knockout animals. More intriguingly, the absence of BRCA1 binding to satellite DNA correlates with a drastic reduction of ubiquitinated histone H2A at the constitutive heterochromatin region. Because BRCA1 contains an E3 ubiquitin ligase activity which is enhanced by BARD1 and the complex ubiquitinate histone H2A in vitro, we examined the heterochromatin silencing in cells with different BRCA1 mutants. These experiments showed that the E3 ligase activity of BRCA1 is required for repression of satellite DNA transcription. Conclusions: Our working model is that mutations of BRCA1 may destabilize the protein complex and lead to defects in the heterochromatin integrity, thereby affecting the stability of the genome which is a hallmark of cancer. Given that mutants in the RING domains are the same as in women who have pathological diseases, we hypothesize that deregulation of satellite DNA transcription can be explored as an early detection marker for breast cancer progression in BRCA1 patients.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant disease that represents approximately 5-10% of all patients with breast cancer. This syndrome is mainly associated to high-risk pathogenic alleles in BRCA1 and BRCA2 genes, but only for 25% of HBOC cases. This work aimed to find new pathogenic alleles in a panel of 143 cancer-predisposing genes in 300 Mexican cancer patients with suspicion of HBOC and 31 non-cancer patients with a severe family history of cancer, using massive parallel sequencing. We found 15% (45/300) patients with pathogenic variants in the group of cancer patients; 12% (35/300) harbored variants with unknown clinical significance (VUS) and 73% (220/300) were negative. The non-cancer group had a 32% (10/31) of patients with pathogenic variants, 3% (1/31) had VUS and 65% (20/31) were negative. Moreover, the most recurrent mutation was the Mexican founder deletion of exons 9-12 in BRCA1, found in 5 of 16 cancer patients with alterations in this gene. Private or rare VUS variants with potential impact at protein level were found in 22 genes, being CHEK2 the one with most VUS (6/39). Noteworthy, our results show for the first time in the Mexican population an equal contribution of pathogenic alleles in other susceptibility cancer genes (50%) as in BRCA1/2 (50%). This highlights the high locus heterogeneity of HBOC and the necessity of expanding genetic tests for this disease to broader gene panels. Further studies need to be conducted to define the clinical impact of the pathogenic alleles and VUS identified. Citation Format: Felipe Vaca-Paniagua, Rosalía Quezada-Urban, Clara E. Díaz-Velásquez, Rina Gitler, María P. Rojo-Castillo, Max Sirota-Toporek, Andrea Figueroa-Morales, Oscar Moreno-García, Lizbeth García Esquivel, Gabriela Torres-Mejía, Michael Dean, Ivan Delgado-Enciso, Héctor Ochoa-Díaz-López, Fernando Rodriguez-León, Virginia Jan, Victor H. Hugo Garzón-Barrientos, Pablo Ruiz-Flores, Perla K. Espino-Silva, Jorge Haro-Santa Cruz, Héctor Martínez-Gregorio, Ernesto Rojas-Jiménez, Rosa M. Álvarez-Gómez, Luis A. Herrera, Isabelle Romieu, Luis I. Terrazas, Yolanda I. Chirino, Cecilia Frecha, Javier Oliver, Sandra Perdomo. Comprehensive analysis of germline variants in Mexican patients with hereditary breast and ovarian cancer susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1240.

Cancer is a dreadful disease, with genetics being a significant causative etiological factor. About 5–10% of the cancers arise through the congenital alteration of a group of genes which are predisposition genetic factor. Inherited mutation can play a role in leading or receding fashion that can deliberate varied degree of penetrance. The risk for causing hereditary cancer mainly focused on identification of BRCA1 and BRCA2, which are high-penetrance breast cancer genes linked with the highest lifetime cancer risks. The hereditary breast and ovarian cancer is also attributed to alterations in various other breast and ovarian cancer susceptibility genes. In this review, a comprehensive information of genes associated with HBOC is discussed. Extensive data are gathered using different search tools for Indian studies related to HBOC. It revealed that genetic prevalence of hereditary breast and ovarian cancer in Indian population is less explored. There are very few large-scale studies in Indian population related to HBOC. Further, there is lack of studies that analyzed various other high-penetrance gene except BRCA1/BRCA2 in Indian HBOC patients. There are various limiting factors like ethnicity, clinical history for inclusion and exclusion, inconsistency in method used for analysis, etc. The study suggested that a strategic approach is required for Indian hereditary breast and ovarian cancer patients.

Identification of deleterious variants in hereditary breast and ovarian cancer (HBOC) susceptibility genes allows for increased clinical surveillance and early detection, and could predict the response to poly (ADP‐ribose) polymerase (PARP) inhibitor in patients with advanced ovarian carcinomas. To determine the prevalence and clinical prediction factors for HBOC syndrome, 882 selected individuals underwent multigene panel testing for HBOC risk assessment during the period from January 2015 to March 2018. Overall, 176 deleterious mutations were observed in 19.50% (n = 172) of individuals. Twenty‐six of 176 mutations could not be retrieved in related public databases and were considered to be novel. Among patients with ovarian cancer, 115 deleterious mutations were identified in 429 patients (48.6%) with significant enrichment for a family history of breast or ovarian cancer syndrome (P < .05). In the breast cancer subgroup, 31 deleterious mutations were identified in 261 patients. Besides BRCA1 (8; 25.8%) and BRCA2 (11; 35.5%), the most frequently occurring genes, an additional 12 deleterious mutations (38.7%) were found in seven other susceptibility genes. Higher mutation incidence (57.9%) was observed in subjects with histories of breast and ovarian cancer. Our results highlighted the genetic heterogeneity of HBOC and the efficiency of a multigene panel in carrying out risk assessment.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant disease mainly associated to high-risk pathogenic alleles in the BRCA1 and BRCA2 genes, but only in ~25% of HBOC cases. With the emergence of broader multi-gene panels, population-based studies have revealed that 3-4% of high-risk individuals have germline pathogenic variants in cancer risk genes other than BRCA1 and BRCA2, including ATM, CHEK2, PALB2, PTEN, TP53, and others. One of the understudied population with HBOC is the Latin American, and in this study, we aimed to identify the prevalence of high, moderate and new pathogenic alleles within two panels of cancer predisposition genes (143 and 86 genes) in 217 Latino breast cancer patients that met the NCCN criteria for panel testing in 4 countries. The Latino population was integrated by 68 Mexicans (31.3%), 19 Guatemalan (8.8%), 56 Argentinians (25.8%) and 74 Colombians (34.1%). We detected (i) 19.4% patients with pathogenic variants (42/217; Mexicans: 12, 5.5%; Guatemalans: 3, 1.4%; Argentinians: 10, 4.6%; Colombians: 17, 7.8%); (ii) 36.9% harbored variants with unknown clinical significance (VUS) (80/217; Mexicans: 24, 11.1%; Guatemalans: 6, 2.7%; Argentines: 26, 12%; Colombians: 24, 11.1%) and (iii) 43.7% were negative (95/217; Mexicans: 32, 14.7%; Guatemalans: 10, 4.6%; Argentines: 20, 9.2%; Colombians: 33, 15.2%). Moreover, most of the pathogenic variants (66%, 28/42) were found in high-risk genes including BRCA1/2, MSH2, MSH6 and PALB2. Our findings show the high locus heterogeneity of HBOC in the Latin American population. To establish the level of risk and the ultimate clinical utility of the variants detected in expanded panels tests, further international efforts are necessary in a population-based context. Citation Format: Felipe Vaca-Paniagua, Rosalia Quezada-Urban, Clara Estela Díaz Velásquez, Eva María Gómez García, Claudia Fabiola Méndez Catalá, Javier Oliver, Alejandra Franco, Cecilia Frecha, Cecilia Riggi, Claudia Carranza, Carlos Castañeda Altamirano, Ana Milena Gómez, Ana Lorena Montealegre, Sandra Gaitán Chaparro, Juan Javier López, Sandra Perdomo. Comprehensive analysis of germline variants in patients with hereditary breast and ovarian cancer susceptibility from 4 countries of Latin America [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4164.

Clinical validity assessment of genes frequently tested on hereditary breast and ovarian cancer susceptibility sequencing panels

While germline mutations in BRCA1 and BRCA2 account for most, if not all families with autosomal dominant transmission of susceptibility to both breast and ovarian cancer, it has become clear that together these genes only account for a small proportion of hereditary site-specific breast cancer susceptibility. However, difficulties due to genetic heterogeneity, reduced penetrance and perhaps gene mutation frequency complicate ongoing efforts to identify additional susceptibility genes. Therefore, multiple approaches are being used to identify additional high and low penetrance genes. Families with three or more breast cancer cases are being used in traditional linkage studies, which are expected to yield only moderate or high penetrance susceptibility genes. Breast cancer case-control studies are being used to look for genetic variants or polymorphisms that confer an increased risk of breast cancer in a wide variety of cellular pathways, ranging from the detoxification of environmental carcinogens to steroid hormone metabolism, DNA damage repair and immune surveillance, an approach useful primarily to identify low penetrance susceptibililty genes. However, neither approach has yielded convincing results to date. A third approach, using BRCA1 and BRCA2 mutation carriers to identify genes that are associated with modification of breast cancer risk has met with some limited success, perhaps because effects on breast cancer risk in BRCA1 and BRCA2 mutation carriers are more readily detected in smaller studies, given the much higher number of events in these cohorts at very high risk of breast cancer. Clearly, hereditary breast cancer susceptibility is a complex phenomenon, in which multiple genes may play a role. It will be necessary to use all of these approaches, as well as more comprehensive genomic studies, to identify additional breast cancer-related genes.

NGS-based multiple gene panel resequencing in combination with a high resolution CGH-array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.