BRCA1 Variants Research Articles

Characteristics of Chinese breast cancer patients with double heterozygosity for BRCA1 and BRCA2 germline pathogenic variants.

Despite of very rare, breast cancer patients with double heterozygosity (DH) variants in BRCA1 and BRCA2 genes have been identified in other ethnic groups and seem to be associated with distinctive phenotypes. However, little is known about the frequency and clinical characteristics of Chinese breast cancer patients with BRCA1/2 DH variants. Four hundred and eleven unrelated patients with BRCA1 or BRCA2 pathogenic variants (PVs) were identified in a large series of unselected breast cancer patients. Another two siblings with metachronous bilateral breast cancer were referred for genetic counseling, after which BRCA1/2 DH variants were detected. Four unrelated breast cancer patients with BRCA1/2 DH were identified in the cohort of 411 patients with BRCA1 or BRCA2 PVs, the frequency of BRCA1/2 DH was 0.97%. In total, six BRCA1/2 DH patients from five families were found in this study. In two families, the hereditary pattern of DH was speculated to have originated from both sides of the family. BRCA1/2 DH patients were more likely to have a family history of breast cancer than patients with a BRCA1 (100% vs. 29.2%, P = 0.004) or BRCA2 (100% vs. 29.6%, P = 0.004) single PV. BRCA1/2 DH patients were more likely to be triple-negative breast tumors than patients with single BRCA2 PVs (66.7% vs. 14.1%, P = 0.020), which was comparable to the findings in patients with single BRCA1 PVs (66.7% vs. 56.9%, P = 1.00). Chinese patients with BRCA1/2 DH exhibit a high percentage of family history of breast cancer. The tumor pathological features of BRCA1/2 DH carriers are similar to those of BRCA1 PV carriers.

Exploring the impact of BRCA1 and BRCA2 mutation type and location on Olaparib maintenance therapy in platinum-sensitive relapsed ovarian Cancer patients: A single center report

ObjectiveIn patients with platinum-sensitive relapsed ovarian cancer (PSROC) harboring pathogenic/likely pathogenic variants (PV) in BRCA1 and BRCA2 genes, olaparib maintenance monotherapy (OMT) is a viable option. Our study aimed to evaluate the impact of different BRCA1/2 PV in survival outcomes and safety of OMT in BRCA1/2-mutated PSROC patients, focusing on the type and location of PV. MethodsWe assessed the outcomes of 100 BRCA1/2-mutated PSROC patients treated at our institute, analyzing progression-free survival (PFS) and overall survival (OS). Germline and tumor BRCA1/2 genotyping was conducted using Illumina's next-generation sequencing (NGS). ResultsPFS and OS were significantly shorter in PSROC patients with PV in BRCA1 compared to those with PV in BRCA2 (PFS:14.0 vs. 38.8 months, p = 0.007, OS: 21.8 vs. 62.0 months, p = 0.011). Notably, there was a significant difference in PFS based on the intragenic location of BRCA1 PV, with shorter PFS in patients with 1st/2nd relapse, harboring PV in BRCA1 RING domain compared to those with PV in the DNA binding domain (DBD) and BRCT domains (12.4 vs. 23.0 months, p = 0.046). No differences in PFS and OS were observed between patients with germline versus somatic BRCA1/2 PV (PFS:14.9 vs.19.3, p = 0.316, OS: not reached vs. 25.8 months; p = 0.224). However, there were significant differences in the reasons for OMT discontinuation between patients with germline and somatic BRCA1/2 PV, primarily due to adverse side effects. ConclusionsIn summary, the type and location of BRCA1 and BRCA2 PV provide additional insight into the expected survival outcomes of olaparib MT in PSROC patients.Trial registration number: ISRCTN42408038, Name of registry: ISRCTN registry, Date of registration: 24/11/2015.

The discrepancy of somatic BRCA1/2 pathogenic variants from two different platforms in epithelial ovarian, fallopian tube, and peritoneal cancer

The somatic BRCA1 or BRCA2 Pathogenic Variant (PV)/Likely PV (LPV) from Next Generation Sequencing (NGS) is the most important biomarker for PARP inhibitor use and maintenance-targeted therapies. A discrepancy in the detection rates of BRCA1 and BRCA2 PV/LPV was identified among the NGS platforms. The objective of this study was to compare the somatic BRCA results from two distinct platforms using the same cohort and to identify the causes of these differences. Patients with epithelial ovarian cancer who concurrently underwent tumor NGS using two different platforms between January 2022 and June 2023 were included in this study. The two platforms used were in-house tumor NGS (Illumina NextSeq 550Dx, SureSelectXT library kit, and datasets from 1000 Genomes, ESP6500, ExAC, and ClinVar) and GreenPlan homologous recombination deficiency (HRD) test (Illumina NextSeq 550Dx, customized Twist Bioscience library kit, and datasets from COSMIC and OncoKB). The results of somatic mutations in BRCA1 and BRCA2 were compared between the two platforms. Of the 118 patients, 11.9% (n = 14) exhibited a discordant interpretation of BRCA1 or BRCA2 between the two platforms. Eleven patients (9.3%) exhibited negative results in the in-house platform but positive results (eight seven as PV of BRCA1, one as PV of BRCA2, one as LPV of BRCA1, and two as LPV of BRCA2) in the GreenPlan HRD test, while three patients (2.6%) had positive BRCA pathogenic variants (two as PV of BRCA1 [c.3340G > T, c.5152 + 3 A > C], one as LPV of BRCA2 [c.8174G > T], and one as LPV of BRCA1 [c.5017_5019delCAC]) in the in-house platform but a negative result in the GreenPlan HRD test. The discordance rate of somatic BRCA1 or BRCA2 mutations from different platforms was approximately 12%. In the case of the strong implication of BRCA PV/LPV with a negative result with one genetic test, different platforms could be considered in limited cases. Careful interpretation and further studies for the cross-validation of gene analysis platforms are needed.

Germline DNA damage repair variants and prognosis of patients with high-risk or metastatic prostate cancer.

Deleterious germline variants in certain DNA repair genes are risk factors for developing aggressive prostate cancer. The objective was to quantify their prognostic impact after prostate cancer diagnosis. Men with prostate cancer, predominantly of European ancestry, were included from four cohorts with long-term follow-up. Pathogenic or likely pathogenic germline variants in 26 DNA repair genes were assessed in relation to metastasis-free survival in high-risk, localized prostate cancer and to overall survival in metastatic castration-sensitive prostate cancer (mCSPC) and metastatic castration-resistant prostate cancer (mCRPC). Among 3,525 patients initially diagnosed with non-metastatic prostate cancer, 2,594 had high-risk localized prostate cancer, 429 mCSPC, and 502 mCRPC at inclusion. BRCA2 variant carriers did not have worse metastasis-free survival in high-risk localized prostate cancer (hazard ratio [HR] 1.01, 95% confidence interval [CI] 0.69, 1.46) or overall survival in mCSPC (HR 0.46, 95% CI 0.14, 1.45) or mCRPC (HR 0.60, 95% CI 0.31, 1.17) compared to non-carriers of DNA repair variants. Among 868 additional patients with de novo metastatic (M1) prostate cancer, BRCA2 variant carriers tended to have worse overall survival (HR 1.59, 95% CI 1.01, 2.51). BRCA2 prognostic associations were not explained by radiation, PARP inhibitor, or platinum therapy. Results for other genes were limited in precision because variants were less common. Among patients with high-risk or metastatic prostate cancer who were initially diagnosed with and treated for non-metastatic tumors, germline DNA repair variants in BRCA2 do not confer a substantially worse prognosis.

Establishment of a clinical cancer genetics program for breast cancer in a resource-limited country; challenges and opportunities

Breast cancer is the most common cancer among women worldwide, and its incidence rate is still increasing, especially among younger women. Nationally, it constitutes one-fifth of all cancer cases and almost 40% of all female cancers. With a median age of 51 years, breast cancer is diagnosed at least a decade earlier, and at more advanced stages compared to Western societies. Hereditary cancers account for 10% or more of all cancer burden worldwide. With expanded indications, increased number of genes tested, and significant decline in cost of testing, such proportion will probably increase. Individuals with pathogenic variants of BRCA1 and BRCA2 are at higher risk of breast, ovarian, pancreatic and many other cancers. Over the past two decades, several highly penetrant cancer-susceptibility genes were identified across almost all tumor sites, thus increasing the need for comprehensive cancer genetic programs that address the testing process, counselling patients and at-risk family members, and then deal with all testing results and its consequences. In addition to its important role in preventing more cancers in index patients themselves and among their close relatives, identification of pathogenic or likely pathogenic variants, mostly in BRCA1 or BRCA2, may inform therapeutic decisions in common cancers including breast, ovarian, prostate and pancreatic cancers. In this manuscript, we describe the experience of a comprehensive cancer center, in a resource-limited country in establishing a comprehensive clinical cancer genetics program that can serve as an example for others who share similar demographic and financial restrains.

Molecular analysis of BRCA1 and BRCA2 genes in La Rioja (Spain): five new variants.

To study BRCA1/2 gene variants in La Rioja in the northcentral area of Spain. We performed a molecular analysis of BRCA1 and BRCA2 in 642 individuals from 427 different families from June 2008 to December 2019. We identified 71 families with pathogenic variants in these genes, 32 families with BRCA1 variants and 39 families with BRCA2 variants. The pathogenic variants c.959delG in BRCA1 and c.1363_1369delTCAGAGA, c.1397dupA, c.4234_4236delACTinsC and c.8387delC in BRCA2 have not been previously described. The c.81-2A > T variant in BRCA1, detected in two unrelated families, has not been reported previously in the Spanish population. Two large genomic deletions were found in the BRCA1 gene in exons (Ex) 23-24 and Ex1A-1B-2, and one deletion was found in the BRCA2 gene in Ex2. The pathogenic variant c.5123C > A in BRCA1 was detected in 8 unrelated families and was the most frequent pathogenic variant in our population. The c.6024dupG mutation in BRCA2 was detected in 6 unrelated families; the c.2808_2011delACAA mutation in BRCA2 was found in 5 different families; the c.211A > G mutation in BRCA1 was found in three different families; and the c.68_69delAG, c81-2A > T, c.4038_4039delAA, and c.5266dupC variants in BRCA1 and the c.2457delA, c.2701delC, c.5116_5119delAATA, c.6275delTT, c.7558C > T and c.7617 + 1G > A variants in BRCA2 were found in two different families. The spectrum of pathogenic variants in the BRCA1/2 genes in La Rioja is similar to that in other Spanish regions, with some unique characteristics. The pathogenic c.6024dupG variant in the BRCA2 gene was detected in a large number of families and could have a founding effect in the Ebro riverside areas in the regions of La Rioja and Navarra. Not applicable.

Two founder variants account for over 90% of pathogenic BRCA alleles in theOrkney and Shetland Isles in Scotland.

For breast and ovarian cancer risk assessment in the isolated populations of the Northern Isles of Orkney and Shetland (in Scotland, UK) and their diasporas, quantifying genetically drifted BRCA1 and BRCA2 pathogenic variants is important. Two actionable variants in these genes have reached much higher frequencies than in cosmopolitan UK populations. Here, we report a BRCA2 splice acceptor variant, c.517-2A>G, found in breast and ovarian cancer families from Shetland. We investigated the frequency and origin of this variant in a population-based research cohort of people of Shetland ancestry, VIKING I. The variant segregates with female breast and ovarian cancer in diagnosed cases and is classified as pathogenic. Exome sequence data from 2108 VIKING I participants with three or more Shetlandic grandparents was used to estimate the population prevalence of c.517-2A>G in Shetlanders. Nine VIKING I research volunteers carry this variant, on a shared haplotype (carrier frequency 0.4%). This frequency is ~130-fold higher than in UK Biobank, where the small group of carriers has a different haplotype. Records of birth, marriage and death indicate genealogical linkage of VIKING I carriers to a founder from the Isle of Whalsay, Shetland, similar to our observations for the BRCA1 founder variant c.5207T>C from Westray, Orkney. In total, 93.5% of pathogenic BRCA variant carriers in Northern Isles exomes are accounted for by these two drifted variants. We thus provide the scientific evidence of an opportunity for screening people of Orcadian and Shetlandic origins for each drifted pathogenic variant, particularly women with Westray or Whalsay ancestry.

Phenotypic evaluation of deep learning models for classifying germline variant pathogenicity.

Deep learning models for predicting variant pathogenicity have not been thoroughly evaluated on real-world clinical phenotypes. Here, we apply state-of-the-art pathogenicity prediction models to hereditary breast cancer gene variants in UK Biobank participants. Model predictions for missense variants in BRCA1, BRCA2 and PALB2, but not ATM and CHEK2, were associated with breast cancer risk. However, deep learning models had limited clinical utility when specifically applied to variants of uncertain significance.

Using AI-predicted protein structures as a reference to predict loss-of-function activity in tumor suppressor breast cancer genes

BackgroundThe loss-of-function (LOF) classification of most missense variants in tumor suppressor breast cancer genes BRCA1, BRCA2, PALB2, and RAD51C remains unclassified and confounds clinical actionability. Classifying these variants is challenging due to their rarity, leading clinicians to rely on in silico predictive methods. Protein stability changes are associated with function, making stability predictors valuable. Stability predictions upon missense variant perturbations require high-resolution protein structures. However, the availability of these high-resolution structures is lacking. This study explores using generative AI to predict high-resolution protein structures, which can then be analyzed with in silico protein stability prediction methods to assess LOF activity in ordered regions of the protein. This study also determines the appropriate in silico protein stability and dedicated in silico missense prediction methods in dbNSFP v4.7 database to predict LOF activity in ordered regions of these four genes. Functional classifications from homology recombination DNA repair (HDR) assays and variant classifications from the ClinVar database provide a reliable dataset for evaluating the performance of these in silico prediction methods. ResultsComplex AlphaFold2 structures of the BRCA1-C terminal (BRCT) domain and the DNA-binding (DB) domain of BRCA2, analyzed using protein stability tool FoldX predicts LOF activity from missense variants significantly better than experimentally-derived structures in ordered regions. The BRCT domain achieved an Area Under the Curve (AUC)= 0.861 (95 % CI:0.858–0.863) and AUC= 0.842 (95 % CI:0.840–0.845), while the DB domain achieved an AUC= 0.836 (95 % CI:0.8322–0.841), compared to AUC= 0.847 (95 % CI:0.844–0.850) and AUC= 0.835 (95 % CI:0.832–0.837) from the BRCT domain, and AUC= 0.830 (95 % CI:0.821–0.8320) from the DB domain from experimentally-derived structures. Protein stability does not predict LOF activity from missense variants better than dedicated in silico missense predictors. Overall, we find that AlphaMissense ranks highly, with an average AUC= 0.890 (95 % CI 0.886–0.895) from ordered regions across these four cancer genes, compared to all other in silico missense predictors present in the dbNSFP database. ConclusionsThe study reveals that generative AI protein predicted structures can outperform experimentally-derived structures in evaluating LOF activity from predicted protein stability in ordered regions of genes BRCA1, BRCA2, PALB2 and RAD51C. The study also highlights the predictive performance of AlphaMissense as the premier in silico missense prediction method to predict LOF activity from missense variants in these four tumor suppressor breast cancer genes. The code for this study can be downloaded for free on GitHub (https://github.com/rohandavidg/CarePred)

The implementation and outcome of a breast cancer genetic counselling service at Potchefstroom Hospital: a model for outreach

Aim Breast cancer is the most prevalent cancer in South African females, making up 27.1% of all histologically diagnosed cancers in 2020. Although genetic technology and awareness of genetic counselling have improved, genetic counselling services in South Africa remain largely inaccessible. Clinical genetic services are only formally available in four South African provinces and few outreach programmes exist for small towns and cities. Until 2019, genetic counselling services were unavailable in the North West province; however, since then, genetic counsellors from the National Health Laboratory Service and the University of the Witwatersrand have provided genetic counselling services to patients at the Breast Clinic at Potchefstroom Hospital regularly each year. Method The aim of this pilot study was to perform a retrospective file review and report on the implementation and outcomes of the genetic counselling service at the Breast Clinic at Potchefstroom Hospital, from its inception in 2019, until November 2022. Fifty-two patients attended a genetic counselling consultation during that period. Results The majority of patients (83.7%) were diagnosed with an invasive ductal carcinoma, and 57.7% of the patients had a family history of cancer. A total of 62.8% of patients had a histologic grade 3 tumour. A total of 25.5% (12/47) of patients tested positive for a pathogenic variant in BRCA1 or BRCA2. A total of 45 at-risk first-degree relatives were identified who could benefit from predictive testing. Conclusions This study highlights the benefit of offering clinical genetics services through outreach clinics. Being able to offer this service is not only beneficial for the management of the affected individuals, but also for their at-risk relatives. The initiative serves as a positive example of how limited resources can be extended to benefit patients.

Pathogenic Germline Variants in Uveal Melanoma Driver and BAP1-Associated Genes in Finnish Patients with Uveal Melanoma.

Uveal melanoma (UM) is a rare yet aggressive eye cancer causing over 50% mortality from metastasis. Familial UM, amounting to 1%-6% of patients in Finland and the United States, mostly lack identified genetic cause, while 8% show associations with other cancer syndromes. We searched novel genetic associations for predisposition to UM, additional to already studied BAP1 and MBD4, by using targeted amplicon sequencing of 19 genes associated with UM, BAP1, or renal cell carcinoma in 270 consecutively enrolled Finnish patients with UM. Key UM drivers GNAQ, GNA11, CYSLTR2, PLCB4, EIF1AX, and SF3B1 lacked pathogenic germline variants. One patient carried the pathogenic BRCA1 variant c.3626del p.(Leu1209*), and one harbored a novel truncating MET variant c.252C > G p.(Tyr84*), classified as likely pathogenic. FLCN and BRCA2, previously identified with pathogenic variants in patients with UM, did not have such variants in our cohort. Two patients were heterozygous for a pathogenic recessive BLM variant c.2824-2A > T. None of the carriers of identified variants had familial UM. We identified BRCA1 and MET as genes with pathogenic germline variants in Finnish UM patients, each with a frequency of 0.4% (95% confidence interval, 0-2).

The role of RAD51 regulators and variants in primary ovarian insufficiency, endometriosis,and polycystic ovary syndrome.

The study of RAD51 regulators in female reproductive diseases has novel biomarker potential and implications for therapeutic advancement. Regulators of RAD51 play important roles in maintaining genome integrity and variations in these genes have been identified in female reproductive diseases including primary ovarian insufficiency (POI), endometriosis, and polycystic ovary syndrome (PCOS). RAD51 modulators change RAD51 activity in homologous recombination, replication stress, and template switching pathways. However, molecular implications of these proteins in primary ovarian insufficiency, endometriosis, and polycystic ovary syndrome have been understudied. For each reproductive disease, we provide its definition, current diagnostic and therapeutic treatment strategies, and associated genetic variations. Variants were discovered in RAD51, and regulators including DMC1, RAD51B, SWS1, SPIDR, XRCC2and BRCA2 linked with POI. Endometriosis is associated with variants in XRCC3, BRCA1and CSB genes. Variants in BRCA1 were associated with PCOS.Our analysis identifiednovel biomarkers for POI (DMC1 and RAD51B) and PCOS (BRCA1). Further biochemical and cellular analyses of RAD51 regulator functions in reproductive disorders will advance our understanding of the pathogenesis of these diseases.

Distinct roles of the two BRCA2 DNA binding domains in DNA damage repair and replication fork preservation.

Homologous recombination (HR) is a highly conserved tool for the removal of DNA double-strand breaks (DSBs) and the preservation of stalled and damaged DNA replication forks. Successful completion of HR requires the tumor suppressor BRCA2. Germline mutations in BRCA2 lead to familial breast, ovarian, and other cancers, underscoring the importance of this protein for maintaining genome stability. BRCA2 harbors two distinct DNA binding domains, one that possesses three oligonucleotide/oligosaccharide binding (OB) folds (known as the OB-DBD), and with the other residing in the C-terminal recombinase binding domain (termed the CTRB-DBD) encoded by the last gene exon. Here, we employ a combination of genetic, biochemical, and cellular approaches to delineate contributions of these two DNA binding domains toward HR and the maintenance of stressed DNA replication forks. We show that OB-DBD and CTRB-DBD confer ssDNA and dsDNA binding capabilities to BRCA2, respectively, and that BRCA2 variants mutated in either DNA binding domain are impaired in the ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected for HR, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DNA binding domains in genome maintenance.

Abstract C158: Contribution of multi-organ cancer predisposition genes for gastric cancer risk of different histological subtypes

Abstract Approximately 10% of gastric cancer (GC) cases exhibit familial clustering, yet only 1-3% can be explained by known hereditary syndromes, being Hereditary Diffuse Gastric Cancer (HDGC) the most well-established disease predisposing for diffuse gastric cancer. Familial intestinal gastric cancer (FIGC) has been defined clinically, but it remains mostly genetically unexplained. Likewise, the heritability of mixed GC remains to be uncovered. We aimed to estimate the frequency of known cancer predisposition gene variants in GC cases with family history of cancer and/or early onset. We evaluated the contribution of pathogenic and likely pathogenic (P/LP) variants in well-established and multi-organ cancer predisposition genes for GC risk in a large multi-center study involving 750 GC patients, either by targeted sequencing or whole exome sequencing. We identified 45 patients (6%) with P/LP variants in ATM (17 cases), BRCA2 (10 cases), MLH1 (five cases), TP53 (three cases), BRCA1, PALB2, RAD51D and CHEK2 (two patients each), and RAD51C and PMS2 (one case each), all mutually exclusive. The P/LP variant prevalence was higher in intestinal (9.8%) than in diffuse (4.3%) or mixed GC (4.5%) (p-value=0.023), with no clear difference per mutated gene by histological subtypes. Only sixteen of the 45 variant carriers fulfilled the criteria for genetic testing of at least one cancer predisposition syndrome. Our findings indicate that multi- organ cancer predisposition genes should be included in gene panels used for investigating germline variants in GC patients, especially when there is family history of cancer but irrespective of histology subtype, as this would increase the chance of identifying patients that might benefit from prevention and targeted treatment strategies. Citation Format: Ana P. Estrada-Florez, Paul C. Lott, Luis G. Carvajal Carmona. Contribution of multi-organ cancer predisposition genes for gastric cancer risk of different histological subtypes [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C158.

The Pivotal Role of Germline BRCA2 Pathogenic Variants in "Apparently Sporadic" Pancreatic Cancer.

In 1996, Goggins and colleagues demonstrated the importance of germline BRCA2 pathogenic variants in the development of apparently sporadic pancreatic ductal adenocarcinoma (PDAC). Previously, the group identified homozygous deletion of the 13q region in PDACs, enabling the identification of the BRCA2 gene. This 1996 article first revealed loss of BRCA2, both germline and somatic, as a key driver of PDAC at a time when there was still doubt if PDAC even had an inherited component. Contrary to the prevailing wisdom, not all individuals with inherited pathogenic BRCA2 variants had a family history of cancer. The innovative bedside-to-bench nature of this work revealed that individuals with these variants would be missed if genetic testing was limited only to those meeting the family history criteria. Therefore, Goggins and colleagues advocated that universal genetic testing may be indicated for pancreatic cancer at a time when genetic testing was in its infancy. Twenty-three years later, in 2019, universal testing for pancreatic cancer became standard of care in the United States. Additionally, this work and future-related publications by the Kern Laboratory set the stage for targeting BRCA2 and related DNA repair mutations in pancreatic cancer via a synthetic lethal therapeutic approach. The provocative discussion initiated by this team in this publication is still inspiring the field today. In this seminal publication, Goggins and colleagues profoundly impacted the direction of pancreatic cancer research, leading to a more sophisticated approach to designing earlier detection and precision treatment strategies for pancreatic cancer. See related article by Goggins and colleagues, Cancer Res 1996;56:5360-4.

Abstract B029: Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma

Abstract Introduction: Germline mutations in DNA repair genes are often associated with increased risk of cancer. Mutations that affect the homologous recombination (HR) DNA repair pathway ( e.g., BRCA 1 and BRCA2) are known drivers of pancreatic adenocarcinoma. Early detection relies on identification of pathogenic mutations in patients with a family history of cancer. Next generation sequencing panels often use a select set of pathogenic variants for diagnosis, meaning that individuals with variants of uncertain significance may be missed, limiting testing of other family members for the potentially disease-causing variant. Here we describe a digital PCR assay developed to detect BRCA1 and BRCA2 variant that may eventually serve as a rapid, cost-effective alternative to sequencing that costs less than $5.00 per sample with results in 24 hours. This study highlights the need for better diagnostic tools, which will become critically important as we begin to study pathogenic mutations in more diverse populations. Methods: We have collected blood from 4 BRCA1 mutants and 5 BRCA2 mutants who have already undergone germline sequencing from commercially available assays. Clinicopathologic data has been collected as well. We designed two fluorescently labelled TaqMan probes, one that recognizes the wild-type BRCA alleles and one specific for mutant BRCA alleles. Briefly, 1 ng of genomic DNA is mixed with amplicon primers and fluorescent Taq-Man probes, then partitioned into 20,000 separate 517 picoliter wells. Quantification and detection are achieved based upon the fluorescent signal measured from each well. Results: We have successfully confirmed pathologic BRCA1 and BRCA2 mutations previously identified with next generation sequencing in 100% of samples analyzed. All three BRCA2 mutations generate a premature stop codon leading to a truncated protein and are known to be pathogenic. Two related patients have the same BRCA1 frameshift mutation that results in a premature stop codon, and one patient has a duplicated cytosine that leads to a frameshift mutation that is one of three characterized Ashkenazi Jewish founder mutations that increases cancer risk in this population . Conclusions: Here we report an innovative assay using digital PCR (dPCR) that provides a cost-effective, time-efficient method to identify MMR mutants and characterize VUS in patients and their families. Citation Format: Jennifer Brooke Valerin, Sophie Hasson, Valeria Rangel, Nicholas Pannunzio. Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B029.

Abstract C006: LODESTAR: A Single Arm Phase II Study of Rucaparib in Solid Tumors with Pathogenic Germline or Somatic Variants in Homologous Recombination Repair (HRR) Genes

Abstract Purpose: Poly (ADP-ribose) polymerase inhibitors (PARPi) have proven clinical utility in platinum-sensitive breast, ovary, pancreas, and prostate cancers with homologous recombination deficiency (HRD). We explored whether PARPi might be efficacious in other solid tumors and for a broader range of genomic variants. Patients and Methods: This single-arm phase II study assessed rucaparib monotherapy in patients with advanced, platinum-sensitive or -resistant solid tumors and pathogenic variants (PVs) in BRCA1, BRCA2, PALB2, RAD51C, RAD51D (Cohort A) or BARD1, BRIP1, FANCA, NBN, RAD51B (Cohort B). The primary endpoint was overall response rate (ORR) in cohort A. Secondary endpoints included disease control rate (DCR), progression free survival (PFS), overall survival (OS) and safety[SS3] [SA4] in the entire study population. Available tumors were analyzed for pre-specified exploratory objective of allele specific loss of heterozygosity (LOH). [SA7] HRD signature (HRDsig) and platinum sensitivity status were explored as biomarkers for rucaparib activity in post hoc exploratory analyses. The study was terminated early by the sponsor due to feasibility concerns. Results: Eighty-three patients were enrolled (Cohort A: 63; Cohort B: 20). Fifty-seven patients in Cohort A and 16 in Cohort B were evaluable for efficacy analysis. The[LM8] [SA9] ORR of cohort A was 16% (95% CI 9-27%) and DCR was 61% (95% CI 48-73%). LOH and HRDsig were available for 48% of Cohort A and 71% of Cohort B. Of 11 patients who had a response to treatment, 6 tumors were HRDsig+, 1 HRDsig-and 4 were unknown. The ORR and DCR were significantly greater in patients with HRDsig+ vs HRDsig-tumors (ORR: HRDsig+ 32% [95% CI 15-54%] vs HRDsig-3% [95% CI 0-15%], p < 0.01; DCR: HRDsig+ 74% [95% CI 51-88%] vs HRDsig-38% [95% CI 24-55%], p = 0.02). mPFS was numerically longer for HRDsig+ vs HRDsig-: 7.0 mos (95% CI, 4.3 – inf), vs. 3.0 mos (95% CI 2.3-5.6), HR 0.57, p = 0.14. mPFS was greater for platinum sensitive vs resistant tumors (7.82 mos [95% CI 5.5-inf] vs. 2.9 mos [95% CI 1.8-inf], HR 0.28, p = 0.02); Neither tumor tissue of origin nor specific mutation was independently predictive of outcome. The HR for progression in tumors that were both HRDsig+ and platinum sensitive versus those that were HRDsig-and platinum resistant was 0.09 (95% CI 0.01-0.55, p = 0.047). The Rucaparib safety profile was consistent with prior studies. Conclusion: Rucaparib has activity across solid tumors with PVs in HRR genes, regardless of tissue of origin. Patients with platinum resistant or HRDsig-tumors had limited clinical benefit from rucaparib. The combined use of HRDsig and platinum sensitivity to predict sensitivity to PARPi is encouraging as a biomarker strategy and warrants prospective study. Citation Format: Sriram Anbil, Nicholas J Seewald, Kevin Lin, Marcia Craib, Christy Connor, Heidi Giordano, Karen McLachlan, Tanya Kwan, Lara Maloney, Hanna Tukachinsky, Alexa Schrock, Shuoguo Wang, Ethan Sokol, Brennan Decker, Katherine Nathanson, Susan Domchek, Kim Reiss. LODESTAR: A Single Arm Phase II Study of Rucaparib in Solid Tumors with Pathogenic Germline or Somatic Variants in Homologous Recombination Repair (HRR) Genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C006.

Long-read DNA and cDNA sequencing identify cancer-predisposing deep intronic variation in tumor-suppressor genes.

The vast majority of deeply intronic genomic variants are benign, but some extremely rare or private deep intronic variants lead to exonification of intronic sequence with abnormal transcriptional consequences. Damaging variants of this class are likely underreported as causes of disease for several reasons: Most clinical DNA and RNA testing does not include full intronic sequences; many of these variants lie in complex repetitive regions that cannot be aligned from short-read whole-genome sequence; and, until recently, consequences of deep intronic variants were not accurately predicted by in silico tools. We evaluated the frequency and consequences of rare deep intronic variants for families severely affected with breast, ovarian, pancreatic, and/or metastatic prostate cancer, but with no causal variant identified by any previous genomic or cDNA-based approach. For 10 tumor-suppressor genes, we used multiplexed adaptive sampling long-read DNA sequencing and cDNA sequencing, based on patient-derived DNA and RNA, to systematically evaluate deep intronic variation. We identified all variants across the full genomic loci of targeted genes, applied the in silico tools SpliceAI and Pangolin to predict variants of functional consequence, and then carried out long-read cDNA sequencing to identify aberrant transcripts. For eight of the 120 (6%) previously unsolved families, rare deep intronic variants in BRCA1, PALB2, and ATM create intronic pseudoexons that are spliced into transcripts, leading to premature truncations. These results suggest that long-read DNA and cDNA sequencing can be integrated into variant discovery, with strategies for accurately characterizing pathogenic variants.

The coexistence of a BRCA2 germline and a DICER1 somatic variant in two first-degree cousins suggests their potential synergic effect

Cancer predisposition syndromes are recognized in about 10% of pediatric malignancies with several genes specifically involved in a subset of pediatric tumors such as DICER1, in pleuropulmonary blastoma, cystic nephroma, and brain sarcomas. By contrast, the role of BRCA1/2 in pediatric cancer predisposition is still under investigation. We present two cases of young first-degree cousins, both carrying a germline BRCA2 variant and developing tumors characterized by somatic DICER1 mutations. Patient 1 presented with a cystic nephroma harboring a somatic DICER1 variant (p.Asp1810Tyr), while patient 2 had a primary intracranial DICER1-mutated sarcoma showing a distinct somatic DICER1 variant (p.Asp1709Glu) as well as biallelic inactivation of TP53 (p.Val173Leu, VAF 91%) and APC (p.Ile1307Lys, VAF 95%) and a pathogenic variant in KRAS (p.Gln61His). Both patients carried the same germline BRCA2 variant (p.Arg2842Cys) of unknown significance. The same variant was found in the mother of patient 2 and in the father of patient 1, who are siblings. A homologous recombination deficiency signature was not identified in any of the two tumors, possibly suggesting a reduction of BRCA2 activity. The association of BRCA2 and DICER1 variants in our cases hints at a potential cooperative role in cancer pathogenesis. Further studies are warranted to elucidate the interplay between BRCA1/2 and DICER1 variants and their implications for cancer predisposition and treatment in pediatric patients.

Colorectal cancer with a germline BRCA1 variant inherited paternally: a case report.

BRCA genes have well-known associations with breast and ovarian cancers. However, variations in the BRCA gene, especially germline variations, have also been reported in colorectal cancer (CRC). We present the case of a rectal cancer with a germline BRCA1 variation inherited from the paternal side. A 39-year-old male was admitted with rectal cancer. The patient underwent surgical resection and the pathologic diagnosis was adenocarcinoma. Next-generation sequencing was performed and a BRCA1 variant was detected. Reviewing the public database and considering the young age of the patient, the variant was suggested to be germline. The patient's father had had prostate cancer and next-generation sequencing testing revealed an identical BRCA1 variant. In the BRCA cancer group, there is relatively little attention paid to male cancers. The accumulation of male CRC cases linked to BRCA variations may help clarify the potential pathological relationship between the two.