Heterozygous BRCA1 Research Articles

Abstract 5610: Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma

Abstract Introduction: In a large-scale exome sequencing study of pediatric rhabdomyosarcoma (RMS) patients, we identified a statistical enrichment of patients with germline variants in BRCA2 (Li, et al. JNCI, 2020), which prompted this study to assess the role of heterozygous BRCA2 variants in the development of pediatric RMS in both precursor cell and mouse models. Methods: We engineered BRCA2 loss of function (LoF) variants in lines of immortalized mesenchymal stem cells, a potential precursor cell to RMS, using CRISPR and Prime Editing (PE). CRISPR was used to induce frameshift alleles in Exons 10 and 11 and with PE, we created two RMS patient specific LOF variants in Exons 5 and 17. We generated a genetically engineered mouse model (GEMM) with heterozygous excision of Exon 11 of Brca2. This allele is now being crossed into two different previously established GEMMs of RMS: a myoblast-specific (MyoD1-Cre) conditional loss of p53, and a gain-of-function mutant p53, LSL-p53R172H (Nakahata, et al. Dis. Model. Mech., 2022), to assess changes in the pace of tumorigenesis and metastatic potential. Through an X01 agreement with the Gabriella Miller Kids First Program, we have submitted paired tumor-normal samples for additional whole-genome sequencing and RNA sequencing (tumor only) on >500 patients with RMS, including those with germline variants in BRCA2. Results: Using CRISPR, we generated cell lines heterozygous for the frameshift variants c.1345dup, c.5680del, and c.5680dup. With Prime Editing, we created heterozygous lines mimicking two specific variants detected in the RMS cohort, c.462_463del and c.7857G>A. Generation of a C57BL6 GEMM with a heterozygous removal of Exon 11 in Brca2 has been successful. No homozygous mice were obtained following 7 heterozygous crosses (50 pups), a departure from Mendelian ratios (p=0.0001) consistent with embryonic lethality. Heterozygous Brca2 mice derived from crosses into two RMS models are being monitored for tumor development. We have completed tumor-normal whole-genome and tumor RNA sequencing for 609 RMS patients, including 228 subjects not previously analyzed. We will directly assess whether there is evidence for mutation or loss of expression of the remaining BRCA2 allele in tumors and whether these tumor genomes exhibit signatures of DNA repair deficiency. Conclusions: The goal of this project is to determine the molecular basis for the enrichment in germline BRCA2 LOF variants in pediatric malignancies, including RMS. Ongoing experiments using the engineered heterozygous BRCA2 in vitro cell line models will assess genomic stability, homologous recombination proficiency, and DNA replication fork stability. Assessment of the effect of heterozygous Brca2 variants in GEMMs of RMS, and subsequent analysis of additional sequencing data, will further elucidate what role this gene may play in increasing risk or progression of pediatric cancers. Citation Format: P. Adam Weinstein, Ryan Zabriskie, Cem Ozdemir, Kyle M. Miller, Olga Medina-Martinez, Jason D. Heaney, Aniko Sabo, Philip J. Lupo, Sharon E. Plon. Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5610.

Abstract 2149: West African genetic ancestry and origin of the BRCA1 locus in Jamaican men with high grade prostate cancer

Abstract Introduction: Men of West African (WA) ancestry are at higher risk for prostate cancer than men of European (EU) or Asian descent. Caribbean populations provide an opportunity to identify potential founder mutations that contribute to cancer risk. Jamaica has among the highest rates of prostate cancer incidence and mortality in the world and the majority of the population are of WA ancestry. However, there is a significant amount of genetic admixture of EU and Asian ancestry from historic migration patterns. Thus, the population provides a unique cohort for studying the complex interplay between genetic ancestry and prostate cancer. In this pilot study, we characterized the overall and gene locus-specific ancestry of Jamaican prostate cancer patients to explore potential associations with clinicopathologic disease features. We specifically focused this analysis on the BRCA1 locus based on its known association with inherited prostate cancer risk and also with breast cancer risk in patients from the Caribbean. Methods: A retrospective cohort of 31 Jamaican men (non-US citizens/residents) who travelled to the University of Miami (2015-2020) specifically for robotic prostatectomy were included in the analysis. DNA was prepared from normal prostate tissue and analyzed using the Illumina Global Screening Array version 3 with Multi-Disease Drop-In that includes more than 650,000 genetic markers. Both overall genetic ancestry and allele specific ancestries were determined for each study subject. Clinical and pathological features (age, PSA, stage, grade group (GG)) were also recorded and analyzed using descriptive statistics. Results: Median age was 67 years, median pre-op PSA was 6.7. The majority of patients were cT1c (91%) and GG 2 (29%) on biopsy. On final pathology most men were ≥pT3 (51%) and the proportion of GG 1, 2 and ≥ 3 was 3%, 52% and 45% respectively. The mean overall West African genetic ancestry was 51.2% (SD 28.4%). Overall % West African ancestry was not associated with pathological GG. In contrast, 50% of the men who were homozygous for African ancestry alleles at the BRCA1 locus had high-grade prostate cancer (GG 3, 4, 5) versus 37% of the men with non-African or heterozygous BRCA1 genotypes. Conclusions: In a select cohort of Jamaican men, we found that half of patients with BRCA1 alleles derived from West African ancestry also have high grade prostate cancer. BRCA1 of African ancestral origin may contribute to the relatively high prevalence of aggressive prostate cancer in men from Jamaica. Work in progress focuses on an expanded study of associations between allele-specific genetic ancestry and prostate cancer in high-risk populations. Citation Format: Sandra M. Gaston, Anthony J. Griswold, Oleksandr N. Kryvenko, Tianjie Gu, Guan-Nan Zhang, Nachiketh S. Prakash, Yuval Avda, Tarek Ajami, Chad R. Ritch. West African genetic ancestry and origin of the BRCA1 locus in Jamaican men with high grade prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2149.

Abstract B011: BRCA2 haploinsufficiency induces P53 degradation in primary mammary epithelial cells – a crucial early event in breast tumor promotion

Abstract The presence of heterozygous germline BRCA2 mutations is associated with an increased risk of breast and ovarian cancer. In order to design effective preventative strategies, it is critical to understand early tumor promoting events that the mammary epithelial cells undergo on their way to tumorigenesis. To investigate the impact of BRCA2 heterozygosity on tumor formation, we utilized primary BRCA2mut/+ mammary epithelial cells obtained from prophylactic breast tissue of women with BRCA2 mutations. Our findings indicate that BRCA2mut/+ cells display haploinsufficiency for repairing stalled replication forks and resolving RPA32 from these sites, indicative of impaired stalled fork repair. Moreover, they exhibit defects in RAD51 recruitment to double strand break sites indicating a defective homologous recombination dependent double strand break repair efficiency. BRCA2mut/+ cells also show increased sensitivity to both replication stress and double strand break-inducing agents. Interestingly, we find that BRCA2 heterozygous cells downregulate P53 protein levels when compared to BRCA2 wild type cells, and that this downregulation is MDM2-dependent. The decreased P53 levels appear to promote the survival of BRCA2 heterozygous cells under endogenous replication stress conditions. Notably, treatment with nutlin, a P53 protein stabilizer, leads to cell death in BRCA2 heterozygous cells but not in BRCA2 wildtype cells, highlighting the sensitivity of BRCA2 heterozygous cells to endogenous P53 protein levels. Moreover, complete loss of P53 in BRCA2 heterozygous cells results in rescue of sensitivity of these cells to both replication stress and double strand break inducing agents. Furthermore, whole genome sequencing analysis of BRCA2 heterozygous cells deleted for P53 reveal DNA mutational signatures similar to those observed in BRCA2 mutant tumors, implying that loss or downregulation of P53 is an early event driving tumorigenesis in the context of BRCA2 heterozygosity. In summary, our study uncovers the significance of BRCA2 haploinsufficiency in replication stress suppression and highlights the role of P53 loss in promoting BRCA2 heterozygosity-driven cancer. These findings contribute to a better understanding of the molecular mechanisms underlying BRCA2-associated tumorigenesis and may have implications for targeted preventative therapies in the future. Citation Format: Haohui Duan, Margaret Gillis, Deboral Dillon, Judy Garber, Shailja Pathania. BRCA2 haploinsufficiency induces P53 degradation in primary mammary epithelial cells – a crucial early event in breast tumor promotion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B011.

Ex Vivo Chromosomal Radiosensitivity Testing in Patients with Pathological Germline Variants in Breast Cancer High-Susceptibility Genes BReast CAncer 1 and BReast CAncer 2.

Individual radiosensitivity is an important factor in the occurrence of undesirable consequences of radiotherapy. The potential for increased radiosensitivity has been linked to highly penetrant heterozygous mutations in DNA repair genes such as BRCA1 and BRCA2. By studying the chromosomal radiosensitivity of BRCA1/2 mutation carriers compared to the general population, we study whether increased chromosomal radiation sensitivity is observed in patients with BRCA1/2 variants. Three-color-fluorescence in situ hybridization was performed on ex vivo-irradiated peripheral blood lymphocytes from 64 female patients with a heterozygous germline BRCA1 or BRCA2 mutation. Aberrations in chromosomes #1, #2 and #4 were analyzed. Mean breaks per metaphase (B/M) served as the parameter for chromosomal radiosensitivity. The results were compared with chromosomal radiosensitivity in a cohort of generally healthy individuals and patients with rectal cancer or breast cancer. Patients with BRCA1/2 mutations (n = 64; B/M 0.47) overall showed a significantly higher chromosomal radiosensitivity than general healthy individuals (n = 211; B/M 0.41) and patients with rectal cancer (n = 379; B/M 0.44) and breast cancer (n = 147; B/M 0.45) without proven germline mutations. Chromosomal radiosensitivity varied depending on the locus of the BRCA1/2 mutation. BRCA1/2 mutations result in slightly increased chromosomal sensitivity to radiation. A few individual patients have a marked increase in radiation sensitivity. Therefore, these patients are at a higher risk for adverse therapeutic consequences.

RE: Heterozygous BRCA1 and BRCA2 and mismatch repair gene pathogenic variants in children and adolescents with cancer.

RE: Heterozygous BRCA1 and BRCA2 and mismatch repair gene pathogenic variants in children and adolescents with cancer.

Heterozygous BRCA1 and BRCA2 and Mismatch Repair Gene Pathogenic Variants in Children and Adolescents With Cancer.

Genetic predisposition is has been identified as a cause of cancer, yet little is known about the role of adult cancer predisposition syndromes in childhood cancer. We examined the extent to which heterozygous pathogenic germline variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, MSH2, MSH6, MLH1, and PMS2 contribute to cancer risk in children and adolescents. We conducted a meta-analysis of 11 studies that incorporated comprehensive germline testing for children and adolescents with cancer. ClinVar pathogenic or likely pathogenic variants (PVs) in genes of interest were compared with 2 control groups. Results were validated in a cohort of mainly European patients and controls. We employed the Proxy External Controls Association Test to account for different pipelines. Among 3975 children and adolescents with cancer, statistically significant associations with cancer risk were observed for PVs in BRCA1 and 2 (26 PVs vs 63 PVs among 27 501 controls, odds ratio = 2.78, 95% confidence interval = 1.69 to 4.45; P < .001) and mismatch repair genes (19 PVs vs 14 PVs among 27 501 controls, odds ratio = 7.33, 95% confidence interval = 3.64 to 14.82; P<.001). Associations were seen in brain and other solid tumors but not in hematologic neoplasms. We confirmed similar findings in 1664 pediatric cancer patients primarily of European descent. These data suggest that heterozygous PVs in BRCA1 and 2 and mismatch repair genes contribute with reduced penetrance to cancer risk in children and adolescents. No changes to predictive genetic testing and surveillance recommendations are required.

Abstract P3-13-03: Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids

Abstract Background: Women carrying a germline BRCA1 mutation, have 60% risk of developing breast cancer during their lifetime and frequently develop triple-negative, basal-like, aggressive breast tumors. Humans produce around 150,000 different proteins from their 25,000-30,000 genes. This is accomplished by alternative splicing (AS). We hypothesized that AS in BRCA1 mutation carriers is different from those women with wild type BRCA1, therefore, we examined AS, expression of genes encoding splicing factors, and enrichment of binding motifs for RNA-binding proteins in BRCA1 carriers (BRCA1mut/+) and controls (BRCA1 normal). Methods: Prophylactic mastectomy (BRCA1mut/+) and reduction mammoplasty (BRCA1 normal) specimens were collected at Prentice Women’s Hospital of Northwestern Medicine under approval by Northwestern’s Institutional Review Board (NU15B07). We isolated organoids from these tissues, and cryopreserved them for future use. We selected organoids of premenopausal women, age-matched (28-46) without oral contraceptive usage within 3 years: BRCA1mut/+ (n=12) and BRCA1 normal (n=4). Organoids were maintained in complete MammoCult medium in ultra-low attachment plates at 37 °C, 5% CO2 for 72 hrs, and harvested for RNA extraction (NucleoSpin RNA Plus, TaKaRa). 100 ng of total RNA (RIN 7+) were used for RNA sequencing assay with the KAPA mRNA Hyper Prep Kit (Roche Corporate) and NovaSeq 6000 for 100b paired-end sequencing (Illumina, Inc). The preprocessed reads were aligned to the human genome (hg38) using STAR. Differential expression analysis (DESeq2, nominal p-value &amp;lt; 0.05) and pathway analysis (GSEA, size ≥30, NES &amp;gt;2, FDR q-value &amp;lt;0.05, and enriched genes ≥ 5) were performed to compare BRCA1mut/+ with BRCA1 normal. We used rMATS- turbo v4.1.0 to perform multivariate analysis of transcript splicing and chose an FDR &amp;lt; 0. 05, to identify significant differential alternative splicing events. To determine which RNA binding proteins and splicing factors were employed in BRCA1mut/+ tissue, we examined the cis-regulatory motifs adjacent to splice sites using rMAPs. Results: BRCA1mut/+ organoids displayed 155 upregulated genes compared to BRCA1 normal organoids (P &amp;lt;0.05). Among top 20 enriched pathways, we identified that expression of mRNA spliceosome (POLR2L, U2AF2, NUP188, SNRPF, CSTF3, HNRNPM, SRSF2), and, in addition, cell cycle, oxidative phosphorylation, HIV infection, and protein metabolism genes were significantly upregulated in BRCA1mut/+ organoids. We identified significant differential AS events (FDR &amp;lt;0.05) between BRCA1mut/+ and BRCA1 normal organoids: 2159 genes (skipped exon), 433 genes (mutually exclusive exon), 325 genes (retained intron), 304 genes (alternative 3’ splice sites), 168 genes (alternative 5’ splice sites). RNA splicing analysis indicated that highly expressed genes (SRSF2, ALG3, AP1B1 and PMF1) in BRCA1mut/+ organoids were also alternatively spliced. In addition, many other serine/arginine-rich splicing factors (SRSF) themselves (SRSF1, 2, 3, 5, 6, 7) were alternatively spliced (skipped exon events). An examination of binding motifs of RNA-binding proteins with significantly increased expression in BRCA1 carriers revealed multiple significantly enhanced and silenced regions flanking exons involved in AS. Conclusions: Our data suggest that AS of BRCA1mut/+ mammary tissue significantly differ from BRCA1 normal tissue, and may alter RNA spliceosome activity, and consequently play a role in malignant transformation and warrant further study. Citation Format: Oukseub Lee, Mi-Ran Choi, Gannon Cottone, Priyam Patel, Susan E Clare, Seema A. Khan. Heterozygous BRCA1 mutation influences alternative splicing in human benign mammary organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-03.

The predictive ability of the 313 variant–based polygenic risk score for contralateral breast cancer risk prediction in women of European ancestry with a heterozygous BRCA1 or BRCA2 pathogenic variant

PurposeTo evaluate the association between a previously published 313 variant–based breast cancer (BC) polygenic risk score (PRS313) and contralateral breast cancer (CBC) risk, in BRCA1 and BRCA2 pathogenic variant heterozygotes. MethodsWe included women of European ancestry with a prevalent first primary invasive BC (BRCA1 = 6,591 with 1,402 prevalent CBC cases; BRCA2 = 4,208 with 647 prevalent CBC cases) from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), a large international retrospective series. Cox regression analysis was performed to assess the association between overall and ER-specific PRS313 and CBC risk. ResultsFor BRCA1 heterozygotes the estrogen receptor (ER)-negative PRS313 showed the largest association with CBC risk, hazard ratio (HR) per SD = 1.12, 95% confidence interval (CI) (1.06–1.18), C-index = 0.53; for BRCA2 heterozygotes, this was the ER-positive PRS313, HR = 1.15, 95% CI (1.07–1.25), C-index = 0.57. Adjusting for family history, age at diagnosis, treatment, or pathological characteristics for the first BC did not change association effect sizes. For women developing first BC < age 40 years, the cumulative PRS313 5th and 95th percentile 10-year CBC risks were 22% and 32% for BRCA1 and 13% and 23% for BRCA2 heterozygotes, respectively. ConclusionThe PRS313 can be used to refine individual CBC risks for BRCA1/2 heterozygotes of European ancestry, however the PRS313 needs to be considered in the context of a multifactorial risk model to evaluate whether it might influence clinical decision-making.

Characteristics and experiences of patients from a community-based and consumer-directed hereditary cancer population screening initiative.

SummaryA clinical hereditary cancer population screening initiative, called Information is Power, began in North Alabama in 2015. After 4 years of the initiative, we were interested in exploring (1) the characteristics and motivations for patients who self-refer to population genetic testing, (2) how patients make decisions on testing, (3) what patients do with results, and (4) patient perceptions of benefits and limitations after undergoing population genetic testing. Patients who consented to research recontact at time of test ordering were sent an electronic survey with the option for a follow-up phone interview. Among the 2,918 eligible patients, 239 responded to the survey and 19 completed an interview. Survey and interview participants were highly educated information seekers motivated by learning more about their health. Those who were previously interested in hereditary cancer testing reported barriers were cost and insurance coverage, access to testing, and uncertainty how results could impact their health. Many participants (77%) communicated with family and friends about their decision to test and communicated about test results. Fewer participants (23%) discussed the decision to test with their healthcare providers; however, 58% of participants discussed their test results with a healthcare provider. Most people (96%) with negative results accurately recalled their results. In contrast, three out of 11 positive results for heterozygous MUTYH, PALB2, and BRCA2 reported receiving negative results. This study contributes to knowledge on population genetic testing and may guide other population genetic testing programs as they develop enrollment materials and educational materials and consider downstream needs of population genetic testing participants.

Mutational Burden before and after Allogeneic Hematopoietic Cell Transplantation (HCT) in Patients with Acute Myeloid Leukemia (AML)

BackgroundAllogeneic HCT remains the only curative option for patients (pts) with high-risk or relapsed AML. Genome sequencing technologies such as next generation targeted deep sequencing (NGS) or whole exome sequencing have identified several recurrent somatic mutations in AML that may be associated with HCT outcomes. Genomic abnormalities may persist in pts who achieve complete remission (CR) after induction chemotherapy; however, the impact of HCT on the mutational burden before and after transplant in such pts has not been described. We explore the dynamic changes of the clonal architecture in serial samples obtained from AML pts before and after HCT.MethodsNGS data on bone marrow and peripheral blood samples obtained at diagnosis, prior to transplant, and at day 30 and day 100 after transplant from pts with AML (according to 2016 WHO criteria) who were treated at our institution between 11/2015 - 1/2017 were analyzed. A total of 170 somatic and germline gene mutations that have been described in hematologic malignancies were sequenced at each time point. The mean depth of the targeted sequencing was 300 fold for the entire cohort. Clonal architecture was defined by using variant allelic frequency (VAF) adjusted for zygosity at each time point.ResultsA total of 28 pts with serial samples were included. The median age at transplant was 57 years (range, 25-74). Cytogenetic analysis at diagnosis included normal karyotype in 15 pts (54%), 9 (32%) with unfavorable risk per Alliance criteria (including 5 pts with complex karyotype), 1 (3%) with t(8,21), and 3 pts (11%) with other (intermediate risk) abnormalities. All pts received induction chemotherapy at diagnosis with cytarabine and anthracycline (7+3) +/- combination except for one pt who received azacitidine. All pts achieved complete remission (CR/CRi) per 2003 IWG criteria prior to transplant. Conditioning regimens were myeloablative (N= 17, 61%) and reduced-intensity (N=11, 39%). Graft sources were bone marrow (N = 17, 61%), and peripheral blood (N =11, 39%). Donor source was matched sibling (N= 9, 32 %), matched unrelated (N= 14, 50 %) or haplo-identical (N= 5, 18%). Among pts with samples available at diagnosis, the most frequently mutated genes were: DNMT3A (46%), NPM1 (21%), FLT3 (ITD/TKD, 20%), TET2 (13%), and WT1 (13%). All pts with abnormal cytogenetics (13 pts) achieved complete cytogenetic remission prior to transplant. Common leukemia-related mutations such as DNMT3A, ASXL1, and TET2 persisted among others prior to transplant even in pts with CR and complete cytogenetic response. Among pts who achieved full engraftment at day 30 and/ or day 100 post-transplant, persistent leukemia-related mutations were still identified. In 2 pts with heterozygous BRCA2 and 1 pt with a heterozygous BRCA1 mutation at diagnosis, mutations persisted at CR, prior to transplant and at day 30 and 100 post-transplant, with a VAF of 50%, suggesting that these likely represented germline mutations. None of these pts had a history of breast or gynecological cancers. The median follow up after transplant was 9.97 months (range, 6.07- 20.17). Among pts with persistent mutations at day 100, 37% relapsed and the rest continued to be in remission until their last follow up.ConclusionGenomic analysis showed persistent leukemia-associated mutations in pts with AML in morphological and cytogenetic CR prior to HCT. Regardless of the conditioning regimen, a significant proportion of pts had persistent mutations at day100 after transplant even in pts with full engraftment. Persistent mutations predicted relapse though not all pts relapsed during this study period. These data suggest that deep genome sequencing may help in identifying higher risk pts of relapse post HCT, though longer follow up is needed to monitor the persistent mutations in pts who did not relapse during study time. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Majhail:Sanofi: Honoraria; Anthem, Inc.: Consultancy.

The Fanconi Pathway in Pediatric T-Cell Acute Lymphoblastic Leukemia

BRCA2 (also known as FANCD1), a core component of the Fanconi pathway, suppresses transformation of immature T-cell progenitors in mice. However, whether the Fanconi pathway contributes to human T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis is unknown. Using targeted exon sequencing of childhood T-ALL diagnostic specimens, we found heterozygous mutations of Fanconi pathway genes in 23% of 40 cases analyzed. Array comparative genomic hybridization (CGH) analysis revealed that an additional 15% of these cases harbored large heterozygous deletions involving Fanconi pathway genes. The majority of these mutations appear to be somatic although in 2 of 8 available remission samples, the mutations were present, suggestive that these mutations are likely to be germline. Fanconi genes are typically considered two-hit tumor suppressors, where breast or ovarian cancers arising in heterozygous carriers often lose activity of the wild-type allele, leading to genomic instability. However, T-ALL cases with heterozygous Fanconi mutations lacked evidence of increased genomic instability based on array CGH analysis, were not hypersensitive to the PARP inhibitor olaparib, and did not demonstrate increased radial chromosome formation in response to mitomycin C (MMC), indicating that these cases retained activity of the wild-type allele. Complementation studies of Fanconi mutant cells revealed that most of the identified Fanconi mutations encoded pathogenic alleles, as evidenced by their impaired ability to rescue Fanconi-deficient cells from mitomycin C-induced radial chromosome formation and cytotoxicity. Treatment of a patient-derived xenograft sample harboring a heterozygous BRCA2 mutation with mitomycin C revealed that these cells had an intermediate increase in sensitivity to mitomycin C-induced cytotoxicity compared to Fanconi wild-type T-ALLs, suggesting that Fanconi haploinsufficiency is pathogenic in T-ALL. Together, these findings implicate a tumor suppressor role for Fanconi pathway haploinsufficiency in childhood T-ALL. DisclosuresHunger:Jazz Pharmaceuticals: Honoraria; Novartis: Consultancy; Amgen: Consultancy, Equity Ownership; Erytech Pharmaceuticals: Consultancy. Neuberg:Synta Pharmaceuticals: Other: Stock shares.

Leptin Mediates Obesity-Induced DNA Damage in BRCA1 Breast Epithelial Cells

Abstract Background: Obesity is a risk factor for breast cancer development in women who carry a mutation in the DNA repair enzymes BRCA1 or BRCA2. Previously, we found that obesity was positively associated with DNA damage in breast epithelium from BRCA mutation carriers. Furthermore, factors secreted by obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells, suggesting a cross-talk between breast epithelial cells and the adipose tissue that surrounds them. We hypothesized that leptin, a hormone secreted in abundance by obese adipose tissue, may be a driver of DNA damage and/or decrease capacity for DNA repair in breast epithelial cells. If true, this would provide a molecular target for intervention to reduce the risk of tumor formation in this high-risk population of women. Methods: RNA-seq followed by Ingenuity Pathway Analysis (IPA) was conducted on primary breast epithelial organoids isolated from lean and obese BRCA mutation carriers. Breast adipose tissue obtained from lean and obese women were cultured as explants for 24 hours to produce lean and obese conditioned media (CM). The effect of leptin on DNA damage was assessed in a non-cancerous breast epithelial cell line (MCF10A) carrying a heterozygous BRCA1 mutation. Immunofluorescence staining of the DNA damage marker ƴH2AX was carried out after treatment with leptin (100ng-800ng), CM, or CM+leptin antibody, used to neutralize leptin. To test whether leptin affects DNA repair capacity, BRCA1+/- MCF10A cells were treated with leptin or vehicle and then irradiated (1Gy) to induce DNA damage. Resolution of damage was quantified at 0, 0.5, 4, 12, and 24 hrs post-irradiation. Results: IPA analysis identified leptin signaling as significantly upregulated in breast epithelial organoids from obese women compared with lean women. Both obese CM and leptin treatment induced DNA damage in BRCA1+/- MCF10A cells while lean CM did not have this effect. Neutralizing leptin in obese CM was sufficient to inhibit obese CM-mediated induction of DNA damage. No significant difference was observed between leptin or vehicle treatments on DNA repair capacity after irradiation of BRCA+/- MCF10A cells. Conclusions: These data identify leptin, an adipose-derived hormone, as a novel driver of DNA damage in breast epithelial cells. To date, no studies have elucidated the molecular mechanisms that explain the increased penetrance of breast cancer in obese BRCA mutation carriers compared to lean BRCA mutation carriers. This work suggests that leptin may be a mediator of the link between obesity and breast cancer development in this population. Further studies are warranted to determine if targeting the leptin signaling axis will be an effective risk reduction strategy in BRCA mutation carriers who have excess adiposity.

Spontaneous Development of Hepatocellular Carcinoma and B-Cell Lymphoma in Mosaic and Heterozygous Brca2 and Cdkn1a Interacting Protein Knockout Mice

Hepatocellular carcinoma (HCC) is the most common form of liver tumors. Although HCC is associated with chronic viral infections, alcoholic cirrhosis, and nonalcoholic fatty liver disease, genetic factors that contribute to the HCC risk remain unknown. The BRCA2 DNA repair associated (BRCA2) and cyclin-dependent kinase inhibitor 1A (CDKN1A) interacting protein, known as BCCIP, are essential for cell viability and maintenance of genomic stability. In this study, we established a new genetically engineered mouse model with Bccip deficiency. Mosaic or heterozygous Bccip deletion conferred an increased risk of spontaneous liver tumorigenesis and B-cell lymphoma development at old age. These abnormalities are accompanied with chronic inflammation, histologic features of nonalcoholic steatohepatitis, keratin and ubiquitin aggregates within cytoplasmic Mallory-Denk bodies, and changes of the intracellular distribution of high-mobility group box 1 protein. Our study suggests BCCIP dysregulation as a risk factor for HCC and offers a novel mouse model for future investigations of nonviral or nonalcoholic causes of HCC development.

Abstract P4-09-13: Spectrum analysis of the inherited mutations of BRCA1/-2 genes that associated with high risk breast cancer in Saudi population

Abstract Background: Breast cancer (BC) is the most common prevalent cancer among female worldwide, despite the advances in molecular biology. In Saudi Arabia, the disease represents 19.9% of the total cancer-related deaths in women. Inherited mutations of BRCA1/-2 are well documented to be associated with higher risk of breast cancer. Aim: To investigate the role of inherited mutation in BRCA1/-2 genes that associated with breast cancer in Saudi population. Methodology: A total of 41 FFPE samples were collected from BC patients admitted to KFSHRC-Jeddah from 2013 to 2018. Genomic DNA was extracted using the Qiagen FFPE kit. A100ng of each DNA samples were used for library preparation using Ion AmpliSeq™ Library Kit Plus with Saudi Inherited Cancer Panel. Ion Torrent Proton platforms were used for DNA sequencing on Ion Torrent PGM using Ion PI™ Hi-Q™ Sequencing 200 Kit. Results: A total of 26 distinct heterozygous BRCA1/-2 variants were identified in Saudi population, 15 variants in BRCA1 gene and 11 in BRCA2 gene. In BRCA1, one variant was nonframeshift deletion and 11 were missense, 3 variants likely benign, while in BRCA 2, one was stopgain and 6 were missense and 4 variants likely benign. Conclusion: Screening of BRCA1/-2 genes mutations helps in understanding the risk associated with mutations of other forms of cancer in Saudi population. The preliminary data is used as an establishing of a high-throughput NGS molecular profiling for Saudi breast cancer patients Citation Format: Yousef MohammedRabaa Hawsawi, Abdelrazak Meliti, Edward Cupler, Turki Sobahy, Samar Zailaie, Mohammed Baghdadi, Hosam Alardati. Spectrum analysis of the inherited mutations of BRCA1/-2 genes that associated with high risk breast cancer in Saudi population [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-13.

Analysis of Heterozygous BRCA1 5382ins Founder Mutation in a Cohort of Egyptian Breast Cancer Female Patients Using Pyrosequencing Technique.

Background:Up to half of the heritable mutations in breast cancer (BC) are attributed to BRCA1 and BRCA2 genes. The mutation prevalence is variable based on ethnicity and may be influenced by founder mutations. The aim of this pilot study is to determine for the first time, the prevalence of BRCA1 5382insC founder mutation in a cohort of Egyptian familial breast cancer patients (FBC). Methods:Female patients were selected to have familial type of breast cancer. Twenty healthy females were included as a control group. Peripheral blood samples were withdrawn from all studied females and were analyzed for BRCA1 5382insC founder mutation detection using pyrosequencing technique. Results:Eighty Egyptian FBC females were eligible to be enrolled in the study with a mean age of 48.31 ± 10.97years.We found a BRCA1 5382insC mutation carrier frequency of 5% of total studied FBC patients (4 out of 80 patients) with 95% confidence interval (1.61-12.99). There was a high statistical significant difference between carriers and non-carriers concerning the number of affected family members by BC, (p=0.001). Conclusion: BRCA1 5382insC founder mutation is not uncommon among Egyptian FBC females. The carrier frequency is comparable to that reported worldwide; however it is lower than those from previous Egyptian studies using different molecular techniques. The strong association between the mutation and the number of affected family members suggest wider screening of the mutation among high risk families using the reliable pyrosequencing technique.

Abstract 2723: Defective homologous recombination DNA repair as therapeutic target in advanced chordoma

Abstract Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown. We performed whole-exome and genome sequencing of tumor and matched germline control samples from 11 patients with locally advanced or metastatic chordoma within the MASTER program, a prospective clinical sequencing program of the German Cancer Consortium. All patients were pretreated and had progressive disease prior to molecular analysis. Genomic profiling showed that advanced chordomas are frequently characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair. First, DNA copy number profiles showed high numbers of structural variants greater than 10 million base pairs in size in the majority of cases. Second, all patients harbored somatic aberrations of at least 2 genes known to be involved in HR, and 10/11 cases harbored somatic alterations in 3 or more HR pathway genes. For example, 8 patients showed heterozygous BRCA2 deletions, which were associated with heterozygous deletions of ERCC6 in 6 patients and RAD54L in 7 patients, as well as PTEN alterations (heterozygous deletion, heterozygous mutation and deletion of the wildtype allele or loss of heterozygosity). Other recurrently altered HR genes included ATR, CHEK2, FANCC, FANCD2, FANCG, RAD18, RAD51B, and XRCC3. Third, pathogenic germline alterations were detected in 3 patients. A heterozygous BRCA2 frameshift mutation (p.T3085fs*26; ACMG Class 5), a heterozygous NBN frameshift mutation (p.K219Nfs*16; ACMG Class 5), and a heterozygous CHEK2 missense mutation (p.R145W; ACMG Class 4) were accompanied by somatic deletion of the respective wildtype alleles. Fourth, a mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and coincided with genomic instability. The high prevalence of an HR deficiency “footprint” in chordoma patients prompted us to explore the clinical efficacy of the poly(ADP-ribose) polymerase(PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells. Olaparib treatment of a patient whose tumor showed a prominent exposure to an HR deficiency-associated mutational signature, a high degree of genomic instability, and 13 heterozygous HR gene alterations halted tumor growth for 10 months. Whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain, providing novel insight into the mechanisms of PARP inhibitor resistance. In summary, our study has uncovered a key biological feature of advanced chordoma that represents an immediately actionable therapeutic target and provides a rationale for genomics-guided clinical trials of PARP inhibition in this intractable tumor entity. Citation Format: Stefan Gröschel, Daniel Hübschmann, Francesco Raimondi, Peter Horak, Gregor Warsow, Martina Fröhlich, Barbara Klink, Laura Gieldon, Barbara Hutter, Kortine Kleinhenz, David Bonekamp, Oliver Marschal, Priya Chudasama, Jagoda Mika, Marie Groth, Sebastian Uhrig, Stephen Krämer, Christoph Heining, Christoph Heilig, Daniela Richter, Eva Reisinger, Katrin Pfütze, Roland Eils, Stephan Wolf, Christof von Kalle, Christian Brandts, Claudia Scholl, Wilko Weichert, Stephan Richter, Sebastian Bauer, Roland Penzel, Evelin Schröck, Albrecht Stenzinger, Richard Schlenk, Benedikt Brors, Robert Russell, Hanno Glimm, Matthias Schlesner, Stefan Fröhling. Defective homologous recombination DNA repair as therapeutic target in advanced chordoma [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 2723.

SAT-339 Cross-Talk with Breast Adipose Tissue Contributes to Obesity-induced DNA Damage in BRCA Mutant Breast Epithelial Cells

Background: Obese women who carry a mutation in the DNA repair enzymes BRCA1 and/or BRCA2 are at a greater risk of developing breast cancer compared with lean BRCA1/2 mutation carriers. Causative factors that drive the increased penetrance of breast cancer in obese women are poorly characterized. Previously, we showed that BMI was positively correlated with DNA damage in breast epithelium of BRCA mutation carriers. We hypothesized that obesity-associated dysfunctional adipose tissue may cause DNA damage in neighboring breast epithelial cells. If true, this would provide evidence for the initial insults associated with increased risk of tumor formation in obese women. Methods: To test the effect of locally secreted factors on DNA damage induction, conditioned media was collected over 24 hours from primary breast adipose explants obtained from lean and obese women. Conditioned media was used to treat normal breast epithelial cells (MCF10A) harboring a heterozygous BRCA1 mutation or wildtype for BRCA. Immunofluorescence (IF) staining of the DNA damage marker ƴH2AX was carried out after treatment. Cell line findings were validated in primary breast epithelial organoids isolated by collagenase digestion of breast tissue obtained from the clinic. Additionally, RNA-seq was conducted on primary breast epithelial organoids isolated from lean and obese women to identify potential drivers of gene expression differences associated with obesity and DNA damage. Results: Conditioned media from obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells while lean conditioned media did not have this effect. Similar findings were made in primary breast epithelial organoids, suggesting a role for locally secreted factors in mediating DNA damage. Ingenuity Pathway Analysis of RNA-seq data from lean and obese primary breast organoids identified several upstream regulators of gene expression differences, including signaling by estradiol and pro-inflammatory cytokines, both of which are elevated in obese breast tissue and are known inducers of DNA damage. Conclusions: These data show for the first time that local factors produced by obese breast adipose tissue induce breast epithelial cell DNA damage. Our results suggest that therapies targeting weight or dysfunctional breast adipose tissue may reduce the increased DNA damage observed in obese BRCA mutation carriers and thereby decrease tumor formation in this high-risk population of women.

DOPlify, Target Sequence Enrichment and Allele Drop-Out – is there a benefit?

Introduction The ability to combine whole genome amplification (WGA) with gene-specific primers in a single PCR reaction to allow both aneuploidy detection (PGT-A) and monogenic disease detection (PGT-M) is an advantage of DOPlify® and the RHS targeted sequence enrichment (TSE) protocol. This study aims to compare Allele Drop-Out (ADO) rates by comparing three different approaches; gene specific PCR only, WGA and WGA with TSE. Material and Methods Thirty 5-cells aliquots were manually sorted from a cell line containing a 2bp heterozygous BRCA1 mutation (GM14090, Coriell Institute). To allow an assessment of aneuploidy detection, thirty 5-cell aliquots from an aneuploid cell line (GM04965, 48,XXY,+21, Coriell Institute) were also used. In the TSE approach, cells were amplified using DOPlify® (RHS Ltd) with the inclusion of three BRCA1 primers. One BRCA1 primer set was designed to specifically amplify the region on chromosome 17 containing the deletion site in GM14090 and other two primer sets to amplify BRCA1 linker markers. The resulting WGA-TSE product was diluted 1/10 and used as template for a second gene specific multiplex PCR (Qiagen) containing the same BRCA1 primers. The BRCA1 PCR products were then pooled back into the WGA-TSE product before library preparation and analysed using a single library index (WGA-TSE+GS). Secondly, for PGT-M only, cells were subjected to two rounds of gene specific PCR using multiplex PCR (Qiagen) and the same BRCA1 primers (GS+GS). Lastly, cells were amplified according to standard DOPlify® protocol (RHS Ltd) with no BRCA1 primers added. The resulting WGA product was diluted 1/10 and seeded into the BRCA1 multiplex PCR reaction (Qiagen) (WGA+GS). PCR products from the WGA-TSE+GS condition were sequenced according to standard PG-Seq™ protocol (RHS Ltd). Amplicons from the GS+GS and WGA+GS conditions were diluted 1/10 before final library pooling then were sequenced with the Illumina MiSeq. Results The 2bp heterozygous BRCA1 deletion was accurately detected in 10/10 of the WGA-TSE+GS samples, 10/10 of the GS+GS samples and 7/10 of the WGA+GS samples with average allele frequencies of 41%, 47% and 53%, respectively. The average read depth across the 2bp deletion site was 120, 129 and 138, respectively. One GM04965 sample from the GS+GS condition failed to amplify the region. Correct aneuploidy results were obtained for 9/9 BRCA1 cells and 10/10 GM04965 cells processed with the WGA-TSE+GS protocol with one BRCA1 sample excluded after failing quality control (weak amplification). The GS+GS protocol does not allow for PGT-A assessment. Conclusions The DOPlify® TSE protocol is able to reliably generate both PGT-A and PGT-M results from a cell sample representing a single embryo biopsy. The PGT-A results were not compromised, and PGT-M results showed no ADO and were comparable to the gene specific PCR protocol. These results highlight the benefit of the DOPlify® TSE protocol, which is now being clinically validated.

Abstract ES12-1: BRCA2 and control of transcription: R-loop dynamics

Abstract Pathogenic germline mutations affecting the breast cancer genes, BRCA1 and BRCA2, predispose to cancers of the breast, ovary, and other tissues with a high penetrance. Cancer pathogenesis in mutation carriers has been linked to genomic instability following inactivation of the essential cellular functions of the encoded BRCA1 and BRCA2 proteins in DNA repair and the restoration of stalled DNA replication (reviewed in Venkitaraman, Science 343, 1470-5 (2014)). Here, I will discuss emerging evidence that links previously unrecognized functions of the BRCA proteins to carcinogenesis, focusing on BRCA2. Increasing evidence indicates that heterozygosity for pathogenic BRCA2 mutations suffices for carcinogenesis in experimental models and in human mutation carriers (eg., Skoulidis et al., Cancer Cell 18, 499-509 (2010); Maxwell et al., Nature Commun 8, 319 (2017); Huang et al., Cell 173, 355-370 (2018)). Heterozygous mis-sense mutations in BRCA2 may act trans-dominantly to interfere with the nuclear transport of BRCA2/RAD51 complexes (Jeyasekharan et al., Nature Str Mol Biol 20, 1191-8 (2013)). We have reported recently a gene-environment interaction that may modify carcinogenesis in BRCA2 mutation carriers, wherein exposure to aldehydes, a pervasive class of endogenous and environmental toxins, selectively proteolyzes BRCA2, inducing haploinsufficiency for DNA repair and replication fork stability in cells bearing heterozygous truncating BRCA2 mutations (Tan et al., Cell 169, 1105-18 (2017)). BRCA2-deficient cells accumulate unscheduled RNA-DNA hybrids (R-loops) and genomic damage through an unexpected role of BRCA2 in promoting the switch from promoter-proximal pausing to transcription elongation by RNA polymerase II (Shivji et al., Cell Rep 22, 1031-1039 (2018)). These emerging functions have implications for the pathogenesis and therapy of cancers arising in patients who carry BRCA2 mutations. Citation Format: Venkitaraman A. BRCA2 and control of transcription: R-loop dynamics [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES12-1.

Accurate combined preimplantation genetic testing with doplify and target sequence enrichment

Allele Drop Out (ADO), or failed PCR amplification of one or both alleles, presents a significant risk of misdiagnosis or the return of a “no result” for preimplantation genetic testing of monogenetic diseases (PGT-M). Although a PGT-M result may indicate that an embryo is unaffected by a monogenic disease, standard PGT-M methods are unable to assess aneuploidy and up to 50% of these transferred embryos may be aneuploid1. Combining Whole Genome Amplification (WGA) along with gene-specific primers in a single reaction to allow both aneuploidy detection (PGT-A) and monogenic disease detection (PGT-M) is an advantage of DOPlify and RHS’ Target Sequence Enrichment (TSE) protocol. This study aims to compare ADO rates across three different approaches; Gene Specific PCR only, DOPlify WGA only and DOPlify WGA with TSE. Thirty 5-cell aliquots were manually sorted from cell line GM14090 containing a 2bp heterozygous BRCA1 mutation on chromosome 17 and aneuploid cell line GM04965, 48,XXY,+21 (Coriell Institute). For the Gene Specific PCR only approach; cells were subjected to a round of sequence specific PCR using multiplex PCR (Qiagen) and three sets of BRCA1 primers. One BRCA1 primer set amplifies the region containing the 2bp deletion and the other two primer sets amplify linked markers. For the DOPlify WGA only approach; cells were amplified according to standard protocol with no BRCA1 primers added. For the DOPlify WGA with TSE approach, cells were amplified with the inclusion of the three BRCA1 primer sets according to the DOPlify TSE protocol. PCR products from the three approaches were then diluted 1/10 and seeded into a second sequence specific multiplex PCR using the three sets of BRCA1 primers to generate sufficient DNA for sequencing. Presence, allele frequency and read depth of the 2bp heterozygous deletion and the linked markers were measured to assess ADO. The allele frequency and read depth for the three PCR approaches for GM14090 are listed in the table below. The linked marker primer sets were less efficient in the multiplex PCR, as evidenced by their lower read depth. These results may be improved with further multiplex optimisation and deeper sequencing. Correct aneuploidy results were obtained for all cells processed with the DOPlify TSE protocol which passed sequencing quality control.Tabled 1Allele Frequency and Read DepthGene Specific PCR Only (PGT-M)DOPlify Only (PGT-A)DOPlify with TSE (PGT-M and PGT-A)Presence of both alleles/number passed QC (read depth >10)BRCA1 2bp Deletion10/107/910/10D17S8559/99/910/10D17S11858/82/210/10Allele frequency (%)BRCA1 2bp Deletion47.153.741.6D17S85536.350.438.9D17S118576.274.380.2Read DepthBRCA1 2bp Deletion129±38138±12120±13D17S855113±4066±4469±12D17S118577±4914±3628±8 Open table in a new tab DOPlify with TSE enables a sensitive and reliable PGT-M result; showing no ADO in any of the three regions amplified by the sequence specific primers and linked markers, while also providing accurate PGT-A information from a single embryo biopsy.