Reis‐Filho, JS

Abstract Introduction:Pathogenic and/or founder germline variants in the ataxia-telangiectasia mutated (ATM) gene confer an increased breast cancer (BC) risk. The protein kinase ATM plays a central role inDNA double-strand break-repair and in the activation of downstream targets such as p53 and BRCA1. We sought to define the repertoire of somatic genetic alterations of BCs from patients with pathogenic germline ATM mutations and whether somatic loss of heterozygosity (LOH) of ATM would be present in these cancers. Methods: 21 BCs from ATM germline mutation carriers were microdissected. Tumor and normal DNA samples were subjected to whole-exome sequencing (WES, n=12) or massively parallel sequencing targeting all coding regions and selected intronic and regulatory regions of 410 key cancer genes (n=9). Somatic mutations, copy number alterations, cancer cell fractions, large-scale state transitions (LSTs) and mutational signatures were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to assess LOH of the wild-type allele of ATM. Results: Of the patients included in this study, 71%, 24% and 5% of cases harbored ATM missense (all but one p.V2424G), frame-shift and nonsense germline mutations, respectively. All tumors were ER-positive and four (19%) were HER2-positive. The median age of the patients was 46 years (32–79 years). Our analyses revealed biallelic inactivation of ATM through LOH of the wild-type allele in 16 of 21 cases (76%), and second somatic ATM mutations were not found. The median number of non-synonymous somatic mutations was 38 (range 15-113) and 2 (range 0-8)in tumors subjected to WES and targeted sequencing, respectively. The repertoire of somatic genetic alterations of ATM-associated BCs was found to be heterogeneous, including clonal PIK3CA mutations (24%), GATA3 mutations (19%), FANCI amplifications (19%) and CCND1 amplifications (14%). Importantly, however, no somatic mutations affecting TP53 were found. Analysis of the WES data revealed that 5 (42%) ATM-associated BCs displayed high LST scores, all of which harbored bi-allelic ATM inactivation. In contrast to BRCA1- and BRCA2-associated BCs, which frequently display the mutational signature 3 associated with defective homologous recombination DNA repair, the ATM-associated BCs studied displayed the ageing mutational signature (i.e. signature 1). Comparison of the mutational profiles of the ATM--associated BCs subjected to WES (n=12) with those of BRCA1- (n=11) and BRCA2-associated (n=10) BCs from The Cancer Genome Atlas revealed that TP53 was more frequently mutated in BCs from BRCA1 germline mutation carriers (0% vs 72%, P<0.001), while no differences with BRCA2-associated BCs were found. Conclusion: ATM-associated BCs frequently display bi-allelic ATM inactivation through LOH of the wild-type allele and a subset of these cases displayed high levels of LSTs. These findings suggest that at least in a subset of ATM-associated BCs, biallelic inactivation of ATM rather than a dominant negative effect of the germline mutation may be the mechanism of inactivation of this tumor suppressor gene. The repertoire of somatic genetic alterations of ATM-associated BCs is heterogeneous, with a noticeable lack of TP53 somatic mutations. Citation Format: Weigelt B, Bi R, Kumar R, James PA, Thorne H, Couch FJ, Eccles DM, Blows F, Geyer FC, Li A, Selenica P, Lim RS, Blecua P, Shen R, Wen H, Robson ME, Reis-Filho JS, Chenevix-Trench G. The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-15.

Abstract Background: Germline mutations in the BRCA1 and BRCA2 genes lead to hereditary breast cancers that are defective in homologous recombination (HR) repair and sensitive to DNA damaging agents. HR deficiency (HRD) also occurs in sporadic breast cancers, but its incidence and etiology are unclear. Genomic signatures of HRD recently were employed as biomarkers in clinical trials with modest success. We posited that sporadic breast cancers displaying functional HR deficiency would harbor genetic alterations affecting HR DNA repair genes. To test this hypothesis, we applied a functional assay to define lack of competent HR DNA repair and sequenced the exomes of consecutive sporadic breast cancers. Methods: We developed an assay to assess the ability of cancer cells to localize RAD51 into sub nuclear foci in response to ex-vivo irradiation (IR) in fresh sporadic breast cancer tissue specimens from 60 patients. RAD51 focus formation was compared between mock and IR conditions to determine relative fold induction. Twenty-nine tumors with sufficient DNA underwent whole-exome sequencing. Structural genomic signatures of HRD (i.e. large state transitions (LST), telomeric imbalance (NtAI), loss-of-heterozygositiy (LOH)) and a previously reported mutational signature related to BRCA1/2 hereditary breast cancers were assessed. HR deficient tumors were defined as those with both RAD51 foci defects and a genomic signature of HR deficiency (LST>15 or presence of a BRCA mutational signature). Somatic, germ-line, and copy number changes in HR genes were investigated. BRCA1 methylation status was determined. Results: Seventeen of 60 (28%) tumors displayed defective RAD51 recruitment following ex-vivo IR (RAD51-DEF). RAD51-DEF was seen in all breast cancer subtypes , including 7 of 33 (21%) ER+/HER2-, 4 of 14 (29%) HER2+, and 6 of 13 (48%) triple-negative cases. Of the 29 sequenced tumors, 13 (45%) were RAD51-DEF and 16 (55%) were competent for inducing RAD51 foci. LST was elevated in 10 tumors (LST >15) and associated with RAD51-DEF (p=0.02), whereas NtAI (p=0.10) and LOH (p=0.052) did not show a significant association with RAD51-DEF. The BRCA1/2 mutational signature was evident in 4 tumors, all were RAD51-DEF (p=0.03) and 2 were BRCA2 mutated. Nine of 29 (31%) sequenced tumors were determined to have HRD by the RAD51 assay and presence of a genomic scar. Eight of these 9 (88%) cases with HRD had a genetic alteration of both alleles of a bona fide HR gene due to a pathogenic mutation (somatic or germline) coupled with loss of heterozygosity or a homozygous deletion compared to 1 (5%) tumor without HRD (p<0.001). Both alleles of a gene were affected for BRCA2 (n=4), FAAP100 (n=2), CHEK2 (n=1), TP53BP1 (n=1) and BRCA1 (n=1). BRCA1 gene promoter methylation was found not to be significantly associated with HRD. Conclusion: Combined functional and genomic analyses of breast tumors demonstrated that genetic loss of an HR gene may underpin HRD in sporadic breast cancers. Our findings warrant further comprehensive genetic assessment (somatic, germline, and copy number) of HR genes as potential biomarker for HR-directed therapies. Citation Format: Powell SN, Riaz N, Mutter RW, Ng CKY, Delsite R, Piscuoglio S, King TA, Martelotto L, Sakr R, Brogi E, Edelweiss M, Lim R, Higginson D, Weigelt B, Lee W, Reis-Filho JS. A functional assay for homologous recombination (HR) DNA repair and whole exome sequencing reveal that HR-defective sporadic breast cancers are enriched for genetic alterations in DNA repair genes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-03.