Background Loss-of-function variants in RAD51C are associated with familial ovarian cancer, but its role in hereditary breast cancer remains unclear. The aim of this study was to couple breast tumor sequencing with case-control data to clarify the contribution of RAD51C to hereditary breast cancer. Methods RAD51C was sequenced in 3080 breast cancer index cases that were negative in BRCA1/2 clinical tests and 4840 population-matched cancer-free controls. Pedigree and pathology data were analyzed. Nine breast cancers and one ovarian cancer from RAD51C variant carriers were sequenced to identify biallelic inactivation of RAD51C, copy number variation, mutational signatures, and the spectrum of somatic mutations in breast cancer driver genes. The promoter of RAD51C was analyzed for DNA methylation. Results A statistically significant excess of loss-of-function variants was identified in 3080 cases (0.4%) compared with 2 among 4840 controls (0.04%; odds ratio = 8.67, 95% confidence interval = 1.89 to 80.52, P< .001), with more than half of the carriers having no personal or family history of ovarian cancer. In addition, the association was highly statistically significant among cases with estrogen-negative (P <. 001) or triple-negative cancer (P < .001), but not in estrogen-positive cases. Tumor sequencing from carriers confirmed bi-allelic inactivation in all the triple-negative cases and was associated with high homologous recombination deficiency scores and mutational signature 3 indicating homologous recombination repair deficiency. Conclusions This study provides evidence that germline loss-of-function variants of RAD51C are associated with hereditary breast cancer, particularly triple-negative type. RAD51C-null breast cancers possess similar genomic and clinical features to BRCA1-null cancers and may also be vulnerable to DNA double-strand break inducing chemotherapies and poly ADP-ribose polymerase inhibitors.

Purpose: Advanced stage MOC have poor chemotherapy response and prognosis and lack biomarkers to aid Stage I adjuvant treatment. Differentiating primary mucinous ovarian carcinoma (MOC) from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathological and gene expression data were analysed to identify prognostic and diagnostic features. Experimental Design: Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n=333), mucinous borderline ovarian tumors (MBOT, n=151), upper GI (n=65), and lower GI tumors (n=55). Results: Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2-years from diagnosis, compared with expansile pattern in Stage I MOC (hazard ratio HR 2.77 (1.04-7.41, p=0.042). Increased expression of THBS2 and TAGLN were associated with shorter OS in MOC patients, (HR 1.25 (95% CI 1.04-1.51, p=0.016)) and (1.21 (1.01-1.45, p=0.043)) respectively. ERBB2 (HER2)-amplification or high mRNA expression was evident in 64/243 (26%) of MOCs, but only 8/243 (3%) were also infiltrative (4/39, 10%) or Stage III/IV (4/31, 13%). Conclusions: An infiltrative growth pattern infers poor prognosis within 2-years from diagnosis and may help select Stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confer an adverse prognosis and is upregulated in the infiltrative subtype which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

Breast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon–intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10−9) and missense (OR 1.27, p = 3.96 × 10−73) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2–4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

Background Breast cancers (BC) that arise in individuals heterozygous for a germline pathogenic variant in a susceptibility gene, such as BRCA1/2, PALB2 and RAD51C, have been shown to exhibit bi-allelic loss in the respective genes, and be associated with triple-negative (TN) BC and distinctive somatic mutational signatures. Tumour sequencing thus presents an orthogonal approach to assess the role of candidate genes in BC development. Methods Exome sequencing was performed on paired normal-breast tumour DNA from 124 carriers of germline loss-of-function (LoF) or missense (MS) mutations carriers in 15 known and candidate BC predisposition genes identified in the BEACCON case-control study. Bi-allelic inactivation and association with tumour genome features including mutational signatures and homologous recombination deficiency (HRD) score were investigated. Results BARD1-carrying TN BC (4/5) displayed bi-allelic loss and associated high HRD scores and mutational signature 3, as did a RAD51D-carrying TN BC and ovarian cancer. Bi-allelic loss was less frequent in BRIP1 BCs (4/13) and had low HRD scores. In contrast to other established BC genes, BCs from carriers of CHEK2 LoF (6/17) or MS (2/20) carriers had low rates of bi-allelic loss. Exploratory analysis of BC from carriers of LoF mutations in candidate genes such as BLM, FANCM, PARP2 and RAD50 found little evidence of bi-allelic inactivation. Conclusions BARD1 and RAD51D behave as classic BRCA-like genes with bi-allelic inactivation but this was not observed for any of the candidate genes. However, as demonstrated for CHEK2, the absence of bi-allelic inactivation does not provide definitive evidence against the gene’s involvement in BC predisposition.

For genes with reliable estimates of disease risk associated with loss-of-function variants, case-control data can be used to estimate the proportion of variants of typical risk effect for defined groups of variants, of relevance for variant classification. A calculation was derived for a maximum likelihood estimate of the proportion of pathogenic variants of typical effect from case-control data and applied to rare variant counts for ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, and RAD51D from published breast cancer studies: BEACCON (5770 familial cases and 5741 controls) and breast cancer risk after diagnostic sequencing (60,466 familial and population-based cases and 53,461

Mammographic density (MD) influences breast cancer risk, but how this is mediated is unknown. Molecular differences between breast cancers arising in the context of the lowest and highest quintiles of mammographic density may identify the mechanism through which MD drives breast cancer development. Women diagnosed with invasive or in situ breast cancer where MD measurement was also available (n = 842) were identified from the Lifepool cohort of >54,000 women participating in populationbased mammographic screening. This group included 142 carcinomas in the lowest quintile of MD and 119 carcinomas in the highest quintile. Clinico-pathological and family history information were recorded. Tumor DNA was collected where available (n = 56) and sequenced for breast cancer predisposition and driver gene mutations, including copy number alterations. Compared to carcinomas from low-MD breasts, those from high-MD breasts were significantly associated with a younger age at diagnosis and features associated with poor prognosis. Low-and high-MD carcinomas matched for grade, histological subtype, and hormone receptor status were compared for somatic genetic features. Low-MD carcinomas had a significantly increased frequency of TP53 mutations, higher homologous recombination deficiency, higher fraction of the genome altered, and more copy number gains on chromosome 1q and losses on 17p. While high-MD carcinomas showed enrichment of tumor-infiltrating lymphocytes in the stroma. The data demonstrate that when tumors were matched for confounding clinico-pathological features, a proportion in the lowest quintile of MD appear biologically distinct, reflective of microenvironment differences between the lowest and highest quintiles of MD.

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<p>Supplementary Methods</p>