Heather Thorne

Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers

Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell–enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

Whole–genome characterization of chemoresistant ovarian cancer

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

Analysis of cancer risk and BRCA1 and BRCA2 mutation prevalence in the kConFab familial breast cancer resource.

IntroductionThe Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is a multidisciplinary, collaborative framework for the investigation of familial breast cancer. Based in Australia, the primary aim of kConFab is to facilitate high-quality research by amassing a large and comprehensive resource of epidemiological and clinical data with biospecimens from individuals at high risk of breast and/or ovarian cancer, and from their close relatives.MethodsEpidemiological, family history and lifestyle data, as well as biospecimens, are collected from multiple-case breast cancer families ascertained through family cancer clinics in Australia and New Zealand. We used the Tyrer-Cuzick algorithms to assess the prospective risk of breast cancer in women in the kConFab cohort who were unaffected with breast cancer at the time of enrolment in the study.ResultsOf kConFabs first 822 families, 518 families had multiple cases of female breast cancer alone, 239 had cases of female breast and ovarian cancer, 37 had cases of female and male breast cancer, and 14 had both ovarian cancer as well as male and female breast cancer. Data are currently held for 11,422 people and germline DNAs for 7,389. Among the 812 families with at least one germline sample collected, the mean number of germline DNA samples collected per family is nine. Of the 747 families that have undergone some form of mutation screening, 229 (31%) carry a pathogenic or splice-site mutation in BRCA1 or BRCA2. Germline DNAs and data are stored from 773 proven carriers of BRCA1 or BRCA1 mutations. kConFabs fresh tissue bank includes 253 specimens of breast or ovarian tissue – both normal and malignant – including 126 from carriers of BRCA1 or BRCA2 mutations.ConclusionThese kConFab resources are available to researchers anywhere in the world, who may apply to kConFab for biospecimens and data for use in ethically approved, peer-reviewed projects. A high calculated risk from the Tyrer-Cuzick algorithms correlated closely with the subsequent occurrence of breast cancer in BRCA1 and BRCA2 mutation positive families, but this was less evident in families in which no pathogenic BRCA1 or BRCA2 mutation has been detected.

Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

Intragenic ATM Methylation in Peripheral Blood DNA as a Biomarker of Breast Cancer Risk

Few studies have evaluated the association between DNA methylation in white blood cells (WBC) and the risk of breast cancer. The evaluation of WBC DNA methylation as a biomarker of cancer risk is of particular importance as peripheral blood is often available in prospective cohorts and easier to obtain than tumor or normal tissues. Here, we used prediagnostic blood samples from three studies to analyze WBC DNA methylation of two ATM intragenic loci (ATMmvp2a and ATMmvp2b) and genome-wide DNA methylation in long interspersed nuclear element-1 (LINE1) repetitive elements. Samples were from a case-control study derived from a cohort of high-risk breast cancer families (KConFab) and nested case-control studies in two prospective cohorts: Breakthrough Generations Study (BGS) and European Prospective Investigation into Cancer and Nutrition (EPIC). Bisulfite pyrosequencing was used to quantify methylation from 640 incident cases of invasive breast cancer and 741 controls. Quintile analyses for ATMmvp2a showed an increased risk of breast cancer limited to women in the highest quintile [OR, 1.89; 95% confidence interval (CI), 1.36-2.64; P = 1.64 × 10(-4)]. We found no significant differences in estimates across studies or in analyses stratified by family history or menopausal status. However, a more consistent association was observed in younger than in older women and individually significant in KConFab and BGS, but not EPIC. We observed no differences in LINE1 or ATMmvp2b methylation between cases and controls. Together, our findings indicate that WBC DNA methylation levels at ATM could be a marker of breast cancer risk and further support the pursuit of epigenome-wide association studies of peripheral blood DNA methylation.

Loss of Heterozygosity at the BRCA2 Locus Detected by Multiplex Ligation-Dependent Probe Amplification is Common in Prostate Cancers from Men with a Germline BRCA2 Mutation

Purpose: Prostate cancer risk is increased for men carrying a pathogenic germline mutation in BRCA2, and perhaps BRCA1. Our primary aim was to test for loss of heterozygosity (LOH) at the locus of the mutation in prostate cancers from men who a carry pathogenic germline mutation in BRCA1 or BRCA2, and to assess clinical and pathologic features of these tumors. Experimental Design: From 1,243 kConFab families: (a) 215 families carried a pathogenic BRCA1 mutation, whereas 188 families carried a pathogenic BRCA2 mutation; (b) of the 158 men diagnosed with prostate cancer (from 137 families), 8 were confirmed to carry the family-specific BRCA1 mutation, whereas 20 were confirmed to carry the family-specific BRCA2 mutation; and (c) 10 cases were eliminated from analysis because no archival material was available. The final cohort comprised 4 and 14 men with a BRCA1 and BRCA2 mutation, respectively. We examined LOH at the BRCA1 and BRCA2 genes using multiplex ligation-dependent probe amplification of DNA from microdissected tumor. Results: LOH at BRCA2 was observed in 10 of 14 tumors from BRCA2 mutation carriers (71%), whereas no LOH at BRCA1 was observed in four tumors from BRCA1 mutation carriers (P = 0.02). Under the assumption that LOH occurs only because the cancer was caused by the germline mutation, carriers of BRCA2 mutations are at 3.5-fold (95% confidence interval, 1.8-12) increased risk of prostate cancer. A high Gleason was the only distinct clinical feature. Conclusions: These observations are consistent with the idea that BRCA2, but not BRCA1, is a tumor suppressor of prostate cancer.

Patient-derived Xenografts Reveal that Intraductal Carcinoma of the Prostate Is a Prominent Pathology in BRCA2 Mutation Carriers with Prostate Cancer and Correlates with Poor Prognosis

BACKGROUND Intraductal carcinoma of the prostate (IDC-P) is a distinct clinicopathologic entity associated with aggressive prostate cancer (PCa). PCa patients carrying a breast cancer 2, early onset (BRCA2) germline mutation exhibit highly aggressive tumours with poor prognosis. OBJECTIVE To investigate the presence and implications of IDC-P in men with a strong family history of PCa who either carry a BRCA2 pathogenic mutation or do not carry the mutation (BRCAX). DESIGN, SETTING, AND PARTICIPANTS Patient-derived xenografts (PDXs) were generated from three germline BRCA2 mutation carriers and one BRCAX patient. Specimens were examined for histologic evidence of IDC-P. Whole-genome copy number analysis (WG-CNA) was performed on IDC-P from a primary and a matched PDX specimen. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The incidence of IDC-P and association with overall survival for BRCA2 and BRCAX patients were determined using Kaplan-Meier analysis. RESULTS AND LIMITATIONS PDXs from BRCA2 tumours showed increased incidence of IDC-P compared with sporadic PCa (p=0.015). WG-CNA confirmed that the genetic profile of IDC-P from a matched (primary and PDX) BRCA2 tumour was similar. The incidence of IDC-P was significantly increased in BRCA2 carriers (42%, n=33, p=0.004) but not in BRCAX patients (25.8%, n=62, p=0.102) when both groups were compared with sporadic cases (9%, n=32). BRCA2 carriers and BRCAX patients with IDC-P had significantly worse overall and PCa-specific survival compared with BRCA2 carriers and BRCAX patients without IDC-P (hazard ratio [HR]: 16.9, p=0.0064 and HR: 3.57, p=0.0086, respectively). CONCLUSIONS PDXs revealed IDC-P in patients with germline BRCA2 mutations or BRCAX classification, identifying aggressive tumours with poor survival even when the stage and grade of cancer at diagnosis were similar. Further studies of the prognostic significance of IDC-P in sporadic PCa are warranted. PATIENT SUMMARY Intraductal carcinoma of the prostate is common in patients with familial prostate cancer and is associated with poor outcomes. This finding affects genetic counselling and identifies patients in whom earlier multimodality treatment may be required.

Germline BRCA2 mutations drive prostate cancers with distinct evolutionary trajectories

Germline mutations in the BRCA2 tumour suppressor are associated with both an increased lifetime risk of developing prostate cancer (PCa) and increased risk of aggressive disease. To understand this aggression, here we profile the genomes and methylomes of localized PCa from 14 carriers of deleterious germline BRCA2 mutations (BRCA2-mutant PCa). We show that BRCA2-mutant PCa harbour increased genomic instability and a mutational profile that more closely resembles metastastic than localized disease. BRCA2-mutant PCa shows genomic and epigenomic dysregulation of the MED12L/MED12 axis, which is frequently dysregulated in metastatic castration-resistant prostate cancer (mCRPC). This dysregulation is enriched in BRCA2-mutant PCa harbouring intraductal carcinoma (IDC). Microdissection and sequencing of IDC and juxtaposed adjacent non-IDC invasive carcinoma in 10 patients demonstrates a common ancestor to both histopathologies. Overall we show that localized castration-sensitive BRCA2-mutant tumours are uniquely aggressive, due to de novo aberration in genes usually associated with metastatic disease, justifying aggressive initial treatment.

Targeted massively parallel sequencing of a panel of putative breast cancer susceptibility genes in a large cohort of multiple-case breast and ovarian cancer families

Introduction Gene panel testing for breast cancer susceptibility has become relatively cheap and accessible. However, the breast cancer risks associated with mutations in many genes included in these panels are unknown. Methods We performed custom-designed targeted sequencing covering the coding exons of 17 known and putative breast cancer susceptibility genes in 660 non-BRCA1/2 women with familial breast cancer. Putative deleterious mutations were genotyped in relevant family members to assess co-segregation of each variant with disease. We used maximum likelihood models to estimate the breast cancer risks associated with mutations in each of the genes. Results We found 31 putative deleterious mutations in 7 known breast cancer susceptibility genes (TP53, PALB2, ATM, CHEK2, CDH1, PTEN and STK11) in 45 cases, and 22 potential deleterious mutations in 31 cases in 8 other genes (BARD1, BRIP1, MRE11, NBN, RAD50, RAD51C, RAD51D and CDK4). The relevant variants were then genotyped in 558 family members. Assuming a constant relative risk of breast cancer across age groups, only variants in CDH1, CHEK2, PALB2 and TP53 showed evidence of a significantly increased risk of breast cancer, with some supportive evidence that mutations in ATM confer moderate risk. Conclusions Panel testing for these breast cancer families provided additional relevant clinical information for <2% of families. We demonstrated that segregation analysis has some potential to help estimate the breast cancer risks associated with mutations in breast cancer susceptibility genes, but very large case–control sequencing studies and/or larger family-based studies will be needed to define the risks more accurately.

Clinical, pathological and genetic features of women at high familial risk of breast cancer undergoing prophylactic mastectomy

Prophylactic mastectomy (PM) is a risk‐management option for women at high familial risk of breast cancer (BC). This study describes the PM experience of women enrolled in a large observational cohort study involving families with a history of hereditary breast cancer. Within 357 multiple‐case BC families [119 (33%) BRCA1 or BRCA2 mutation positive], identified via family cancer clinics, 49 cases of PM [21 (43%) BRCA1 or BRCA2 mutation positive] were identified and their clinical, pathological and genetic features reviewed. Families with at least one incidence of PM displayed stronger breast/ovarian cancer histories than did families without PM. Median age at time of PM was 45 years (range 28–58). Ten cases (21%) were bilateral PMs in unaffected women and 39 cases were contralateral PMs in women with prior invasive BC (71%) or ductal carcinoma in situ (DCIS) (8%). Most (88%) underwent total mastectomy. Unnecessary axillary surgery occurred in eight subjects (16%). Malignant histology was found in three PM specimens (6%). Prior to genetic testing, PM was performed in two women who were subsequently shown not to carry the mutation specific to their family. Optimal utilization of genetic testing to guide surgical decision making, appropriate surgical technique and careful pathology examination of PM specimens, are important issues to consider prior to PM in women at high familial risk of BC.