Genomic and phenotypic analysis of BRCA2 mutated breast cancers reveals co-occurring changes linked to progression

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.Inherited mutations in the BRCA2 gene greatly increase the risk of developing breast cancer. Consistent with an important role for BRCA2 in error-free DNA repair, complex genomic changes are frequently observed in tumors derived from BRCA2 mutation carriers. Here, we explore the impact of DNA copy-number changes in BRCA2 tumors with respect to phenotype and clinical staging of the disease. METHODS: Breast tumors (n = 33) derived from BRCA2 999del5 mutation carriers were examined in terms of copy-number changes with high-resolution aCGH (array comparative genomic hybridization) containing 385 thousand probes (about one for each 7 kbp) and expression of phenotypic markers on TMAs (tissue microarrays). The data were examined with respect to clinical parameters including TNM staging, histologic grade, S phase, and ploidy. RESULTS: Tumors from BRCA2 carriers of luminal and basal/triple-negative phenotypes (TNPs) differ with respect to patterns of DNA copy-number changes. The basal/TNP subtype was characterized by lack of pRb (RB1) coupled with high/intense expression of p16 (CDKN2A) gene products. We found increased proportions of Ki-67-positive cells to be significantly associated with loss of the wild-type (wt) BRCA2 allele in luminal types, whereas BRCA2wt loss was less frequent in BRCA2 tumors displaying basal/TNP phenotypes. Furthermore, we show that deletions at 13q13.1, involving the BRCA2wt allele, represents a part of a larger network of co-occurring genetic changes, including deletions at 6q22.32-q22.33, 11q14.2-q24.1, and gains at 17q24.1. Importantly, copy-number changes at these BRCA2-linked networking regions coincide with those associated with advanced progression, involving the capacity to metastasize to the nodes or more-distant sites at diagnosis. CONCLUSIONS: The results presented here demonstrate divergent paths of tumor evolution in BRCA2 carriers and that deletion of the wild-type BRCA2 allele, together with co-occurring changes at 6 q, 11 q, and 17 q, are important events in progression toward advanced disease.Eimskipafelag University Minningarsjodur Bergthoru Magnusdottur and Jakobs J Bjarnasonar Gongum Saman Icelandic Cancer Research Fund SKI Icelandic Centre for Research RANNIS The University of Icelan

BRCA1 Promoter Methylation Status in 1031 Primary Breast Cancers Predicts Favorable Outcomes Following Chemotherapy.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadBackground: Breast Cancer 1 gene (BRCA1) is known to be inactivated in breast tumors by promoter methylation. Tumor cells in patients carrying a germline mutation in BRCA1 are sensitive to cytotoxic drugs that cause DNA double strand breaks. However, very little is known on whether patients with BRCA1 promoter methylated tumors are similarly sensitive to cytotoxic drugs. In this study, we address this by making use of extensive follow-up data on patients treated with cyclophosphamide, methotrexate, and fluorouracil in Iceland between 1976 and 2007. Methods: We analyzed BRCA1 promoter methylation by pyrosequencing DNA from tumor samples from 1031 patients with primary breast cancer. Of those, 965 were sporadic cases, 61 were BRCA2, and five were BRCA1 germline mutation carriers. All cases were examined with respect to clinicopathological parameters and breast cancer-specific survival in patients treated with cytotoxic drugs. Information on chemotherapy treatment in noncarriers was available for 26 BRCA1 methylated tumors and 857 unmethylated tumors. Results: BRCA1 was promoter methylated in 29 sporadic tumors or in 3.0% of cases (29 of 965), whereas none of the tumors derived from BRCA germline mutation carriers were promoter methylated. Important to note, patients with BRCA1 promoter methylation receiving chemotherapeutic drug treatment show highly improved breast cancer-specific survival compared with unmethylated controls (hazard ratio = 0.10, 95% confidence interval = 0.01 to 0.75, two-sided P = .02). Conclusions: BRCA1 promoter methylation is predictive of improved disease outcome in patients receiving cyclophosphamide, methotrexate, and fluorouracil drug treatment. Our results support the use of markers indicative of "BRCAness" in sporadic breast cancers to identify patients that are likely to benefit from the use of DNA-damaging agents.Icelandic Research Fund Gongum sama

The Icelandic founder mutation BRCA2 999del5: analysis of expression

INTRODUCTION: A founder mutation in the BRCA2 gene (BRCA2 999del5) accounts for 7–8% of female breast cancers and for 40% of male breast cancers in Iceland. If expressed, the mutant gene would encode a protein consisting of the first 256 amino acids of the BRCA2 protein. The purpose of this study was to determine whether this mutant protein is produced in heterozygous individuals and, if so, what might be the functional consequences of mutant protein production. METHODS: The presence of BRCA2 999del5 transcripts in fibroblasts from heterozygous individuals was assayed by cDNA synthesis and sequencing. The potential protein-coding portion of BRCA2 999del5 was cloned into the pIND(SP1)/V5-His vector and expressed in COS7 cells. The presence of the mutant protein in cell lysates from heterozygous fibroblasts and from COS7 cells was tested by a number of methods including immunoprecipitation, affinity purification with nickel-coated agarose beads, Western blotting and ELISA, using antibodies to the N-terminal end of BRCA2, antiserum specific for the 16 nonrelevant amino acids at the carboxyl end and antibodies to fusion partners of recombinant proteins. RESULTS: The frequency of the BRCA2 999del5 transcript in heterozygous fibroblasts was about one-fifth of the wild-type transcript; however, no mutant protein could be detected. Overexpression of BRCA2 999del5 mRNA in COS7 cells failed to produce a mutant protein unless degradation by proteasomes was blocked. CONCLUSION: Our results show that the protein product of BRCA2 999del5 is extremely unstable. Therefore, an increase in breast cancer risk in BRCA2 999del5 carriers is due to haploinsufficiency at the BRCA2 locus

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, CK8, CK18, EGFR, MUC1, and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response

HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.

Approximately 1-5% of breast cancers are attributed to inherited mutations in BRCA1 or BRCA2 and are selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. In other cancer types, germline and/or somatic mutations in BRCA1 and/or BRCA2 (BRCA1/BRCA2) also confer selective sensitivity to PARP inhibitors. Thus, assays to detect BRCA1/BRCA2-deficient tumors have been sought. Recently, somatic substitution, insertion/deletion and rearrangement patterns, or 'mutational signatures', were associated with BRCA1/BRCA2 dysfunction. Herein we used a lasso logistic regression model to identify six distinguishing mutational signatures predictive of BRCA1/BRCA2 deficiency. A weighted model called HRDetect was developed to accurately detect BRCA1/BRCA2-deficient samples. HRDetect identifies BRCA1/BRCA2-deficient tumors with 98.7% sensitivity (area under the curve (AUC) = 0.98). Application of this model in a cohort of 560 individuals with breast cancer, of whom 22 were known to carry a germline BRCA1 or BRCA2 mutation, allowed us to identify an additional 22 tumors with somatic loss of BRCA1 or BRCA2 and 47 tumors with functional BRCA1/BRCA2 deficiency where no mutation was detected. We validated HRDetect on independent cohorts of breast, ovarian and pancreatic cancers and demonstrated its efficacy in alternative sequencing strategies. Integrating all of the classes of mutational signatures thus reveals a larger proportion of individuals with breast cancer harboring BRCA1/BRCA2 deficiency (up to 22%) than hitherto appreciated (∼1-5%) who could have selective therapeutic sensitivity to PARP inhibition

Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

MYC amplification and TERT expression in breast tumor progression

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldThe complex roles of genomic instability, MYC oncogene amplification, activation of telomerase, and p53 function still remain to be fully described in breast tumors. MYC stimulates the telomerase catalytic subunit, TERT, which interacts with p53. Oncogene MYC amplification analysis was performed on 27 paraffin-embedded breast tumor samples by fluorescence in situ hybridization, selected on the basis of chromosomal instability. TERT immunostaining was performed on a larger group of breast tumor sections. All tumor samples were analyzed for TP53 mutation, genomic index, S-phase fraction, and pathological stages. Amplification of MYC was detected in 16 of 27 tumors (59%) and found to be associated with TNM stages I and II (P = 0.018), genomic index > 1.5 (P = 0.033), and S-phase fraction > 5% (P = 0.020). No association was found between MYC amplification and TERT immunostaining or TP53 mutations. Analysis of TERT in 103 primary breast tumors showed > 50% nuclei immunostaining in 58% of cases. High TERT immunostaining associated with genomic index > 1.5 (P = 0.017), high S-phase fraction (P = 0.056), and TP53 mutations (P = 0.030). No association was found between TERT staining and TNM stages. This study supports early involvement of MYC amplification in breast tumor progression. Both MYC amplification and TERT expression appear to be associated with high genomic instability and proliferation. TERT association with TP53 mutations indicates that TERT activity is downregulated by functional p53 protein in breast tumors

West Australian Airways (?) de Havilland DH66 Hercules airliner G-AUJP (?) in hangar, ca. 1935 [picture]

Part of collection: E.A. Crome collection of photographs on aviation.; Title devised by cataloguer based on reference sources.; Also available in an electronic version via the internet at: http://nla.gov.au/nla.pic-vn3797545; E.A. Crome collection relating to aviation in Australia, 1914-2005; located at; National Library of Australia Manuscript collection MS 1925