Olafur A. Stefansson

Genomic profiling of breast tumours in relation to BRCA abnormalities and phenotypes

IntroductionGermline mutations in the BRCA1 and BRCA2 genes account for a considerable fraction of familial predisposition to breast cancer. Somatic mutations in BRCA1 and BRCA2 have not been found and the involvement of these genes in sporadic tumour development therefore remains unclear.MethodsThe study group consisted of 67 primary breast tumours with and without BRCA1 or BRCA2 abnormalities. Genomic alterations were profiled by high-resolution (~7 kbp) comparative genome hybridisation (CGH) microarrays. Tumour phenotypes were analysed by immunohistochemistry on tissue microarrays using selected biomarkers (ER, PR, HER-2, EGFR, CK5/6, CK8, CK18).ResultsClassification of genomic profiles through cluster analysis revealed four subgroups, three of which displayed high genomic instability indices (GII). Two of these GII-high subgroups were enriched with either BRCA1- or BRCA2-related tumours whereas the third was not BRCA-related. The BRCA1-related subgroup mostly displayed non-luminal phenotypes, of which basal-like were most prominent, whereas the other two genomic instability subgroups BRCA2- and GII-high-III (non-BRCA), were almost entirely of luminal phenotype. Analysis of genome architecture patterns revealed similarities between the BRCA1- and BRCA2 subgroups, with long deletions being prominent. This contrasts with the third instability subgroup, not BRCA-related, where small gains were more prominent.ConclusionsThe results suggest that BRCA1- and BRCA2-related tumours develop largely through distinct genetic pathways in terms of the regions altered while also displaying distinct phenotypes. Importantly, we show that the development of a subset of sporadic tumours is similar to that of either familial BRCA1- or BRCA2 tumours. Despite their differences, we observed clear similarities between the BRCA1- and BRCA2-related subgroups reflected in the type of genomic alterations acquired with deletions of long DNA segments being prominent. This suggests similarities in the mechanisms promoting genomic instability for BRCA1- and BRCA2-associated tumours, possibly relating to deficiency in DNA repair through homologous recombination. Indeed, this feature characterized both familial and sporadic tumours displaying BRCA1- or BRCA2-like spectrums of genomic alterations. The importance of these findings lies in the potential benefit from targeted therapy, through the use of agents leading to DNA double-strand breaks such as PARP inhibitors (olaparib) and cisplatin, for a much larger group of patients than the few BRCA1 and BRCA2 germline mutation carriers.

DNA methylation profiling in breast cancer discordant identical twins identifies DOK7 as novel epigenetic biomarker

Using whole blood from 15 twin pairs discordant for breast cancer and high-resolution (450K) DNA methylation analysis, we identified 403 differentially methylated CpG sites including known and novel potential breast cancer genes. Confirming the results in an independent validation cohort of 21 twin pairs determined the docking protein DOK7 as a candidate for blood-based cancer diagnosis. DNA hypermethylation of the promoter region was also seen in primary breast cancer tissues and cancer cell lines. Hypermethylation of DOK7 occurs years before tumor diagnosis, suggesting a role as a powerful epigenetic blood-based biomarker as well as providing insights into breast cancer pathogenesis.

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, 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.

BRCA1 epigenetic inactivation predicts sensitivity to platinum-based chemotherapy in breast and ovarian cancer.

Germline mutations in the BRCA1 or BRCA2 genes are associated with an increased risk of breast and ovarian cancer development. Both genes are involved in DNA repair, and tumors harboring genetic defects in them are thought to be more sensitive to DNA-damaging agents used in chemotherapy. However, as only a minority of breast and ovarian cancer patients carry BRCA1 or BRCA2 mutations, few patients are likely to benefit from these pharmacogenetic biomarkers. Herein, we show that, in cancer cell lines and xenografted tumors, BRCA1 CpG island promoter hypermethylation-associated silencing also predicts enhanced sensitivity to platinum-derived drugs to the same extent as BRCA1 mutations. Most importantly, BRCA1 hypermethylation proves to be a predictor of longer time to relapse and improved overall survival in ovarian cancer patients undergoing chemotherapy with cisplatin.

A DNA methylation-based definition of biologically distinct breast cancer subtypes

In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation‐based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype‐specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal‐B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal‐like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation‐based subtypes, Epi‐LumB and Epi‐Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation‐based subtypes thereby providing valuable information on disease prognosis.

Linkage of DNA methylation quantitative trait loci to human cancer risk.

Epigenetic regulation and, in particular, DNA methylation have been linked to the underlying genetic sequence. DNA methylation quantitative trait loci (meQTL) have been identified through significant associations between the genetic and epigenetic codes in physiological and pathological contexts. We propose that interrogating the interplay between polymorphic alleles and DNA methylation is a powerful method for improving our interpretation of risk alleles identified in genome-wide association studies that otherwise lack mechanistic explanation. We integrated patient cancer risk genotype data and genome-scale DNA methylation profiles of 3,649 primary human tumors, representing 13 solid cancer types. We provide a comprehensive meQTL catalog containing DNA methylation associations for 21% of interrogated cancer risk polymorphisms. Differentially methylated loci harbor previously reported and as-yet-unidentified cancer genes. We suggest that such regulation at the DNA level can provide a considerable amount of new information about the biology of cancer-risk alleles.

Epigenetic Modifications in Breast Cancer and Their Role in Personalized Medicine

In cancer, the overall patterns of epigenetic marks are severely distorted from the corresponding normal cell type. It is now well established that these changes can contribute to cancer development through inactivation of tumor suppressor genes and, conversely, through activation of oncogenes. Recent technological advances have enabled epigenome-wide analyses of cancers that are yielding unexpected findings. The study of cancer epigenetics holds great promise for expanding the range of therapeutic opportunities for personalized medicine. Here, we focus on DNA methylation in breast cancer and the potential implications for clinical management of patients.

A Comprehensive DNA Methylation Profile of Epithelial-to-Mesenchymal Transition

Epithelial-to-mesenchymal transition (EMT) is a plastic process in which fully differentiated epithelial cells are converted into poorly differentiated, migratory and invasive mesenchymal cells, and it has been related to the metastasis potential of tumors. This is a reversible process and cells can also eventually undergo mesenchymal-to-epithelial transition. The existence of a dynamic EMT process suggests the involvement of epigenetic shifts in the phenotype. Herein, we obtained the DNA methylomes at single-base resolution of Madin-Darby canine kidney cells undergoing EMT and translated the identified differentially methylated regions to human breast cancer cells undergoing a gain of migratory and invasive capabilities associated with the EMT phenotype. We noticed dynamic and reversible changes of DNA methylation, both on promoter sequences and gene-bodies in association with transcription regulation of EMT-related genes. Most importantly, the identified DNA methylation markers of EMT were present in primary mammary tumors in association with the epithelial or the mesenchymal phenotype of the studied breast cancer samples.

EZH2-mediated epigenetic repression of DNA repair in promoting breast tumor initiating cells

Members of the Polycomb-group (PcG) family of proteins, including EZH2 (enhancer of zeste homolog 2), are involved in establishing epigenetic silencing of developmental genes in adult and embryonic stem cells, and their deregulation has been implicated in cancer. In a recent report, EZH2-mediated epigenetic repression of DNA damage repair in breast tumor initiating cells (BTICs) was identified as a mechanism that could promote expansion of BTICs, and may contribute to cancer progression.

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

BackgroundInherited 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.MethodsBreast 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.ResultsTumors 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.ConclusionsThe 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.