Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

The circular RNome of primary breast cancer.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (R < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of CNOT2, CREBBP, and RERE in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of circCNOT2 was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, CNOT2 levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that circCNOT2 is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.

Signatures of mutational processes in human cancer.

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, 'kataegis', is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells.

Oestrogen receptor status, treatment and breast cancer prognosis in Icelandic BRCA2 mutation carriers.

BACKGROUND: The impact of an inherited BRCA2 mutation on the prognosis of women with breast cancer has not been well documented. We studied the effects of oestrogen receptor (ER) status, other prognostic factors and treatments on survival in a large cohort of BRCA2 mutation carriers. METHODS: We identified 285 breast cancer patients with a 999del5 BRCA2 mutation and matched them with 570 non-carrier patients. Clinical information was abstracted from patient charts and pathology records and supplemented by evaluation of tumour grade and ER status using archived tissue specimens. Univariate and multivariate hazard ratios (HR) were estimated for breast cancer-specific survival using Cox regression. The effects of various therapies were studied in patients treated from 1980 to 2012. RESULTS: Among mutation carriers, positive ER status was associated with higher risk of death than negative ER status (HR=1.94; 95% CI=1.22-3.07, P=0.005). The reverse association was seen for non-carriers (HR=0.71; 95% CI: 0.51-0.97; P=0.03). CONCLUSIONS: Among BRCA2 carriers, ER-positive status is an adverse prognostic factor. BRCA2 carrier status should be known at the time when treatment decisions are made.

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.

Tumour diploidy and survival in breast cancer patients with BRCA2 mutations.

It is not well known to what extent carrying a BRCA2 mutation affects the survival of women with breast cancer and prognostic factors among BRCA2-positive women warrant investigation. Using a record linkage approach we compared the long-term survival in carriers and noncarriers of an inherited BRCA2 founder mutation (999del5), and sought to identify prognostic factors among the BRCA2 mutation-positive subset, including markers of genetic instability (aneuploidy) and mitotic activity (S-phase fraction). We established the genetic status of 2,967 Icelandic breast cancer patients (215 mutation carriers and 2,752 noncarriers) diagnosed from 1955 to 2004, representing 72 % of all cases diagnosed in the country during this period. Tumour ploidy and S-phase fraction were assessed on tumour cells by DNA flow cytometry. Prognostic factors were assessed blindly with respect to mutation status. Univariate and multivariate hazard ratios (HR) were estimated for breast cancer-specific survival by BRCA2 status, using Cox regression. After a median follow-up of 9.5 years, BRCA2 mutation carriers had a higher risk of death from breast cancer than noncarriers (HR 1.64, 95 % CI 1.24-2.16, p < 0.001). The risk increase was restricted to women with diploid tumours (HR 3.03, 95 % CI 1.91-4.79, p < 0.001). Among breast cancer patients with aneuploid tumours, survival of carriers was similar to that of noncarriers (HR 0.76, 95 % CI 0.41-1.41, p = 0.38). Increased tumour size and a positive nodal status predicted worse prognosis in all patients, whereas the highly correlated prognostic factors diploidy, low proliferative activity and a positive estrogen receptor status had reverse effects in mutation carriers and noncarriers. Breast cancer patients who carry the Icelandic founder BRCA2 mutation have inferior long-term survival than noncarriers, but the adverse prognosis is restricted to mutation carriers with diploid, slowly proliferating tumours.

Obesity-associated changes in molecular biology of primary breast cancer.

Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients' body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Sensitivity of BRCA2 mutated human cell lines to Aurora kinase inhibition.

Aurora kinases play a vital part in successful mitosis and cell division. Aberrant Aurora-A and -B expression is commonly seen in various types of tumors. Small molecule Aurora inhibitors have already entered clinical trials. Aurora-A amplification has been shown to be associated with breast tumors from BRCA2-mutation carriers and such patients might therefore be candidates for treatment with Aurora kinase inhibitors. There is a need to identify markers that can predict sensitivity to Aurora inhibition. In this study sensitivity to the inhibitor ZM447439 was tested on a panel of 15 non-malignant and malignant epithelial cell lines that differed with respect to BRCA2 and p53 status and related to level of Aurora kinase expression. The IC(50) value for cell survival ranged from 1.9-8.1 µM and was not related to presence or absence of BRCA2 mutation. The levels of Aurora-A and -B expression correlated with each other but sensitivity towards ZM447439 did not correlate with levels of Aurora-A and -B mRNA expression, alone. Cells treated with the Aurora kinase inhibitor completed mitosis but cytokinesis was inhibited resulting in polyploidy and multinucleation. Different levels of polyploidy could not be fully explained by defects in p53. Only cell lines with a combination of high Aurora-A and -B expression, BRCA2 mutation and p53 defects showed more sensitivity towards Aurora inhibition than other cell lines. In conclusion, BRCA2-mutated cells showed variable sensitivity towards Aurora kinase inhibition. The level of sensitivity could not be predicted by Aurora expression levels alone but BRCA2 mutated tumors with high Aurora expression and non-functional p53 are likely candidates for treatment with Aurora inhibitors.

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines.

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

Interactive effect of genetic susceptibility with height, body mass index, and hormone replacement therapy on the risk of breast cancer.

BACKGROUND: Breast cancer today has many established risk factors, both genetic and environmental, but these risk factors by themselves explain only part of the total cancer incidence. We have investigated potential interactions between certain known genetic and phenotypic risk factors, specifically nine single nucleotide polymorphisms (SNPs) and height, body mass index (BMI) and hormone replacement therapy (HRT). METHODS: We analyzed samples from three different study populations: two prospectively followed Swedish cohorts and one Icelandic case-control study. Totally 2884 invasive breast cancer cases and 4508 controls were analysed in the study. Genotypes were determined using Mass spectrometry-Maldi-TOF and phenotypic variables were derived from measurements and/or questionnaires. Odds Ratios and 95% confidence intervals were calculated using unconditional logistic regression with the inclusion of an interaction term in the logistic regression model. RESULTS: One SNP (rs851987 in ESR1) tended to interact with height, with an increasingly protective effect of the major allele in taller women (p = 0.007) and rs13281615 (on 8q24) tended to confer risk only in non users of HRT (p-for interaction = 0.03). There were no significant interactions after correction for multiple testing. CONCLUSIONS: We conclude that much larger sample sets would be necessary to demonstrate interactions between low-risk genetic polymorphisms and the phenotypic variables height, BMI and HRT on the risk for breast cancer. However the present hypothesis-generating study has identified tendencies that would be of interest to evaluate for gene-environment interactions in independent materials.

Cytogenetic polyclonality of breast carcinomas: association with clinico-pathological characteristics and outcome.

Routinely used prognostic factors fail to predict clinical outcome in a significant proportion of breast cancer patients, implying that they can not detect some important biological characteristics. Chromosomal changes have been described in breast carcinomas for many years but their significance is not clear. We compared chromosomal changes with clinico-pathological characteristics and clinical outcome in 203 breast cancer patients with a follow-up of 9-18 years. Combining data from classical cytogenetics and flow cytometry revealed chromosomal abnormalities in 142 cases (70%). Of these, 51 (35.9%) contained two or more cytogenetically abnormal clones. Polyclonality was significantly associated with poor breast-cancer-specific survival (P = 0.03) within 5 years, independent of tumor size, lymph node metastases, and hormone receptors. Specific changes were similar to those previously described, but a new finding was a significant association between del 3p12p21 and poor survival. Polyclonality was significantly associated with TP53-mutations but not with a germline BRCA2 mutation. Less than one third of the polyclonal samples were identified by flow cytometry alone. Cytogenetic changes were detected in 17 out of 114 samples from non-tumorous tissue (15%), two of them identical with a clone in the corresponding tumor. Several samples contained clearly unrelated clones within the tumor and outside, implying either multifocal origin or early divergence. In conclusion, the common deletion on Chromosome 3p12p21 was associated with poor clinical outcome. Chromosomal polyclonality is common in breast carcinomas and predicts poor survival. Polyclonality was poorly detected by one-sample flow cytometry. Multiple sampling might improve the detection rate.

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

BACKGROUND: 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.

A candidate CpG SNP approach identifies a breast cancer associated ESR1-SNP.

Altered DNA methylation is often seen in malignant cells, potentially contributing to carcinogenesis by suppressing gene expression. We hypothesized that heritable methylation potential might be a risk factor for breast cancer and evaluated possible association with breast cancer for single nucleotide polymorphisms (SNPs) either involving CpG sequences in extended 5'-regulatory regions of candidate genes (ESR1, ESR2, PGR, and SHBG) or CpG and missense coding SNPs in genes involved in methylation (MBD1, MECP2, DNMT1, MGMT, MTHFR, MTR, MTRR, MTHFD1, MTHFD2, BHMT, DCTD, and SLC19A1). Genome-wide searches for genetic risk factors for breast cancers have in general not investigated these SNPs, because of low minor allele frequency or weak haplotype associations. Genotyping was performed using Mass spectrometry-Maldi-Tof in a screening panel of 538 cases and 1,067 controls. Potential association to breast cancer was identified for 15 SNPs and one of these SNPs (rs7766585 in ESR1) was found to associate strongly with breast cancer, OR 1.30 (95% CI 1.17-1.45; p-value 2.1 × 10(-6)), when tested in a verification panel consisting of 3,211 unique breast cancer cases and 4,223 unique controls from five European biobank cohorts. In conclusion, a candidate gene search strategy focusing on methylation-related SNPs did identify a SNP that associated with breast cancer at high significance.

Single nucleotide polymorphisms in the 20q13 amplicon genes in relation to breast cancer risk and clinical outcome.

The 20q13 region is frequently amplified/overexpressed in breast tumours. However, the nature of this amplification/overexpression is unknown. Here, we investigated genetic variation in five 20q13 amplicon genes (MYBL2, AURKA, ZNF217, STK4 and PTPN1) and its impact on breast cancer (BC) susceptibility and clinical outcome. As a novel finding, four polymorphisms in STK4 (rs6017452, rs7271519) and AURKA (rs2273535, rs8173) associated with steroid hormone receptor status both in a Swedish population-based cohort of 783 BC cases and in a Polish familial/early onset cohort of 506 BC cases. In the joint analysis, the minor allele carriers of rs6017452 had more often hormone receptor positive tumours (OR 0.57, 95% CI 0.40-0.81), while homozygotes for the minor allele of rs7271519, rs2273535 and rs8173 had more often hormone receptor negative tumours (2.26, 1.30-3.39; 2.39, 1.14-5.01; 2.39, 1.19-4.80, respectively) than homozygotes for the common allele. BC-specific survival analysis of AURKA suggested that the Swedish carriers of the minor allele of rs16979877, rs2273535 and rs8173 might have a worse survival compared with the major homozygotes. The survival probabilities associated with the AURKA genotypes depended on the tumour phenotype. In the Swedish case-control study, associations with BC susceptibility were observed in a dominant model for three MYBL2 promoter polymorphisms (rs619289, P = 0.02; rs826943, P = 0.03 and rs826944, P = 0.02), two AURKA promoter polymorphisms (rs6064389, P = 0.04 and rs16979877, P = 0.02) and one 3'UTR polymorphism in ZNF217 (rs1056948, P = 0.01). In conclusion, our data confirmed the impact of the previously identified susceptibility locus and provided preliminary evidence for novel susceptibility variants in BC. We provided evidence for the first time that genetic variants at 20q13 may affect hormone receptor status in breast tumours and influence tumour aggressiveness and survival of the patients. Future studies are needed to confirm the prognostic value of our findings in the clinic.

BRCA2 Haploinsufficiency in Telomere Maintenance.

Our previous studies showed an association between monoallelic BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.

Centriole movements in mammalian epithelial cells during cytokinesis.

BACKGROUND: In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and alpha-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules. RESULTS: Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent. CONCLUSIONS: These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.

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

BACKGROUND: 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.

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

INTRODUCTION: Germline 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. METHODS: The 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). RESULTS: Classification 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. CONCLUSIONS: The 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.