The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ∼40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.

Genome-wide mapping of oestrogen receptor-α binding sites in primary breast cancer tissues shows that oestrogen receptor binding is dynamically regulated and that the expression of genes near differentially bound regulatory regions is associated with clinical outcome. Most breast tumours express the oestrogen receptor-α (ER), and this transcription factor is seen as an important drug target for the treatment of breast cancer. Jason Carroll and colleagues have mapped ER binding sites in solid primary cancers for the first time, and find distinct ER-binding profiles that are mediated in part by the FoxA1 DNA-binding protein and are associated with better or worse clinical outcome. As well as providing a potentially useful method of prognosis, this work may help to answer one of the major questions in breast cancer biology: how some ER+ tumours become drug resistant. Oestrogen receptor-α (ER) is the defining and driving transcription factor in the majority of breast cancers and its target genes dictate cell growth and endocrine response, yet genomic understanding of ER function has been restricted to model systems1,2,3. Here we map genome-wide ER-binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in primary breast cancers from patients with different clinical outcomes and in distant ER-positive metastases. We find that drug-resistant cancers still recruit ER to the chromatin, but that ER binding is a dynamic process, with the acquisition of unique ER-binding regions in tumours from patients that are likely to relapse. The acquired ER regulatory regions associated with poor clinical outcome observed in primary tumours reveal gene signatures that predict clinical outcome in ER-positive disease exclusively. We find that the differential ER-binding programme observed in tumours from patients with poor outcome is not due to the selection of a rare subpopulation of cells, but is due to the FOXA1-mediated reprogramming of ER binding on a rapid timescale. The parallel redistribution of ER and FOXA1 binding events in drug-resistant cellular contexts is supported by histological co-expression of ER and FOXA1 in metastatic samples. By establishing transcription-factor mapping in primary tumour material, we show that there is plasticity in ER-binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes.

The genomic landscape of breast cancer is complex, and inter- and intra-tumour heterogeneity are important challenges in treating the disease. In this study, we sequence 173 genes in 2,433 primary breast tumours that have copy number aberration (CNA), gene expression and long-term clinical follow-up data. We identify 40 mutation-driver (Mut-driver) genes, and determine associations between mutations, driver CNA profiles, clinical-pathological parameters and survival. We assess the clonal states of Mut-driver mutations, and estimate levels of intra-tumour heterogeneity using mutant-allele fractions. Associations between PIK3CA mutations and reduced survival are identified in three subgroups of ER-positive cancer (defined by amplification of 17q23, 11q13–14 or 8q24). High levels of intra-tumour heterogeneity are in general associated with a worse outcome, but highly aggressive tumours with 11q13–14 amplification have low levels of intra-tumour heterogeneity. These results emphasize the importance of genome-based stratification of breast cancer, and have important implications for designing therapeutic strategies.

Breast carcinomas are often infiltrated by inflammatory cells, particularly macrophages and T lymphocytes, but the significance of these cells remains unclear. One possible role of these inflammatory cells is that they represent a cell-mediated immune response against the carcinoma. CD8⁺lymphocytes are a known crucial component of cell-mediated immunity. The purpose of this study was to explore the prognostic value of tumor-infiltrating CD8⁺cytotoxic lymphocytes in breast cancer. Tumor-infiltrating CD8⁺lymphocytes were assessed by immunohistochemical staining of tissue microarray cores from 1,334 unselected breast tumors from patients with long-term follow-up. The number of CD8⁺T cells was counted in tumor nests (intratumoral), in stroma adjacent to tumor cells, and in stroma distant to tumor cells, and their relationship with clinical outcome was determined. The total number of CD8⁺cells was positively correlated with tumor grade (r_s= 0.20; P < .001) and inversely correlated with patient's age at diagnosis, estrogen receptor–alpha (ER-α), and progesterone receptor (PgR) expression (Mann-Whitney U test, P < .001). The total patient cohort was randomly divided into two separate training and validation sets before performing univariate survival analysis. Total number and distant stromal CD8⁺lymphocytes were associated with better patient survival (P = .041 and P < .001, respectively) in the training set. In multivariate analysis, total CD8⁺T-cell count was an independent prognostic factor in both training and validation sets. These results suggest that tumor-infiltrating CD8⁺T lymphocytes have antitumor activity as judged by their favorable effect on patients' survival and could potentially be exploited in the treatment of breast cancer.

BACKGROUND.: Triple‐negative breast cancer (estrogen receptor‐negative, progesterone receptor‐negative, and HER2‐negative) is a high risk breast cancer that lacks the benefit of specific therapy that targets these proteins.METHODS.: In this study, the authors examined a large and well characterized series of invasive breast carcinoma (n = 1944) with a long‐term clinical follow‐up (median, 56 months) by using tissue microarray. The series were also stained with concurrent immunohistochemical prognostic panels (estrogen receptor, progesterone receptor, HER‐2, androgen receptor, epidermal growth factor receptor (EGFR), P‐cadherin, E‐cadherin, and basal (CK5/6, CK14), and p53), to characterize this specific subgroup of breast cancer and to identify prognostic markers that can identify tumors with more aggressive behavior.RESULTS.: Of informative cases, 16.3% were of the triple‐negative phenotype. The majority of these tumors were grade 3, ductal/no‐specific‐type carcinomas. There were positive associations with larger size, pushing margins, poorer Nottingham Prognostic Index, development of recurrence and distant metastasis, and poorer outcome. In addition, associations were found with loss of expression of androgen receptor and E‐cadherin, and positive expression of basal cytokeratins (basal phenotype), P‐cadherin, p53, and EGFR. In all tumors, tumor size, lymph node stage, and androgen receptor were the most useful prognostic markers. In the lymph node‐positive subgroup, both size and androgen receptor retained their prognostic significance. However, in the lymph node‐negative tumors, basal phenotype was the sole prognostic marker identified in this subgroup. Other parameters including age, histological grade, tumor size, vascular invasion or other biomarkers included in the current study were not significant.CONCLUSIONS.: The authors concluded that assessment of androgen receptor and basal phenotype, in addition to the established pathologic variables, mainly lymph node status and tumor size, can be used to select high‐risk and low‐risk patients at the time of primary surgery and can provide valuable information on treatment options in these triple‐negative tumors. Cancer 2007. © 2006 American Cancer Society

Background: MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression. Results: Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed. Conclusion: This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.