Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these "liquid biopsies" are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease.

BACKGROUND:Breast cancer outcome, including response to therapy, risk of metastasis and survival, is difficult to predict using currently available methods, highlighting the urgent need for more informative biomarkers. Androgen receptor (AR) has been implicated in breast carcinogenesis however its potential to be an informative biomarker has yet to be fully explored. In this study, AR protein levels were determined in a cohort of 73 Grade III invasive breast ductal adenocarcinomas.METHODS:The levels of Androgen receptor protein in a cohort of breast tumour samples was determined by immunohistochemistry and the results were compared with clinical characteristics, including survival. The role of defects in the regulation of Androgen receptor gene expression were examined by mutation and methylation screening of the 5' end of the gene, reporter assays of the 5' and 3' end of the AR gene, and searching for miRNAs that may regulate AR gene expression.RESULTS:AR was expressed in 56% of tumours and expression was significantly inversely associated with 10-year survival (P = 0.004). An investigation into the mechanisms responsible for the loss of AR expression revealed that hypermethylation of the AR promoter is associated with loss of AR expression in breast cancer cells but not in primary breast tumours. In AR negative breast tumours, mutation screening identified the same mutation (T105A) in the 5'UTR of two AR negative breast cancer patients but not reported in the normal human population. Reporter assay analysis of this mutation however found no evidence for a negative impact on AR 5'UTR activity. The role of miR-124 in regulating AR expression was also investigated, however no evidence for this was found.CONCLUSION:This study highlights the potential for AR expression to be an informative biomarker for breast cancer survival and sets the scene for a more comprehensive investigation of the molecular basis of this phenomenon.

Aim: HER-2/neu amplification occurs in 15-25% of breast carcinomas. This oncogene, also referred to as c-erbB-2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER-2 over-expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti-oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)-based chemotherapy compared with anthracycline-based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER-2 positive patients.Methods: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method.Results: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER-2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/ PRp+, seven (7.7%) were ERp+/PR-, six (6.6%) were ERp-/PRp+ and 37 (40.7%) were ERp-/PR-. Of HER-2 positive tumours, 5.5% showed > 80% 3+ staining for ERp compared with 31.8% of 0-2+ HER-2 tumours; 24.2% of HER-2-positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER-2 status and tamoxifen.Conclusion: Oestrogen receptor status cannot be used to select tumours for evaluation of HER-2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER-2 status. There is a higher proportion of ERp negative tumours associated with HER-2 positivity, however, more than 20% of HER-2 positive tumours show moderate or strong staining for ERp. HER-2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.

9683 Background: More than 100 variables have been described to predict prognosis in breast cancer but only maximum tumour size (T), histological grade, nodal status, ER and HER-2 are routinely incorporated into clinical practice. In this study both classic histological features and novel biomarkers using immunohistochemistry (IHC) were evaluated to assess overall survival. Methods: Formalin fixed paraffin embedded tissue was collected from 414 breast cancer patients with invasive ductal carcinoma. 247 patients were node negative. Median follow-up was 110 months (Range 1–148 months). Routine histological parameters were assessed and a number of antigens including ER, PR, HER-2, p53, Ki67 and microvessel density were evaluated using IHC. ER, PR, HER-2 and p53 were assessed semiquantitatively using the Quickscore method. Microvessel density was counted over a 200x field and adjusted to mm2, and 500 tumour cells were counted for assessment of Ki67. Tumour size was estimated using T while volume was modeled as an ellipsoid. Results: With univariate analysis, grade (p <0.001), nodal status (p <0.001), tumour volume (p <0.01), HER-2 (p = 0.006), Ki67 (p <0.001), mitotic rate (p <0.001) and T (p <0.001) were statistically significant. For node negative patients grade (p = 0.02), tumour volume (p = 0.03) and mitotic rate (p = 0.009) were significant. Using multivariate analysis, mitotic rate (p = 0.0005) and tumour volume (p <0.001) predicted nodal involvement, and grade (p = 0.002), nodal status (p = 0.001) and T (p <0.001) predicted overall survival. Conclusions: This analysis validates the current staging system. Additional IHC markers failed to predict overall survival when standard histological features were utilised. Mitotic rate and tumour volume were independent predictors of nodal metastases. Tumour volume estimation may be superior to T in predicting nodal metastases and requires further evaluation to determine its potential role in staging. No significant financial relationships to disclose.