Sir Peter MacCallum Department of Oncology - Research Publications
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ItemMethylation profiling of ductal carcinoma in situ and its relationship to histopathological features(BMC, 2014)INTRODUCTION: DNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material. METHODS: Promoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction. RESULTS: Methylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification. CONCLUSIONS: DNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.
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ItemBRCA2 carriers with male breast cancer show elevated tumour methylation(BIOMED CENTRAL LTD, 2017-09-11)BACKGROUND: Male breast cancer (MBC) represents a poorly characterised group of tumours, the management of which is largely based on practices established for female breast cancer. However, recent studies demonstrate biological and molecular differences likely to impact on tumour behaviour and therefore patient outcome. The aim of this study was to investigate methylation of a panel of commonly methylated breast cancer genes in familial MBCs. METHODS: 60 tumours from 3 BRCA1 and 25 BRCA2 male mutation carriers and 32 males from BRCAX families were assessed for promoter methylation by methylation-sensitive high resolution melting in a panel of 10 genes (RASSF1A, TWIST1, APC, WIF1, MAL, RARβ, CDH1, RUNX3, FOXC1 and GSTP1). An average methylation index (AMI) was calculated for each case comprising the average of the methylation of the 10 genes tested as an indicator of overall tumour promoter region methylation. Promoter hypermethylation and AMI were correlated with BRCA carrier mutation status and clinicopathological parameters including tumour stage, grade, histological subtype and disease specific survival. RESULTS: Tumours arising in BRCA2 mutation carriers showed significantly higher methylation of candidate genes, than those arising in non-BRCA2 familial MBCs (average AMI 23.6 vs 16.6, p = 0.01, 45% of genes hypermethylated vs 34%, p < 0.01). RARβ methylation and AMI-high status were significantly associated with tumour size (p = 0.01 and p = 0.02 respectively), RUNX3 methylation with invasive carcinoma of no special type (94% vs 69%, p = 0.046) and RASSF1A methylation with coexistence of high grade ductal carcinoma in situ (33% vs 6%, p = 0.02). Cluster analysis showed MBCs arising in BRCA2 mutation carriers were characterised by RASSF1A, WIF1, RARβ and GTSP1 methylation (p = 0.02) whereas methylation in BRCAX tumours showed no clear clustering to particular genes. TWIST1 methylation (p = 0.001) and AMI (p = 0.01) were prognostic for disease specific survival. CONCLUSIONS: Increased methylation defines a subset of familial MBC and with AMI may be a useful prognostic marker. Methylation might be predictive of response to novel therapeutics that are currently under investigation in other cancer types.