DNA methylation profiling in the Carolina Breast Cancer Study. Homo sapiens

A microarray targeting promoters of cancer-related genes was used to evaluate DNA methylation at 935 CpG sites in 517 invasive breast tumors from the Carolina Breast Cancer Study (CBCS), a population-based study of invasive breast cancer. Concensus clustering using methylation (β) values for the 167 most variant CpG loci defined 4 clusters differing most distinctly in hormone receptor (HR) status, intrinsic subtype (luminal versus basal-like) and p53 mutation status. Supervised analyses for HR status, subtype, and p53 status identified differentially methylated CpG loci with considerable overlap (n=266). Concensus clustering also defined a hypermethylated luminal-enriched tumor cluster 3; gene ontology analysis of cluster 3 hypermethylated loci revealed enrichment for developmental genes, including homeobox domain genes (HOXB13, PAX6, IPF1, EYA4, DLK1, IHH, ISL1, TBX1, SOX1, SOX17). The hypermethylated luminal-enriched cluster 3 independently predicted poorer survival in multivariate Cox proportional hazard analysis, and this finding was confirmed in analysis of luminal A tumors. This study demonstrates epigenetic heterogeneity among breast tumors of a single intrinsic subtype, and shows that epigenetic patterns are strongly associated with HR status, subtype, and p53 mutation status. Among HR+ tumors, a gene signature characterized by hypermethylation of developmental genes may have prognostic value. Genes differentially methylated between clinically-important tumor subsets have roles in differentiation, development, and tumor growth and may be critical to inducing and maintaining tumor phenotypes and clinical outcomes. Overall design: 517 breast tumors, 9 normal breast tissues

本研究采用靶向癌症相关基因启动子的微阵列（microarray），对卡罗莱纳乳腺癌研究（Carolina Breast Cancer Study, CBCS）中的517例浸润性乳腺肿瘤的935个CpG位点的DNA甲基化水平进行检测。该研究是一项基于人群的浸润性乳腺癌研究。针对167个变异程度最高的CpG位点的甲基化（β）值进行共识聚类，得到4个聚类簇，其在激素受体（HR）状态、固有亚型（管腔型与基底样型）及p53突变状态上差异最为显著。针对HR状态、肿瘤亚型及p53突变状态的有监督分析共识别出266个存在显著重叠的差异甲基化CpG位点。共识聚类同时鉴定出一个以高甲基化为特征、富含管腔型肿瘤的聚类簇3；对聚类3的高甲基化位点进行基因本体（gene ontology, GO）分析显示，其显著富集于发育相关基因，包括同源框结构域基因（HOXB13、PAX6、IPF1、EYA4、DLK1、IHH、ISL1、TBX1、SOX1、SOX17）。该高甲基化管腔富集型聚类簇3在多因素Cox比例风险回归分析中可独立预测不良生存结局，这一结果在管腔A型肿瘤亚组分析中得到验证。本研究揭示了同一固有亚型乳腺肿瘤之间的表观遗传异质性，并表明表观遗传模式与HR状态、肿瘤亚型及p53突变状态密切相关。在HR阳性肿瘤中，以发育基因高甲基化为特征的基因特征可能具备预后价值。临床重要肿瘤亚组间存在差异甲基化的基因参与细胞分化、发育及肿瘤生长过程，可能在肿瘤表型与临床结局的诱导与维持中发挥关键作用。整体实验设计：纳入517例乳腺肿瘤与9例正常乳腺组织。