DNA methylation profiling in the Carolina Breast Cancer Study defines cancer subclasses differing in clinicopathologic characteristics and survival

Kathleen Conway, Sharon N Edmiston, Ryan May, Pei Fen Kuan, Haitao Chu, Christopher Bryant, Chiu-Kit Tse, Theresa Swift-Scanlan, Joseph Geradts, Melissa A Troester, Robert C Millikan

IntroductionBreast cancer is a heterogeneous disease, with several intrinsic subtypes differing by hormone receptor (HR) status, molecular profiles and prognosis. However, the role of DNA methylation in breast cancer development and progression, and its relationship with the intrinsic tumor subtypes is not fully understood.MethodsA microarray targeting promoters of cancer-related genes was used to evaluate DNA methylation at 935 CpG sites in 517 breast tumors from the Carolina Breast Cancer Study (CBCS), a population-based study of invasive breast cancer.ResultsConsensus clustering using methylation (ß) 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 266 differentially methylated CpG loci with considerable overlap. Genes relatively hypermethylated in HR+, luminal A, or p53 wildtype breast cancers included FABP3, FGF2, FZD9, GAS7, HDAC9, HOXA11, MME, PAX6, POMC, PTGS2, RASSF1, RBP1, and SCGB3A1, while those more highly methylated in HR-, basal-like or p53 mutant tumors included BCR, C4B, DAB2IP, MEST, RARA, SEPT5, TFF1, THY1 and SERPINA5. Clustering also defined a hypermethylated luminal-enriched tumor cluster 3 that gene ontology analysis revealed to be enriched for homeobox and other developmental genes (ASCL2, DLK1, EYA4, GAS7, HOXA5, HOXA9, HOXB13, IHH, IPF1, ISL1, PAX6, TBX1, SOX1, SOX17). Although basal-enriched cluster 2 showed worse short-term survival, the luminal-enriched cluster 3 showed worse long-term survival, but was not independently prognostic in multivariate Cox proportional hazard analysis likely due to the mostly early stage cases in this dataset.ConclusionsThis study demonstrates that epigenetic patterns are strongly associated with HR status, subtype, and p53 mutation status, and may show heterogeneity within tumor subclass. Among HR+¿breast tumors, a subset exhibiting a gene signature characterized by hypermethylation of developmental genes and poorer clinicopathologic features may have prognostic value, and requires further study. Genes differentially methylated between clinically-important tumor subsets have roles in differentiation, development, and tumor growth and may be critical to establishing and maintaining tumor phenotypes and clinical outcomes.