Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis

Abstract

Background: Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma. DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development.

Results: We generate genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer, and validate methylation changes between normal and DCIS in an independent dataset of 15 normal and 40 DCIS samples. We also validate a prognostic signature on 583 breast cancer samples from The Cancer Genome Atlas. Our analysis reveals that DNA methylation profiles of DCIS are radically altered compared to normal breast tissue, involving more than 5,000 genes. Changes between DCIS and invasive breast carcinoma involve around 1,000 genes. In tumors, DNA methylation is associated with gene expression of almost 3,000 genes, including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs is associated with survival of breast cancer patients with invasive tumors, as well as with survival of patients with DCIS and mixed lesions of DCIS and invasive breast carcinoma.

Conclusions: This work demonstrates that changes in the epigenome occur early in the neoplastic progression, provides evidence for the possible utilization of DNA methylation-based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis.

Figure 1

Hierarchical clustering of the methylation level of the 500 most variable gene regions. Tissue types (green, healthy breast; blue, DCIS; purple, mixed DCIS-IBC; red, IBC) and PAM50 subtype (dark blue, luminal A; light blue, luminal B; pink, HER2-enriched; red, basal-like; green, normal-like) are indicated.

Figure 2

CpGs whose methylation level significantly correlated with gene expression (Bonferroni corrected P -value <0.05). (A) Significance level of correlation between methylation level and gene expression plotted against distance between the CpG and transcription start site (TSS). Red dots represent negative correlation and blue dots represent positive correlation. (B) Significance level and genome-wide distribution of correlation between methylation level and gene expression.

Figure 3

Significant correlation (Bonferroni corrected P -value <0.05) between gene expression and methylation level of gene regions. Bar plot showing the distribution of negative and positive correlations relative to the functional regions of genes. The distribution is notably different for the negative versus positive correlations.

Figure 4

Application of the DNA methylation-based prognostic signature for patients. (A) In the original data (n = 176); (B) in the TCGA validation (n = 583); (C) with either DCIS or mixed DCIS-IBC (n = 52). (D) Classification with the DNA methylation-based prognostic signature was complementary to classification by lymph node status.