Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

Abstract

Background: Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression.

Methods: Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV.

Results: Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival.

Conclusions: In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.

Figure 1

Average percentage of DNA methylation levels for all CpGs between normal, DCIS and invasive tumor samples (stage I, II, III and IV).

Figure 2

Boxplots illustrating significant association between methylation status and tumor stage and grade. (a) Significantly lower methylation levels in DCIS and stage I samples compared to stage II, III and IV samples was observed. (b) Also grade 1 tumors had significantly lower methylation index compared with grade 2 and grade 3 tumors.

Figure 3

Boxplots illustrating significant association between methylation status and HER2 and TP53 status. (a)TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. (b) HER2 positive tumors had significantly higher methylation index compared to the HER2 negative tumors.

Figure 4

Kaplan-Meier plots of overall survival for patients with normal-like or hyper methylated PPP2R2B promoter. The p value was calculated using a long-rank test.