DNA methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response

Abstract

Background: Breast cancer is the most frequent cancer in women and consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics. In this study, we aimed to identify DNA methylation based biomarkers to distinguish patients with locally advanced breast cancer who may benefit from neoadjuvant doxorubicin treatment.

Results: We investigated quantitatively the methylation patterns in the promoter regions of 14 genes (ABCB1, ATM, BRCA1, CDH3, CDKN2A, CXCR4, ESR1, FBXW7, FOXC1, GSTP1, IGF2, HMLH1, PPP2R2B, and PTEN) in 75 well-described pre-treatment samples from locally advanced breast cancer and correlated the results to the available clinical and molecular parameters. Six normal breast tissues were used as controls and 163 unselected breast cancer cases were used to validate associations with histopathological and clinical parameters.Aberrant methylation was detected in 9 out of the 14 genes including the discovery of methylation at the FOXC1 promoter. Absence of methylation at the ABCB1 promoter correlated with progressive disease during doxorubicin treatment. Most importantly, the DNA methylation status at the promoters of GSTP1, FOXC1 and ABCB1 correlated with survival, whereby the combination of methylated genes improved the subdivision with respect to the survival of the patients. In multivariate analysis GSTP1 and FOXC1 methylation status proved to be independent prognostic markers associated with survival.

Conclusions: Quantitative DNA methylation profiling is a powerful tool to identify molecular changes associated with specific phenotypes. Methylation at the ABCB1 or GSTP1 promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while FOXC1 methylation might be a protective factor against tumour invasiveness. FOXC1 proved to be general prognostic factor, while ABCB1 and GSTP1 might be predictive factors for the response to and efficacy of doxorubicin treatment. Pharmacoepigenetic effects such as the reported associations in this study provide molecular explanations for differential responses to chemotherapy and it might prove valuable to take the methylation status of selected genes into account for patient management and treatment decisions.

Figure 1

Summary of the methylation data. A) Summary of the average DNA methylation values in percentage for the analysis of the fourteen genes (x-axis) in the 75 breast cancer samples, six normal breast tissues (on top) and the six breast cancer cell lines (bottom lines). Absence of methylation over an amplification product is shown in yellow, complete methylation in dark blue; intermediate methylation degrees by the corresponding mixtures of the two colours. B) Nonparametric correlation of methylation levels between genes and between regions within the same gene. The first row and the last column contain the gene name or gene name followed by a number that indicate different genomic regions within the same gene (Additional File 5). Green squares have been assigned to correlations that are non significant. Red square correlations are significant after FDR correction (threshold 10%). For each significant correlation 3 values are given from top to bottom: the correlation coefficient (R² value), the p-value and the number of tested samples.

Figure 2

Kaplan-Meyer plots of overall survival. Kaplan-Meyer plots of overall survival for patients with either methylated or unmethylated GSTP1, FOXC1 or ABCB1 promoter, respectively (left column). Increased differentiation of patients is obtained through the use of two gene methylation panels, having both genes methylated, either of the two genes methylated and the other one unmethylated or both genes unmethylated (right column). The p-value was calculated using a log rank test and are given uncorrected. 'N' is the number of samples in each group. After Bonferroni correction, DNA methylation of GSTP1 and ABCB1 as well as GSTP1/ABCB1 and GSTP1/FOXC1 did reach the level of statistical significance. Deaths due to causes not related to breast cancer were censored.