Identification of breast cancer associated altered DNA methylation in peripheral blood using MALDI-TOF mass spectrometry

Background

Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown.

Methods

Illumina 27K Methylation Array and Illumina 450K Methylation Array for the discovery of BC-related aberrant methylation sites in peripheral blood. The top hints were selected and validated using the MALDI-TOF Mass Spectrometry (MassARRAY, Agena Bioscience, Inc.) in two independent case-control studies with subjects from different centers. Gene expression levels were measured by real-time PCR and correlated with methylation. The clustering of samples by multiple CpG sites from a total of eight genes was realized by logistic regression. Receiver operating characteristic curve analyses was used to determine the discriminatory power.

Results

The methylation of an eight-gene-panel in peripheral blood cells was significantly correlated with BC, and showed outstanding discriminatory power for distinguishing BC cases from non-cancer controls (first validation round with 270 familial BC case and 251 non-cancer controls: AUC = 0.94, 95% C.I. 0.92-0.96; second validation round with 189 sporadic BC case and 189 non-cancer controls: AUC = 0.93, 95% C.I. 0.91-0.96). This panel also shows robust power for the breast cancer at early stage (in the second validation round 101 stage 0&I sporadic BC case vs. 189 non-cancer controls: AUC = 0.93, 95% C.I. 0.90-0.96). In addition, these blood-based DNA methylation signatures were similar among BC patients with differential clinical characteristics regardless of stage, receptor status and menopause status. The expression of four genes were also analysed in the leucocytes from 72 subjects and showed inversely correlation with the methylation levels (p 

Conclusions

This study reveals a strong association between decreased methylation of genes in peripheral blood and BC, and provides a promising blood-based marker panel for the detection of early BC.

Clinical trial identification

Legal entity responsible for the study

University Hospital of Heidelberg

Funding

This work was supported by the Dietmar-Hopp Foundation, University Hospital of Heidelberg, Helmholtz Society and the German Cancer Research Center (DKFZ). The familial BC samples were collected within a project funded by the Deutsche Krebshilfe (Grant number: 107054).

Disclosure

R. Yang, B. Burwinkel: Inventors of a provisional patent application relating to the subject matter of this manuscript and therefore declare a potential conflict of interests.