Methylation signature of lymph node metastases in breast cancer patients

Abstract

Background: Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers.

Methods: The quantitative methylation analysis was performed using the SEQUENOM's EpiTYPER™ assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Results: The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis.

Conclusions: The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis.

Figure 1

Hematotoxylin and eosin staining (H&E) and immunohistochemical staining for estrogen receptor (ER), progesterone receptor (PR) and HER2/neu proteins (400X).(a) Ductal carcinoma. (b) Lobular carcinoma.

Figure 2

a) Double dendrogram presents the methylation profiles of the twelve candidate genes in tumor tissue, matched normal tissue and lymph nodes metastasis (Red clusters indicate 0% methylated, yellow clusters indicate 100% methylated, color gradient between red and yellow indicates methylation ranging from 0-100).b) Comparison between quantitative analysis (0%-100%) of methylation for the candidate genes in the studied cohort (* significant correlation; Mann-Whitney U Test).

Figure 3

Pathway analysis of the hypermethylated genes in lymph node metastasis including their biological processes and association to breast carcinogenesis and neoplasm metastasis.