Gene Promoter Hypermethylation in Tumors and Matched Affected Lymph Nodes of Breast Cancer Patients.

Abstract Gene promoter hypermethylation plays a pivotal role in breast carcinogenesis and breast cancer progression. In this study, we investigated 6 gene promoters (RASSF1A, HIN-1, RARβ2, TWIST, CCND2 and APC) for aberrant DNA methylation by real-time methylation-specific PCR in primary tumors and matched affected lymph nodes from 30 patients with breast cancer. These data were used to explore associations between gene promoter hypermethylation and clinicopathological features, including tumor cell proliferation, angiogenesis and hypoxia marker expression.Overall, gene promoter methylation levels (ratios of DNA copy numbers for methylated and reference genes) were higher in primary tumors than in matched lymph nodes. Notably, we found significant correlations between methylation levels in primary tumors and matched lymph nodes for 5 of 6 genes (P<0.05). An identical gene promoter methylation status (high or low according to the median methylation level) in primary tumors and matched lymph nodes was observed in 67% to 83% of cases. No significant associations between risk factors and gene promoter hypermethylation of the 6 genes was observed, except for the degree of spreading into nearby lymph nodes, which associated with high RASSF1A methylation levels in tumors (P=0.020) and high CCDN2 methylation levels in lymph nodes, and for HER2 expression, which was most frequently observed in tumors with low HIN-1 methylation levels (P=0.039). HIN-1 methylation levels in tumors negatively correlated with the fractions of proliferating tumor and endothelial cells determined by CD34/Ki-67 immunohistochemistry in both primary tumors (r=-0.594, P=0.001 and r=-0.619, P<0.001) and in lymph nodes (r=-0.412, P=0.024 and r=-0.400, P=0.029). Furthermore, high HIN-1 methylation levels in primary tumors tended to associate with breast tumors lacking expression of the hypoxia marker carbonic anhydrase IX in tumor cells (P=0.063) and associated stroma (P=0.086). CCND2 methylation levels in affected lymph nodes positively correlated with the ratios of the fractions of proliferating tumor cells to the fractions of proliferating endothelial cells observed in both primary tumors (r=0.418, P=0.002) and in lymph nodes (r=0.563, P=0.002).In summary, gene promoter hypermethylation in primary breast tumors and matched lymph nodes are highly correlated. Further studies with larger numbers of samples are necessary to investigate the prognostic or predictive potential of these observations. For two genes, HIN-1 and CCDN2, we observed correlations between gene promoter methylation levels and tumor cell proliferation. HIN-1 and CCDN2 are frequently lost in breast cancer and are both involved in cell cycle regulation. The biological significance of these findings needs to be further explored. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1149.