Lymph node metastasis is the most important prognostic indicator for breast cancer patients. Methylation may play an important role in the metastatic process. Our previous study revealed 5 tumor suppressor genes (HIN-1, RASSF1A, RIL, CDH13, RARβ2) were frequently methylated in primary breast cancers. We also defined two panels (HIN-1/ RASSF1A and RIL/ CDH13) of methylation profiles correlated either positively or negatively to ER/PR status. In order to investigate whether methylation of these genes correlate with lymph node metastasis, we collected 21 pairs of primary breast tumors and lymph node metastases from Fondazione IRCCS Istituto Nazionale dei Tumori. Paired samples were defined with comparable tumor cell percentage reviewed by experienced pathologists. Quantitative Pyrosequencing assays were used to study the methylation status of these 5 genes. Our results showed the hypermethylation rate of HIN-1, RASSF1A, RIL, CDH13 and RARβ2 in primary breast tumor tissues was 81%, 62%, 67%, 71% and 43%, respectively. Consistent with our previous studies, HIN-1 hypermethylation was positively correlated to ER expression (p<0.03). Due to the small sample size, no clear correlation was found in the other 3 genes (RASSF1A, RIL, CDH13) between methylation and ER status. Compared with primary tumors, the methylation levels of HIN-1, RASSF1A, RIL, CDH13 and RARβ2 in metastatic lymph nodes were elevated in 38%, 38%, 43%, 14%, and 19%, and were decreased in 38%, 38%, 38%, 24%, and 33%, respectively. We divided the 21 cases into three groups: A-C. Group A included 8 cases with elevated methylation in more than one gene. Most patients in this group showed no evidence of disease in follow-up aside from one death with bone metastasis. Group B included 9 cases with decreased methylation in more than one gene, among which 3 died with liver or lung metastasis. Group C included 4 cases with methylation changed (either elevated or decreased) in only one gene. This group showed no evidence of disease in follow-up. In conclusion, multiple tumor suppressor genes were frequently methylated in metastatic breast tumors. When compared to primary tumor, the methylation profile in metastastic lymph nodes varied. Patients with decreased methylation of multiple genes in lymph nodes had the less favorable outcome.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA