INTRODUCTION: Estrogen receptor (ER)-positive breast cancers are considered 
prognostically more favorable than ER-negative tumors, whereas human epidermal 
growth factor receptor (HER)2/neu-positive breast cancers are associated with 
worse prognosis. The objective of the present study was to determine whether 
ER-positive and ER-negative status relates to epigenetic changes in breast 
cancer-related genes. To evaluate epigenetic differences in tumor-related genes 
relating to ER and HER2/neu status of primary tumors, we examined the promoter 
methylation status of the promoter region CpG islands of eight major breast 
tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RARbeta2, and 
CDH1).
METHODS: Paired ER-positive (n = 65) and ER-negative (n = 65) primary breast 
tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted 
from paraffin-embedded tumor tissue after microdissection, and 
methylation-specific PCR and capillary-array electrophoresis analysis were 
performed.
RESULTS: In early stages of tumor progression (T1 and N0), RASSF1A and CCND2 
were significantly (P < 0.05) more methylated in ER-positive than in ER-negative 
tumors. GSTP1 hypermethylation was more frequent in the lymph node metastasis 
positive group than in the negative group. Double negative (ER-negative, 
HER2/neu-negative) breast cancers had significantly lesser frequencies of 
RASSF1A, GSTP1, and APC methylation (P < 0.0001, P < 0.0001, and P = 0.0035, 
respectively). Both ER and HER2/neu status correlated independently with these 
epigenetic alterations.
CONCLUSION: We demonstrated significant differences in tumor-related gene 
methylation patterns relevant to ER and HER2/neu status of breast tumors. This 
may be of significance in the assessment of targeted therapy resistance related 
to ER and HER2/neu status in breast cancer patients.