Epigenetic silencing of specific genes is associated with cancer progression. CpG islands are present at higher frequency in promoter regions, their methylation leading to gene underexpression. Pyrosequencing provides sequencing analysis of genetic markers, e.g., single nucleotide polymorphisms and DNA methylation. We investigated methylation levels of a spectrum of neoplastic breast lesions, ranging from hyperplasia to invasive carcinoma obtained from the same patient. Assays were designed to analyze promoter regions of RASSF1A, GSTP1, RARbeta, and E-cadherin. Methylation increased from normal to hyperplasia, the increase being significantly higher in invasive and in situ tumors for RASSF1A (p=0.00006 and p=0.009, respectively).