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Background  Circulating hypermethylated  RASSF1A  could be a novel and potential useful marker for monitoring patients with metastatic breast cancer. Technical obstacles include fragmentation of the circulating DNA, fluctuations in the concentration, low concentrations of circulating tumor DNA, and different locations of methylation in the  RASSF1A  gene among patients. One common method for detection of hypermethylated genes is sodium bisulfite conversion of non-methylated cytosine to uracil, followed by detection with PCR. However, the method relies on full conversion of all non-methylated cytosines, cause strand breaks, and loss of DNA. Alternatively, methylation-sensitive restriction enzymes have been used to digest genomic DNA, as well as sodium bisulfite-treated DNA. By flanking different regions of the  RASSF1A  with different PCR primer pairs, we analyzed for methylated genomic regions resistant to cleavage by the methylation-sensitive restriction enzymes HpaII and BstUI. The goal was to find region(s) in  RASSF1A  with high sensitivity and specificity that could be used for monitoring.    Results  The serum was spiked with non-human control DNA. By tracing the spiking control, the isolation procedure of the rare circulating tumor DNA was initially optimized. By analysis of production of PCR amplicons from HpaII- or BstUI-treated DNA isolated from 24 patients with metastatic breast cancer, we located four regions resulting in sensitivities from 63 to 83 %. When examining samples from 24 control subjects, these four regions gave a specificity of 100 %. Among these four regions, the primer pair with the highest PCR efficacy was selected to monitor the  RASSF1A  concentration in 31 collected serum samples. The spiked DNA was then used to calculate the tumor  RASSF1A  concentrations independent of fluctuations in circulating non-tumor DNA. As a proof of principle, there was concordance in the kinetics of the  RASSF1A  and the serological cancer biomarkers CA 15-3, CEA, and TPA.    Conclusions  Methylation-sensitive restriction enzymes may be a useful methodological approach for monitoring circulating hypermethylated  RASSF1A  among patients with metastatic breast cancer.

Detection and monitoring of hypermethylated RASSF1A in serum from patients with metastatic breast cancer