Abstract

BackgroundCirculating 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.ResultsThe 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.ConclusionsMethylation-sensitive restriction enzymes may be a useful methodological approach for monitoring circulating hypermethylated RASSF1A among patients with metastatic breast cancer.

Highlights

  • Circulating hypermethylated RASSF1A could be a novel and potential useful marker for monitoring patients with metastatic breast cancer

  • Methylation profiling of individual tumors further suggests that DNA methylation progressively spreads from the first exon into the promoter area of the RASSF1A gene [11]

  • Serum samples were obtained from 24 patients with metastatic breast cancer and from 24 age-matched healthy female as controls

Read more

Summary

Introduction

Circulating hypermethylated RASSF1A could be a novel and potential useful marker for monitoring patients with metastatic breast cancer. Among patients with metastatic breast cancer, serological tumor markers alone are not recommended for Kristiansen et al Clinical Epigenetics (2016) 8:35 target hypermethylated genes, control genes, or regions not susceptible to methylation by PCR amplification of converted DNA [4]. The sodium bisulfite conversion of the DNA may be incomplete [5] and cause breaks in the DNA strands [6] and critical loss of total sample DNA [7, 8] These critical issues may compromise the analytical detection of hypermethylated genes. Wide clonal intratumor heterogeneity of promoter hypermethylation of RASSF1A has been found in breast cancer [12] There are both intra- and interindividual differences in the methylation profiles of the RASSF1A gene among tumors from different patients. One primer and probe design may not cover all types of RASSF1A methylation profiles, and the patient-to-patient variation may decrease the clinical sensitivity

Methods
Results
Conclusion
Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.