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

TY - JOUR

T1 - Detection and monitoring of hypermethylated RASSF1A in serum from patients with metastatic breast cancer

AU - Kristiansen, Søren

AU - Nielsen, Dorte Lisbet

AU - Søletormos, Gyorgy Tamas Pal

PY - 2016/4/1

Y1 - 2016/4/1

N2 - 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.

AB - 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.

KW - Breast Neoplasms

KW - DNA Methylation

KW - Early Detection of Cancer

KW - Female

KW - Humans

KW - Neoplasm Metastasis

KW - Polymerase Chain Reaction

KW - Promoter Regions, Genetic

KW - Sensitivity and Specificity

KW - Tumor Suppressor Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1186/s13148-016-0199-0

DO - 10.1186/s13148-016-0199-0

M3 - Journal article

C2 - 27042241

SN - 1868-7075

VL - 8

JO - Clinical Epigenetics

JF - Clinical Epigenetics

M1 - 35

ER -