Methylation Analysis of P16, RASSF1A, RPRM, and RUNX3 in Circulating Cell-Free DNA for Detection of Gastric Cancer: A Validation Study

BACKGROUND: Most of Gastric Cancer (GC) patients are diagnosed at an advanced stage with poor prognosis. Hypermethylations of several tumor suppressor genes in cell-free DNA of GC patients have been previously reported. In this study, an attempt was made to investigate the methylation status of P16, RASSF1A, RPRM, and RUNX3 and their potentials for early diagnosis of GC. METHODS: Methylation status of the four tumor suppressor genes in 96 plasma samples from histopathologically confirmed gastric adenocarcinoma patients (Stage I–IV) and 88 healthy controls was determined using methylation-specific PCR method. Receiver operating characteristic curve analysis was performed and Area Under the Curve (AUC) was calculated. Two tailed p<0.05 were considered statistically significant. RESULTS: Methylated P16, RASSF1A, RPRM, and RUNX3 were significantly higher in the GC patients (41.7, 33.3, 66.7, and 58.3%) compared to the controls (15.9, 0.0, 6.8, and 4.5%), respectively (p<0.001). Stratification of patients showed that RPRM (AUC: 0.70, Sensitivity: 0.47, Specificity: 0.93, and p<0.001) and RUNX3 (AUC: 0.77, Sensitivity: 0.59, Specificity: 0.95, and p<0.001) had the highest performances in detection of early-stage (I+II) GC. The combined methylation of RPRM and RUNX3 in detection of early-stage GC had a higher AUC of 0.88 (SE=0.042; 95% CI:0.793–0.957; p<0.001), higher sensitivity of 0.82 and reduced specificity of 0.89. CONCLUSION: Methylation analysis of RPRM and RUNX3 in circulating cell free-DNA of plasma could be suggested as a potential biomarker for detection of GC in early-stages.