Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma

Abstract

Background Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%–80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. Methods We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

Circulating tumor DNA as an emerging liquid biopsy biomarker for early diagnosis and therapeutic monitoring in hepatocellular carcinoma

“…MT1M and MT1G methylation were detected in HCC patients´ serum with a significantly higher frequency compared to patients with CHB or the healthy control group [50]. Occurring early in liver tumor progression with higher levels in HCC patients, INK4A promoter hyper-methylation also showed the potential as a biomarker [45].…”

Circulating Tumor DNA and Hepatocellular Carcinoma

“…67 Results for this single candidate have been mixed. 71,72 Up to 90% of HCC tissues have DNA extracts with hypermethylation of the RASSF1A promoter, 73 were reflected in high frequency of hypermethylation by methylation-specific PCR of cfDNA. [74][75][76][77] Other single methylation marker candidate studies have shown high detection of HCC.…”

Impact of circulating tumor DNA in hepatocellular and pancreatic carcinomas

“…High incidence of p16INK4a promoter hypermethylation in ctDNA with significant decrease in postoperative blood samples was shown to be a useful marker in the detection and monitoring of HCC (Wong et al 2003). Correspondingly, Huang et al demonstrated higher levels of methylated p16INK4a in circulating cfDNA of 66 HCC serum samples versus 43 benign chronic liver diseases (Huang et al 2014). Further common tumor suppressing and cell cycle regulation-related genes with promoter hypermethylation are the adenomatous polyposis coli (APC) on chromosome 5q21 and the Ras association domain family protein 1A (RASSF1A) genes on chromosome 3p21.3 with high frequency of promoter hypermethylation in tumor and blood samples of HCC patients (Hu et al 2010;Huang et al 2011;Yeo et al 2005).…”