Circulating tumour cell combined with DNA methylation for early detection of hepatocellular carcinoma

Abstract

Background: The inadequate early detection strategies makes hepatocellular carcinoma (HCC) patients with poor prognisis. Therefore, more effective detection methods are urgently needed for early detection and early intervention of HCC.Methods: 17 cases of suspected HCC patients and 11 cases of HBV-related decompensated cirrhosis (HBV-DeCi) patients were enrolled. For each patient, 5 ml blood sample was separated into circulating tumor cells (CTCs) and plasma, CTCs were stained with Diff staining for counting. Plasma was used for extracting cell free DNA (cfDNA) and then analyzed by qMSP assay. Ct values were recorded for GNB4 and Riplet as target genes and β-actin as an endogenous reference gene. Finally, clinical efficacy of CTC count combined with GNB4/Riplet methylation detection for early diagnosis of HCC was analyzed.Results: The CTC of HCC patients has pleomorphic characteristics, but it is difficult to distinguish from other blood cells with non-obviously pleomorphic of CTC. Although a small number of CTCs can also be detected in HBV-DeCi patients (control group), the number is significantly lower than that in HCC patients, the sensitivity and specificity of CTC for HCC detection were 70.6% and 90.9% (AUC = 0.81). The Ct values of GNB4 and Riplet methylation were significantly different between HCC patients and control group patients. When CTC combined with two genes, the AUC value was significantly increased to 0.98, the sensitivity was 88.2%, and the specificity was 100%.Conclusion: Our study has developed a novel test that CTC count combined with GNB4/Riplet methylation detection and showed its high performance for early diagnosis of HCC.

Discovery of novel DNA methylation biomarker panels for the diagnosis and differentiation between common adenocarcinomas and their liver metastases

“…Some of the selected regions have already been identified as potential diagnostic methylation biomarkers that differentiate between primary tumors, adjacent normal tissue samples and other tumor types 37 , 62 , 66 , 69 , 70 , 72 – 77 . In addition, the aberrant methylation of annotated genes has been identified as a diagnostic methylation biomarker in liquid biopsies 78 , 79 .…”

Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma

“…The GNB4 and Riplet test was validated in another study. They reported that the sensitivity of the methylated GNB4 and Riplet assay kit for HCC detection provided by Wuhan Ammunition Life-tech Company, Ltd. was 88.9%, with a specificity of 100.0% based on Ct values method on a cohort of 28 samples [ 42 ]. These findings are consistent with our results.…”

“…These findings are consistent with our results. Methylated GNB4 and Riplet have also been reported to improve the diagnostic sensitivity and specificity of circulating tumour cell (CTC) counts for HCC, increasing them from 70.6% and 90.9% to 88.2% and 100%, respectively [ 42 ].…”

Liquid Biopsy in Hepatocellular Carcinoma: The Significance of Circulating Tumor Cells in Diagnosis, Prognosis, and Treatment Monitoring

“…For example, combining total CTCs and AFP was shown to have even higher sensitivity in diagnosing HCC [ 75 ]. Liang et al found that CTC counts, together with guanine nucleotide-binding protein subunit beta-4 (GNB4) and Riplet gene methylation, can improve early diagnosis with a sensitivity of 88.2% and specificity of 100% [ 79 ]. El-Mezayen et al utilized flow cytometry to identify CTCs (CK18 and CK19) and developed a novel score based on five biochemical blood markers (CK18, CK19, AFP, Albumin, Platelets) to predict HCC among HCV-high-risk patients [ 80 ].…”