Analytical validation of MethyLiverDx-Sigma, a plasma cfDNA methylation assay for hepatocellular carcinoma detection
- 1R&D Center, LepiDyne Inc., Seoul, Republic of Korea
- 2R&D Center, LepiDyne Inc., Seoul, Republic of Korea; Interdisciplinary Program of Integrated OMICS for Biomedical Science, Yonsei University, Seoul, Republic of Korea
Abstract
Hepatocellular carcinoma surveillance relies on ultrasonography and alpha-fetoprotein, both limited in sensitivity for early-stage disease; aberrant methylation of circulating cell-free DNA (cfDNA) offers a promising blood-based approach. Reported here is the analytical validation of MethyLiverDx-Sigma (MLDx-s), an assay that detects methylated RNF135 and DNMT3A in bisulfite-converted plasma cfDNA by methylation-specific PCR with high-resolution melting (HRM), with GAPDH as an endogenous control. Performance was evaluated following CLSI EP05-A3, EP07, EP12-A2, and EP37. Precision of quantification cycle (C q ) and melting peak temperature (T m ) was assessed by variance-component analysis within one site and across two sites and two operators. Because precision studies used bulk bisulfite-converted reference DNA, the reported coefficients of variation (CVs) primarily characterize the PCR-HRM detection module; end-to-end variability in clinical plasma specimens may be higher. Interference and cross-reactivity were evaluated in low-positive and contrived-negative samples against reproducibility-derived limits, with dose-response testing of reactive substances. Detection was 100% at both levels, and every run met control-validity criteria. C q repeatability CVs ranged from 0.53% to 1.68%, and total reproducibility CVs ranged from 1.10% to 2.37%; all T m CVs were ≤0.20%. All 18 interferents and 16 microbial cross-reactants evaluated in low-positive samples met the predefined acceptance criteria. In negative samples, hemoglobin, SARS-CoV-2, and measles virus produced concentration-dependent non-specific amplification, and dose-response testing established their respective no-effect concentrations. These findings characterize the analytical precision and multisite reproducibility of the PCR-HRM detection module and the analytical specificity of MLDx-s, complementing its previously reported clinical performance for plasma-based HCC detection.
Related
- Analytical and clinical validation of the EdenDx methylation assay as a noninvasive supportive triage tool for early detection of endometrial cancer in cervical scraping specimens
- Prognostic Significance of Response Dynamics in Atezolizumab plus Bevacizumab-Treated Hepatocellular Carcinoma: A Multicenter Real-World Validation Study.
- Development and internal validation of a perioperative nomogram for predicting moderate-to-severe acute postoperative pain after hepatectomy for hepatocellular carcinoma.
- Validation of Modified Albumin-Bilirubin-Tumor Stage as a Prognostic Model in Early-Stage Hepatocellular Carcinoma Treated With Radiofrequency Ablation.
- Investigating the Expression and Function of PPP2R1A in Hepatocellular Carcinoma: A Multi-Omics Analysis with in vitro Validation.
BibTeX
@article{kim2026analytical,
title = {{Analytical validation of MethyLiverDx-Sigma, a plasma cfDNA methylation assay for hepatocellular carcinoma detection}},
author = {Si-Cho Kim and Hyunwoo Choi and Jiin Chung and Seung-Taek Lee and Young-Joon Kim},
journal = {Molecular and Cellular Probes},
year = {2026},
month = oct,
doi = {10.1016/j.mcp.2026.102090},
url = {https://doi.org/10.1016/j.mcp.2026.102090},
note = {CC BY},
}
RIS
TY - JOUR
TI - Analytical validation of MethyLiverDx-Sigma, a plasma cfDNA methylation assay for hepatocellular carcinoma detection
AU - Si-Cho Kim
AU - Hyunwoo Choi
AU - Jiin Chung
AU - Seung-Taek Lee
AU - Young-Joon Kim
PY - 2026
DA - 2026/10/30
JO - Molecular and Cellular Probes
DO - 10.1016/j.mcp.2026.102090
UR - https://doi.org/10.1016/j.mcp.2026.102090
AB - Hepatocellular carcinoma surveillance relies on ultrasonography and alpha-fetoprotein, both limited in sensitivity for early-stage disease; aberrant methylation of circulating cell-free DNA (cfDNA) offers a promising blood-based approach. Reported here is the analytical validation of MethyLiverDx-Sigma (MLDx-s), an assay that detects methylated RNF135 and DNMT3A in bisulfite-converted plasma cfDNA by methylation-specific PCR with high-resolution melting (HRM), with GAPDH as an endogenous control. Performance was evaluated following CLSI EP05-A3, EP07, EP12-A2, and EP37. Precision of quantification cycle (C q ) and melting peak temperature (T m ) was assessed by variance-component analysis within one site and across two sites and two operators. Because precision studies used bulk bisulfite-converted reference DNA, the reported coefficients of variation (CVs) primarily characterize the PCR-HRM detection module; end-to-end variability in clinical plasma specimens may be higher. Interference and cross-reactivity were evaluated in low-positive and contrived-negative samples against reproducibility-derived limits, with dose-response testing of reactive substances. Detection was 100% at both levels, and every run met control-validity criteria. C q repeatability CVs ranged from 0.53% to 1.68%, and total reproducibility CVs ranged from 1.10% to 2.37%; all T m CVs were ≤0.20%. All 18 interferents and 16 microbial cross-reactants evaluated in low-positive samples met the predefined acceptance criteria. In negative samples, hemoglobin, SARS-CoV-2, and measles virus produced concentration-dependent non-specific amplification, and dose-response testing established their respective no-effect concentrations. These findings characterize the analytical precision and multisite reproducibility of the PCR-HRM detection module and the analytical specificity of MLDx-s, complementing its previously reported clinical performance for plasma-based HCC detection.
SN - 0890-8508
SN - 1096-1194
ER -