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Application of plasma circulating cell-free DNA detection to the molecular diagnosis of hepatocellular carcinoma.

ABSTRACT: Circulating tumor DNA (ctDNA) carries genetic information consistent with tumor cells and has potential value for molecular diagnosis of tumors. The present study analysed the gene mutations of plasma circulating cell-free DNA (cfDNA) and tumor tissue DNA in hepatocellular carcinoma (HCC) patients and explored the clinical application value of plasma cfDNA as a tumor marker in HCC molecular diagnosis. Samples from 29 patients with primary HCC were collected. Hotspot mutations in 50 tumor-associated genes were analysed using amplicon sequencing technology and gene loci with a mutant allele frequency (MAF) >1% were analysed. 35 mutant genes in total were detected by deep sequencing method of which the genes with maximum mutation frequencies were TP53, ATM, and ALK. In addition, a total of 21 patients were found to have a consistent gene mutation in plasma cfDNA and tumor tissue DNA and 17 cases had consistent gene mutations in the paracancerous tissue and tumor tissue DNA. Further analysis showed that the MAFs in the TP53, CTNNB1, PIK3CA, and CDKN2A genes were higher in patients with tumor diameters >5 cm than those with tumor diameters <5 cm. And the MAFs in the TP53, RET, FGFR3 and APC genes were significantly higher in patients with multiple tumors or with metastasis than in single tumor patients. In conclusion, amplicon sequencing technology is highly sensitive for the detection of mutant genes in the plasma cfDNA of HCC patients. Plasma cfDNA might be an effective molecular marker for HCC molecular diagnosis.

SUBMITTER: He G

PROVIDER: S-EPMC6456549 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Application of plasma circulating cell-free DNA detection to the molecular diagnosis of hepatocellular carcinoma.

He Gaixia G Chen Yanhui Y Zhu Chengpei C Zhou Jinxue J Xie Xingwang X Fei Ran R Wei Lai L Zhao Haitao H Chen Hongsong H Zhang Henghui H

American journal of translational research 20190315 3

Circulating tumor DNA (ctDNA) carries genetic information consistent with tumor cells and has potential value for molecular diagnosis of tumors. The present study analysed the gene mutations of plasma circulating cell-free DNA (cfDNA) and tumor tissue DNA in hepatocellular carcinoma (HCC) patients and explored the clinical application value of plasma cfDNA as a tumor marker in HCC molecular diagnosis. Samples from 29 patients with primary HCC were collected. Hotspot mutations in 50 tumor-associa ...[more]

PMID: 30972172

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Project description:BackgroundPatients with cirrhosis are at high risk of hepatocellular carcinoma (HCC). The SEPT9 gene is a key regulator of cell division and tumor suppressor whose hypermethylation is associated with liver carcinogenesis. The primary aim of this study was to evaluate the diagnostic accuracy of a PCR-based assay for the analysis of SEPT9 promoter methylation in circulating cell-free DNA (mSEPT9) for diagnosing HCC among cirrhotic patients.MethodsWe report two phase II biomarker studies that included cirrhotic patients with or without HCC from France (initial study) and Germany (replication study). All patients received clinical and biological evaluations, and liver imaging according to current recommendations. The primary outcome was defined as the presence of HCC according to guidelines from the American Association for the Study of Liver Diseases. The diagnosis of HCC was confirmed by abdominal contrast-enhanced computed tomography scan and systematically discussed in a multidisciplinary consultation meeting. HCC-free cirrhotic patients were recruited if the screening abdominal ultrasound showed no evidence of HCC at the time of blood sampling for the mSEPT9 test and on the next visit six months later. The adjudicating physicians were blinded to patient results associated with the mSEPT9 test.FindingsWe included 289 patients with cirrhosis (initial: 186; replication: 103), among whom 98 had HCC (initial: 51; replication: 47). The mSEPT9 test exhibited high diagnostic accuracy for HCC diagnosis, with an area under the receiver operating characteristic curve (AUROC) of 0.944 (0.900-0.970, p<0.0001) in the initial study (replication: 0.930 [0.862-0.971, p<0.0001]; meta-analysis: AUROC=0.940 [0.910-0.970, p<0.0001], no heterogeneity: I2=0%, p=0.67; and no publication bias). In multivariate logistic regression analysis, the number of positive mSEPT9 triplicates was the only independent variable significantly associated with HCC diagnosis (initial: OR=6.30, for each mSEPT9 positive triplicate [2.92-13.61, p<0.0001]; replication: OR=6.07 [3.25-11.35, p<0.0001]; meta-analysis: OR=6.15 [2.93-9.38, p<0.0001], no heterogeneity: I2=0%, p=0.95; no publication bias). AUROC associated with the discrimination of the logistic regression models in initial and validation studies were 0.969 (0.930-0.989) and 0.942 (0.878-0.978), respectively, with a pooled AUROC of 0.962 ([0.937-0.987, p<0.0001], no heterogeneity: I2=0%, p=0.36; and no publication bias).InterpretationAmong patients with cirrhosis, the mSEPT9 test constitutes a promising circulating epigenetic biomarker for HCC diagnosis at the individual patient level. Future prospective studies should assess the mSEPT9 test in the screening algorithm for cirrhotic patients to improve risk prediction and personalized therapeutic management of HCC.

Project description:BackgroundAberrant DNA methylation may offer opportunities in revolutionizing cancer screening and diagnosis. We sought to identify a non-invasive DNA methylation-based screening approach using cell-free DNA (cfDNA) for early detection of hepatocellular carcinoma (HCC).MethodsDifferentially, DNA methylation blocks were determined by comparing methylation profiles of biopsy-proven HCC, liver cirrhosis, and normal tissue samples with high throughput DNA bisulfite sequencing. A multi-layer HCC screening model was subsequently constructed based on tissue-derived differentially methylated blocks (DMBs). This model was tested in a cohort consisting of 120 HCC, 92 liver cirrhotic, and 290 healthy plasma samples including 65 hepatitis B surface antigen-seropositive (HBsAg+) samples, independently validated in a cohort consisting of 67 HCC, 111 liver cirrhotic, and 242 healthy plasma samples including 56 HBsAg+ samples.ResultsBased on methylation profiling of tissue samples, 2321 DMBs were identified, which were subsequently used to construct a cfDNA-based HCC screening model, achieved a sensitivity of 86% and specificity of 98% in the training cohort and a sensitivity of 84% and specificity of 96% in the independent validation cohort. This model obtained a sensitivity of 76% in 37 early-stage HCC (Barcelona clinical liver cancer [BCLC] stage 0-A) patients. The screening model can effectively discriminate HCC patients from non-HCC controls, including liver cirrhotic patients, asymptomatic HBsAg+ and healthy individuals, achieving an AUC of 0.957(95% CI 0.939-0.975), whereas serum α-fetoprotein (AFP) only achieved an AUC of 0.803 (95% CI 0.758-0.847). Besides detecting patients with early-stage HCC from non-HCC controls, this model showed high capacity for distinguishing early-stage HCC from a high risk population (AUC=0.934; 95% CI 0.905-0.963), also significantly outperforming AFP. Furthermore, our model also showed superior performance in distinguishing HCC with normal AFP (< 20ng ml-1) from high risk population (AUC=0.93; 95% CI 0.892-0.969).ConclusionsWe have developed a sensitive blood-based non-invasive HCC screening model which can effectively distinguish early-stage HCC patients from high risk population and demonstrated its performance through an independent validation cohort.Trial registrationThe study was approved by the ethic committee of The Second Xiangya Hospital of Central South University (KYLL2018072) and Chongqing University Cancer Hospital (2019167). The study is registered at ClinicalTrials.gov(# NCT04383353 ).

Dataset Information

Application of plasma circulating cell-free DNA detection to the molecular diagnosis of hepatocellular carcinoma.

Publications

Application of plasma circulating cell-free DNA detection to the molecular diagnosis of hepatocellular carcinoma.

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