背景：乙型肝炎病毒（HBV）感染是慢性肝病的主要原因之一，从慢性乙型肝炎（CHB）发展到纤维化、肝硬化和肝细胞癌（HCC）。肝细胞癌的早期检测和基于实验室的筛查仍然是重大挑战。本研究旨在确定作为液体活检标志物的被释放到循环中的癌症标志基因特征（即循环肿瘤DNA（ctDNA）），是否可以用于肝细胞癌的筛查、早期检测和预后。 方法：共评估了130名受试者，包括HBV-HCC（n = 80）、HBV肝硬化和非肝硬化（n = 35）以及健康对照组（n = 15），通过基于Sanger循环测序和液滴数字PCR（ddPCR）的方法，检测ctDNA中TP53和β-连环蛋白（CTNNB1）基因热点突变。确定突变检测频率、突变分数百分比以及它们与肿瘤分期、死亡率和吸烟习惯的关联。 结果：针对32名肝细胞癌患者进行了基于Sanger循环测序。Predict SNP Tools分析表明，ctDNA序列中存在几个致病驱动突变，包括TP53基因外显子7中的p.D228N、p.C229R、p.H233R、p.Y234D、p.S240T、p.G245S和p.R249M，以及CTNNB1基因外显子3中的p.S33T。肝细胞癌患者中，p.R249M突变通过测序检测到为主要突变（25％的病例），但并无主导突变在c.747G>T（p.R249S）的位置上，该突变是HBV-HCC患者报道过的。我们开发了双探针ddPCR方法，用于确定TP53（p.R249M和p.R249S）和CTNNB1（p.S45P）基因的突变和野生型拷贝数，以及它们在所有130个受试者中的突变分数百分比。TP53 R249M和CTNNB1 S45P突变分别在31.25％和26.25％的HCC患者中检测到，并伴有较高的突变/野生型分数百分比（1.81％和1.73％），与肝硬化和非肝硬化患者相比具有显著性。合并TP53和CTNNB1基因驱动突变的HCC患者生存率较低。TP53 R249M突变还与吸烟习惯显著相关（p < 0.0001）（OR为11.77，95％CI为3.219-36.20），但TP53 R249S突变的情况则不同。 结论：通过ddPCR筛查ctDNA中的TP53和CTNNB1基因突变可能有助于早期检测和鉴定HCC进展的风险。版权所有© 2023 Kumar, Nadda, Quadri, Kumar, Paul, Tanwar, Gamanagatti, Dash, Saraya, Shalimar和Nayak。

Background: Hepatitis B virus (HBV) infection is one of the major causes of chronic liver disease, which progresses from chronic hepatitis B (CHB) to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Early detection and laboratory-based screening of hepatocellular carcinoma are still major challenges. This study was undertaken to determine whether the cancer hallmark gene signatures that are released into circulation as circulating tumour DNA (ctDNA) can be used as a liquid biopsy marker for screening, early detection, and prognosis of HCC. Methods: A total of 130 subjects, including HBV-HCC (n = 80), HBV-cirrhotic and non-cirrhotic (n = 35), and healthy (n = 15) controls, were evaluated for TP53 and beta-catenin (CTNNB1) gene hotspot mutations in ctDNA by Sanger-based cycle sequencing and droplet digital PCR (ddPCR) assays. Mutation detection frequency, percentage mutant fractions, and their association with tumour stage, mortality, and smoking habits were determined. Results: Sanger-based cycle sequencing was carried out for 32 HCC patients. Predict SNP Tools analysis indicated several pathogenic driver mutations in the ctDNA sequence, which include p.D228N, p.C229R, p.H233R, p.Y234D, p.S240T, p.G245S, and p.R249M for TP53 gene exon 7 and p.S33T for CTNNB1 gene exon 3. The TP53 c.746G>T (p.R249M) mutation was detected predominately (25% cases) by sequencing, but there was no dominant mutation at position c.747G>T (p.R249S) that was reported for HBV-HCC patients. A dual-probe ddPCR assay was developed to determine mutant and wild-type copy numbers of TP53 (p.R249M and p.R249S) and CTNNB1 (p.S45P) and their percentage mutant fraction in all 130 subjects. The TP53 R249M and CTNNB1 S45P mutations were detected in 31.25% and 26.25% of HCC patients, respectively, with a high mutant-to-wild-type fraction percentage (1.81% and 1.73%), which is significant as compared to cirrhotic and non-cirrhotic patients. Poor survival was observed in HCC patients with combined TP53 and CTNNB1 gene driver mutations. The TP53 R249M mutation was also significantly (p < 0.0001) associated with smoking habits (OR, 11.77; 95% CI, 3.219-36.20), but not the same for the TP53 R249S mutation. Conclusion: Screening of ctDNA TP53 and CTNNB1 gene mutations by ddPCR may be helpful for early detection and identifying the risk of HCC progression.Copyright © 2023 Kumar, Nadda, Quadri, Kumar, Paul, Tanwar, Gamanagatti, Dash, Saraya, Shalimar and Nayak.