Optimized digital polymerase chain reaction enables detection of telomerase reverse transcriptase C228T mutation for prognostic assessment in hepatocellular carcinoma

优化的数字聚合酶链式反应可检测端粒酶逆转录酶C228T突变,用于肝细胞癌的预后评估。

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Abstract

BACKGROUND: Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma (HCC), particularly among patients infected with hepatitis B virus (HBV). The telomerase reverse transcriptase (TERT) promoter is the most frequently mutated site in HBV-related HCC; however, its prognostic significance is not fully established. AIM: To evaluate the prognostic impact of TERT promoter mutations and efficiency of digital polymerase chain reaction (dPCR). METHODS: A total of 66 HBV-related HCC patients who underwent hepatectomy were enrolled in this study. DNA extracted from fresh tumor tissues was analyzed for TERT promoter mutations using Sanger sequencing and dPCR. The dPCR assay was optimized by adding 7-deaza-dGTP, CviQ1, and ethylenediaminetetraacetic acid to improve detection sensitivity. Concordance between methods was assessed, and nomogram survival prediction models were developed to evaluate prognostic value based on mutation status. RESULTS: TERT promoter mutations were detected in 26/66 (39.39%) cases by Sanger sequencing and 30/66 (45.45%) by dPCR. The two methods showed high concordance (93.939%, κ = 0.876), with dPCR demonstrating 100% sensitivity and 90% specificity. Patients harboring TERT promoter mutations exhibited reduced overall survival and higher recurrence risk. Nomogram models successfully distinguished mutant from non-mutant cases for both overall survival (C-index: 0.7651) and disease-free survival (C-index: 0.6899). CONCLUSION: TERT promoter mutation predicts poor prognosis in HBV-related HCC and serves as a biomarker for risk stratification. Optimized dPCR outperforms Sanger sequencing, and nomograms with TERT status guide precision therapy.

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