Supplementary Material for: Tumor Mutational Burden as a Prognostic Biomarker for Relapse-Free Survival in Hepatocellular Carcinoma: Insights from Long-Term Follow-Up

Background. Hepatocellular carcinoma (HCC) has a high recurrence rate even after curative resection. Although the tumor mutational burden (TMB) has emerged as a potential biomarker for survival outcomes in HCC, its clinical significance remains unclear.
Methods. A retrospective analysis of 204 patients who underwent an initial liver resection was performed. Patients were classified into TMB-high (≥4.8 mutations/Mb) or TMB-low groups based on whole-exome sequencing. We assessed relapse-free survival (RFS), overall survival (OS), and performed subgroup analyses focusing on patients without recurrence within the first two postoperative years. Gene expression profiling and mutational signature analyses were also conducted.
Results. Patients with TMB-high tumors showed significantly shorter RFS compared to the TMB-low group (median 24.2 vs. 37.1 months; p=0.008), whereas OS was not significantly different. Multivariate analysis identified TMB-high status as an independent prognostic factor for RFS (HR, 1.72; p=0.011). In the subgroup without early recurrence, TMB-high status was the only independent factor associated with late recurrence (HR, 2.45; p=0.005). TMB-high tumors correlated with advanced liver fibrosis and specific somatic mutations (CTNNB1, TTN, MUC16). Additionally, mutational signatures associated with chronic inflammation and alcohol consumption were enriched in the TMB-high group.
Conclusions. High TMB is associated with shorter relapse-free survival in HCC, particularly among patients with long-term follow-up, indicating an increased risk for multicentric recurrence. TMB may serve as a valuable prognostic biomarker for recurrence risk stratification. The associations between TMB, liver fibrosis, and inflammation suggest potential therapeutic strategies targeting the hepatic microenvironment to reduce recurrence risk in patients undergoing liver resection for HCC.

背景：即使接受根治性切除术，肝细胞癌（Hepatocellular carcinoma, HCC）仍具有较高的复发率。尽管肿瘤突变负荷（tumor mutational burden, TMB）已成为肝细胞癌预后结局的潜在生物标志物，但其临床意义仍未明确。
方法：本研究对204例接受首次肝切除术的患者开展回顾性分析。基于全外显子测序（whole-exome sequencing）结果，将患者分为高TMB组（≥4.8突变/Mb）与低TMB组。本研究评估了无复发生存期（relapse-free survival, RFS）与总生存期（overall survival, OS），并针对术后前2年无复发的患者进行亚组分析，同时开展基因表达谱分析与突变特征分析。
结果：高TMB肿瘤患者的无复发生存期显著短于低TMB组（中位24.2 vs 37.1个月；p=0.008），而总生存期无显著差异。多因素分析显示，高TMB状态是无复发生存期的独立预后因素（风险比hazard ratio, HR，1.72；p=0.011）。在无早期复发的亚组中，高TMB状态是与晚期复发相关的唯一独立因素（HR，2.45；p=0.005）。高TMB肿瘤与进展期肝纤维化及特定体细胞突变（CTNNB1、TTN、MUC16）相关。此外，高TMB组中富集了与慢性炎症及酒精摄入相关的突变特征。
结论：高TMB与肝细胞癌患者更短的无复发生存期相关，尤其在长期随访的患者中，提示多中心复发风险升高。TMB可作为复发风险分层的有价值预后生物标志物。TMB与肝纤维化、炎症的关联提示，靶向肝脏微环境的治疗策略或可降低肝细胞癌肝切除术后患者的复发风险。