Supplementary Material for: HBV DNA Integration into Telomerase or MLL4 Genes and TERT Promoter Point Mutation as Three Independent Signatures in Subgrouping HBV-related HCC with distinct features

Introduction: A set of genetic mutations to classify hepatocellular carcinoma (HCC) useful to clinical studies is an unmet need. Hepatitis B virus-related HCC (HBV-HCC) harbors a unique genetic mutation, namely the HBV integration, among other somatic endogenous gene mutations. We explored a combination of HBV DNA integrations and common somatic mutations to classify HBV-HCC by using a capture-sequencing platform. Methods: A total of 153 HBV-HCCs after surgical resection were subjected to capture-sequencing to identify HBV integrations and three common somatic mutations in genomes. Three mutually exclusive mutations, HBV DNA integration into the TERT promoter, HBV DNA integration into MLL4, or TERT promoter point mutation, were identified in HBV-HCC. Results: They were used to classify HBV-HCCs into four groups: G1 with HBV-TERT integration (25.5%); G2 with HBV-MLL4 integration (10.5%); G3 with TERT promoter mutation (30.1%); and G4 without these three mutations (34.0%). Clinically, G3 has the highest male-to-female ratio, cirrhosis rate and associated with higher early recurrence and mortality after resection, but G4 has the best outcome. Transcriptomic analysis revealed a grouping different from the published ones, and G2 with an active immune profile related to immune checkpoint inhibitor response. Analysis of integrated HBV DNA provided clues for HBV genotype and variants in carcinogenesis of different HCC subgroup. This new classification was also validated in another independent cohort. Discussion/Conclusion: A simple and robust genetic classification was developed to aid in understanding HBV-HCC and in harmonizing clinical studies.

引言：适用于临床研究的肝细胞癌（hepatocellular carcinoma, HCC）分类遗传突变标志物集，仍是一项尚未满足的临床需求。乙型肝炎病毒相关肝细胞癌（HBV-HCC）除存在体细胞内源基因突变外，还带有独特的HBV整合突变。本研究采用捕获测序平台，通过联合分析HBV DNA整合事件与常见体细胞突变，实现对HBV-HCC的分类。
方法：本研究共纳入153例手术切除后的HBV-HCC患者样本，通过捕获测序技术鉴定其基因组中的HBV整合事件与3种常见体细胞突变。最终在HBV-HCC中鉴定出3种互斥突变：HBV DNA整合至端粒酶逆转录酶（telomerase reverse transcriptase, TERT）启动子区域、HBV DNA整合至混合谱系白血病基因4（mixed lineage leukemia 4, MLL4），以及TERT启动子点突变。
结果：基于上述3种突变，可将HBV-HCC分为4个亚型：G1型（携带HBV-TERT整合，占比25.5%）、G2型（携带HBV-MLL4整合，占比10.5%）、G3型（携带TERT启动子突变，占比30.1%）以及G4型（不携带上述3种突变，占比34.0%）。临床特征分析显示，G3型的男女比例、肝硬化发生率均为最高，且术后早期复发率与死亡率均更高；而G4型的预后最佳。转录组分析显示，本研究的分型与已发表的分型存在差异，其中G2型具有与免疫检查点抑制剂（immune checkpoint inhibitor）应答相关的活跃免疫表型。对整合HBV DNA的分析还为不同HBV-HCC亚型的致癌机制中HBV基因型与变异株的关联提供了线索。该新型分类体系在另一独立队列中得到了验证。
讨论与结论：本研究建立了一种简便且稳定的遗传分类体系，有助于加深对HBV-HCC的认识，并为临床研究的标准化提供支撑。