[摘 要] 目的：肝细胞癌（HCC）是肝脏最常见的原发性恶性肿瘤。早期HCC诊出率低，多数患者就诊时已经是晚期，预后极 差，因此亟须探索有效的HCC早期诊断标志物及干预治疗靶点。癌症/睾丸抗原1A（CTAG1A）在多种肿瘤中异常表达并且具有 强免疫原性，但其在HCC中的表达特性及免疫原性尚不明确。本研究旨在鉴定HCC组织和细胞中CTAG1A的表达趋势和免疫 原性，为HCC早期诊断提供新的生物标志物，为临床免疫治疗提供新的潜在靶点。方法：通过转录组芯片筛选10对极早期HCC （BCLC 0期HCC）肿瘤与癌旁组织的差异表达基因谱。利用RT-qPCR在独立大样本（BCLC 0、A、B、C期HCC组织及癌旁组织， n = 149）及多种肝癌细胞系中验证CTAG1A的表达。通过生物信息学工具（IEDB数据库的TepiTool和Swiss Model）预测 CTAG1A的MHC-Ⅰ型和MHC-Ⅱ型抗原表位。采用固相多肽合成法合成候选肽段，经HPLC纯化及质谱验证后，使用IFN-γ酶联 免疫斑点法（ELISpot）检测9例HCC患者外周血单个核细胞（PBMC）对各个肽段的特异性T细胞反应。临床样本来源于2015至 2022年海军军医大学第三附属医院（东方肝胆外科医院）收治的HCC患者，所有样本的采集使用均获患者知情同意，并经东方肝 胆外科医院伦理委员会审查批准（EHBHKY2015-01-017），严格按照相关要求和伦理规定严格执行。采用SPSS 30.0软件进行统 计学处理，通过ROC曲线评估诊断效能。结果：转录组芯片筛选结果显示，CTAG1A在极早期HCC（BCLC 0期HCC）中表达显 著上调（|FC| = 99.16，P < 0.000 1），临床独立样本验证显示其在各分期HCC中均呈现出高表达趋势且在早期HCC中呈现出较佳的 诊断效能（BCLC 0期HCC AUC = 0.6893，敏感性 = 85.71%；BCLC A期HCC AUC = 0.8229，敏感性 = 83.33%）；进一步的分析结 果显示，CTAG1A在多种肝癌细胞系中的表达显著高于相对正常肝细胞系（P < 0.001）。与甲胎蛋白（AFP）相比，CTAG1A在 BCLC 0期和A期HCC中的诊断效能更优（AFP在早期HCC中ROC曲线分析无显著差异，P > 0.05）。生物信息学工具预测发现 CTAG1A含8个MHC-Ⅰ型和4个MHC-Ⅱ型抗原表位。IFN-γ ELISpot实验显示，12条合成肽段能不同程度地诱导HCC患者 PBMC的特异性T细胞反应。结论：CTAG1A在HCC早期即显著高表达，且具有多表位免疫原性，可能能够激活CD8?和CD4? T细胞，提示其具有作为HCC免疫治疗靶点的潜力，可望为开发基于mRNA疫苗或过继性细胞疗法的联合免疫治疗策略提供新 的方向。相较于AFP，CTAG1A在早期HCC中展现出更优的诊断效能，提示其作为HCC早期诊断标志物的可能。

[Abstract] Objective: Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. The diagnosis rate of early HCC is low, and most patients are diagnosed at the late stage and have a very poor prognosis. Therefore, it is urgent to explore effective early diagnosis markers and intervention targets for HCC. Cancer/testicular antigen 1A (CTAG1A) is abnormally expressed and highly immunogenic in a variety of tumors, but its expression characteristics and immunogenicity in HCC remain unclear. The aim of this study is to identify the expression and immunogenicity of CTAG1A in HCC tissues and cells, providing a new biomarker for the early diagnosis of HCC and a new potential target for clinical immunotherapy. Methods: This study screened the differentially expressed gene profiles between 10 pairs of very early HCC (BCLC stage 0 HCC) tumors and paracancerous tissues using a transcriptome microarray. The expression of CTAG1A was verified by RT-qPCR in an independent large sample (BCLC stage 0, A, B, C HCC tissues and adjacent non-tumor tissues, n=149) and various hepatocellular carcinoma cell lines. Bioinformatics tools (TepiTool of IEDB database and Swiss Model) were used to predict the MHC-Ⅰ and MHC-Ⅱ epitopes of CTAG1A. The candidate peptides were synthesized by solid-phase polypeptide synthesis method. After purification by HPLC and verification by mass spectrometry assay, the specific T cell responses of peripheral blood mononuclear cells (PBMC) of 9 HCC patients to all peptides were detected by IFN-γ enzyme-linked immunospot assay (ELISpot). The clinical samples were collected from HCC patients admitted to the Third Affiliated Hospital of Naval Medical University (Eastern Hepatobiliary Surgery Hospital) from 2015 to 2022. The collection and usage of all samples were carried out with the consent of the patients, and with the approval of the Ethics Committee of Eastern Hepatobiliary Surgery Hospital (EHBHKY2015-01-017) and in strict accordance with relevant requirements and ethical regulations. Statistical analysis was performed using SPSS 30.0 software, and the diagnostic efficiency was evaluated by ROC curve. Results: Transcriptome chip screening results showed that CTAG1A expression was significantly up-regulated in the very early-stage HCC (BCLC stage 0 HCC) (|FC| = 99.16, P < 0.0001). The verification using the clinical independent samples showed its high expression in all stages of HCC and better diagnostic efficacy in early-stage HCC (BCLC stage 0 HCC AUC=0.6893, sensitivity = 85.71%; BCLC stage A HCC AUC = 0.8229, sensitivity = 83.33%). Furthermore, the expression of CTAG1A was significantly higher in multiple liver cancer cell lines than in relatively normal liver cell lines (P < 0.001). Compared with alpha-fetoprotein (AFP), CTAG1A showed better diagnostic efficacy in BCLC stage 0 and stage A HCC (ROC curve analysis of AFP showed no significant difference in early HCC, P > 0.05). Bioinformatics tools predicted that CTAG1A contained 8 MHC-type I and 4 MHC-type II epitopes. The IFN-γ ELISpot assay showed that 12 synthetic peptides could induce PBMC specific T cell response in HCC patients to varying degrees. Conclusion: CTAG1A is significantly overexpressed in early-stage HCC and has multi-epitope immunogenicity, which may activate CD8? and CD4? T cells, suggesting its potential as a target for HCC immunotherapy. It may provide a new direction for developing combined immunotherapy strategies based on mRNA vaccines or adoptive cell therapy. Compared with AFP, CTAG1A exhibits better diagnostic efficacy in early stage HCC, suggesting its potential as a marker for early diagnosis of HCC.

国家自然科学基金（No. 82172896）；中国科协青年人才托举工程（No. 2023QNRC001）