Supplementary Material for: Immune Gene Expression Profile in Hepatocellular Carcinoma and Surrounding Tissue Predicts Time to Tumor Recurrence

<b><i>Background:</i></b> The antitumor immune response may play a major role in the clinical outcome of hepatocellular carcinoma (HCC). We characterized the liver immune microenvironment by direct hybridization of RNA extracted from HCC and nontumorous tissues. <b><i>Methods:</i></b> RNA was extracted from frozen liver tissue samples of HCC (T; <i>n</i> = 30) and nontumorous tissues (NT; <i>n</i> = 33) obtained from 38 patients. Matched samples were available for 25 patients. The immune gene expression profile was analyzed with the nCounter GX Human Immunology v2 system (NanoString Technologies), which detects the expression levels of 579 immune response-related genes simultaneously. <b><i>Results:</i></b> Since the immune gene expression profile of T and NT tissues was significantly different, the prognostic relevance of the liver immune microenvironment was evaluated in the T and NT samples separately. Unsupervised clustering detected two main clusters of immune gene expression both in T and in NT liver samples. In both cases, the expression clusters identified groups of patients with a significantly different median time to HCC recurrence (TTR) but similar overall survival. Based on T tissue, two groups with median TTR of 19 and 127 months, respectively, were detected (<i>p</i> &lt; 0.005). Expression of genes related to T-cell activation was associated with longer TTR. The analysis of NT tissue discriminated subsets of patients with median TTR of 22 and 68 months (<i>p</i> &lt; 0.05). In contrast to T tissue, a predominant inflammatory immune environment was associated with shorter TTR. <b><i>Conclusions:</i></b> Immune gene expression profiles predictive of TTR could be identified both in HCC and in adjacent cirrhotic tissues. Longer TTR was associated with overexpression in T tissue and downregulation in NT tissue of the immune response and of inflammation-related genes.

<b><i>研究背景：</i></b>
抗肿瘤免疫应答可能在肝细胞癌（hepatocellular carcinoma, HCC）的临床结局中发挥核心作用。本研究通过对肝细胞癌及非肿瘤组织中提取的RNA进行直接杂交实验，对肝脏免疫微环境进行了系统性表征。

<b><i>研究方法：</i></b>
本研究从38例患者的冷冻肝脏组织样本中提取总RNA，样本包含肝细胞癌组织（T组；n=30）与非肿瘤组织（NT组；n=33），其中25例患者可获取配对样本。采用nCounter GX Human Immunology v2系统（NanoString Technologies）分析免疫基因表达谱，该系统可同时检测579个免疫应答相关基因的表达水平。

<b><i>研究结果：</i></b>
鉴于肝细胞癌组织与非肿瘤组织的免疫基因表达谱存在显著差异，本研究分别在癌组织及非肿瘤组织样本中评估了肝脏免疫微环境的预后关联价值。无监督聚类分析在癌组织与非肿瘤肝脏样本中均识别出两类主要的免疫基因表达簇。在两类样本中，该表达簇均可区分出中位肝细胞癌复发时间（time to HCC recurrence, TTR）存在显著差异，但总生存期相似的患者亚组。基于癌组织的分析显示，可划分出中位TTR分别为19个月与127个月的两组患者（p < 0.005）。T细胞活化相关基因的高表达与更长的TTR显著相关。对非肿瘤组织的分析则区分出中位TTR分别为22个月与68个月的患者亚组（p < 0.05）。与癌组织不同，非肿瘤组织中以炎症性免疫环境为主的表型与更短的TTR相关。

<b><i>研究结论：</i></b>
无论是在肝细胞癌组织还是邻近的肝硬化组织中，均可识别出可预测TTR的免疫基因表达谱。癌组织中免疫应答及炎症相关基因的过表达，与非肿瘤组织中此类基因的下调表达，均与更长的TTR显著相关。