作者：许翔，蒋宏，刘钧澄，陈华东，钟志海，张志崇，佘锦标，徐哲，谭立

单位：中山大学附属第一医院 肝胆胰外科中心，广东 广州 510080

Authors: Xu Xiang, Jiang Hong, Liu Juncheng, Chen Huadong, Zhong Zhihai, Zhang Zhichong, She Jinbiao, Xu Zhe, Tan Li

Unit:  Hepatopancreatobiliary Center, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, Guangdong, China

摘要：

目的  分析肝母细胞瘤新辅助化疗对肝母细胞瘤免疫浸润的影响，为免疫治疗在肝母细胞瘤中的应用提供参考。方法  回顾性分析2010年8月至2019年12月在中山大学附属第一医院就诊并接受手术的186例原发肝母细胞瘤患儿，对患儿新辅助化疗前后的肿瘤进行免疫浸润评分（0~3分）评估，利用单因素及多因素Logistic回归分析与免疫浸润评分相关的临床因素，利用Kaplan-Meier法计算并用Log-rank法比较不同免疫浸润评分患儿的无复发生存和总生存，同时采用单因素及多因素Cox回归分析与预后相关的因素。最终采用多重免疫荧光染色分析6例患儿新辅助化疗前后的免疫浸润细胞种类及含量变化。结果  新辅助化疗前，瘤内免疫浸润评分为0分、1分、2分、3 分的患儿比例分别为61.5%、33.0%、3.7%与1.8%。肿瘤边界免疫浸润评分为0分、1分、2分、3分的患儿比例分别为66.7%、28.2%、2.6%与2.6%。新辅助化疗前，无肝细胞癌样病灶P=0.022）、造血组织（P=0.005）是高瘤内免疫浸润评分的影响因素。新辅助化疗前瘤内免疫浸润评分低的患儿3年无复发生存率更高（P=0.003）。无肝细胞癌样病灶是无复发生存的独立保护因素HR=0.26，95%CI 0.10~0.68，P=0.006）。新辅助化疗后，肿瘤内、交界的肿瘤免疫浸润增加的患儿占69.1%和61.5%。新辅助化疗后的肿瘤边界不规则（P=0.021）是高肿瘤边界区免疫浸润评分的影响因素。新辅助化疗后瘤内免疫浸润评分高的患儿的3年无复发生存率更高（P=0.011）。诊断年龄大是无复发生存的独立危险因素（HR=1.17，95%CI 1.02~1.35，P=0.025）。瘤内浸润的CD8+T 细胞（P=0.020）、B细胞（P=0.040）、单核细胞（P=0.017）和调节性T细胞（P=0.027）在新辅助化疗后显著上升。结论  原发肝母细胞瘤淋巴细胞浸润较少，且新辅助化疗前免疫浸润程度高是无复发生存的危险因素，提示未化疗的肝母细胞瘤的高免疫浸润细胞或为免疫抑制细胞。新辅助化疗显著增加肿瘤淋巴细胞浸润，或增加CD8+T细胞计数等。化疗后免疫浸润增多的患儿3年无复发生存率更高。

关键词： 肝母细胞瘤；新辅助化疗；免疫浸润评分；临床预后

Abstract：

Objective  To analyze the effects of neoadjuvant chemotherapy on immune infiltration of hepatoblastoma, and to provide reference for the application of immunotherapy in hepatoblastoma. Method  186 children with primary hepatoblastoma who were admitted and treated in the First Affiliated Hospital of Sun Yat-Sen University from August 2010 to December 2019 were retrospectively analyzed. The immune infiltration score(0-3points) before and after neoadjuvant chemotherapy was evaluated. Clinical factors related to immune infiltration score were analyzed by univariate and multivariate Logistic regression. The Kaplan-Meier method and the Log-rank method were used to calculate and compare the recurrence-free survival (RFS) and overall survival (OS) of patients with different immune infiltration scores. Univariate and multivariate COX regression approach were chosen to analyze factors related to prognosis. Multiple immunofluorescence staining was finally used in 6 patients to evaluate the type and content change of infiltrating immune cells before and after neoadjuvant chemotherapy. Result  Before neoadjuvant chemotherapy, the proportion of patients with intratumoral immune invasion scores of 0, 1, 2, 3 points were 61.5%, 33.0%, 3.7%, and 1.8%, respectively. The proportion of patients with tumor border immune infiltration scores of 0, 1, 2, 3 points were 66.7%, 28.2%, 2.6%, and 2.6%, respectively. Before neoadjuvant chemotherapy, no Hepatocellular carcinoma-like foci (P=0.022) and hematopoietic tissue (P=0.005) were influence factors of high intratumoral immune infiltration score. Patients with low intratumoral immune infiltration scores before neoadjuvant chemotherapy had higher 3-year RFS (P=0.003). No Hepatocellular carcinoma-like foci was an independent protective factor for RFS (HR=0.26, 95%CI 0.10-0.68, P=0.006). After neoadjuvant chemotherapy, the proportions of patients with increased intratumoral and tumor border zone lymphocyte infiltration were 69.1% and 61.5%. Irregular tumor borders after neoadjuvant chemotherapy (P=0.021) was an influence factor of high tumor border zone immune infiltration score. Patients with high intratumoral immune infiltration scores after neoadjuvant chemotherapy had higher 3 -year RFS (P=0.011). Older age at diagnosis was an independent risk factor for RFS (HR=1.17, 95%CI 1.02-1.35, P=0.025). Infiltrating CD8+T cells (P=0.020), B cells (P=0.040), monocytes (P=0.017) and regulatory T cells (P=0.027) in the tumor were increased significantly after neoadjuvant chemotherapy. Conclusion  Primary hepatoblastoma had less lymphocyte infiltration, and higher immune infiltration in primary hepatoblastoma before neoadjuvant chemotherapy was the risk factor of RFS, indicating the highly enriched cells in non-treated hepatoblastom were immune suppressive cells. Lymphocyte infiltration in hepatoblastoma was significantly increased after neoadjuvant chemotherapy, which may indicate CD8+T cell counts increase. And patients with increased immune infiltration after chemotherapy had a higher 3-year RFS.

Key Words:  Hepatoblastoma；Neoadjuvant chemotherapy；Immune infiltration score；Clinical prognosis