Late-stage tertiary lymphoid structures in hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade [scRNA-seq]. Late-stage tertiary lymphoid structures in hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade [scRNA-seq]

Tertiary lymphoid structures (TLS) are associated with improved response in solid tumors treated with immune checkpoint blockade (ICB), but understanding of their clinical significance and the circumstances of their resolution remains incomplete. Here, we found that in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy, high intratumoral TLS density at the time of surgery is associated with pathologic response and improved relapse free survival. In areas of tumor regression, we further identified a non-canonical involuted morphology of TLS marked by dispersion of the B cell follicle, persistence of a T cell zone enriched for T cell-mature dendritic cell interactions, and increased expression of T cell memory markers. Collectively, these data suggest that TLS may serve be both a prognostic and predictive marker of response to immunotherapy in HCC and suggest a functional role for late-stage TLS T cell memory formation after elimination of viable tumor. Overall design: We utilized surgical resection specimens and peripheral blood obtained from patients with locally advanced hepatocellular carcinoma receiving neoadjuvant immune checkpoint blockade based therapy. Bulk RNA sequencing was performed on post-treatment tumor resection specimens to characterize differences in gene expression between tumors with high and low density of tertiary lymphoid structures. Tumor associated tertiary lymphoid structures were microdissected and subjected to bulk TCR and BCR sequencing using the Adaptive Biotechnologies ImmunoSEQ platform (shared separately). Single cell RNA, TCR, and BCR sequencing was performed on matched peripheral blood and tumor specimens to infer the identity of T and B cells identified in tertiary lymphoid structures. *** Submitters state that raw data is being submitted to dbGap (phs003672.v1.p1)***

三级淋巴结构（tertiary lymphoid structures, TLS）与接受免疫检查点阻断（immune checkpoint blockade, ICB）治疗的实体瘤患者的更佳临床应答相关，但目前学界对其临床意义及消退相关机制的认知仍存在不足。本研究发现，在接受新辅助免疫治疗的肝细胞癌（hepatocellular carcinoma, HCC）患者中，手术时瘤内三级淋巴结构的高密度与病理应答及更长的无复发生存期显著相关。在肿瘤消退区域内，我们进一步鉴定出一种非典型的退缩型三级淋巴结构形态：其特征为B细胞滤泡弥散分布、富含T细胞与成熟树突状细胞相互作用的T细胞区得以保留，且T细胞记忆标志物的表达水平显著升高。综上，本研究数据提示，三级淋巴结构可作为肝细胞癌患者免疫治疗应答的预后及预测标志物，并提示在清除存活肿瘤后，晚期三级淋巴结构的T细胞记忆形成发挥了功能性作用。

整体实验设计：我们纳入了接受基于免疫检查点阻断的新辅助治疗的局部晚期肝细胞癌患者，收集其手术切除标本及外周血。对治疗后的肿瘤切除标本进行批量RNA测序，以鉴定高、低三级淋巴结构密度肿瘤之间的基因表达差异。对肿瘤相关三级淋巴结构进行显微切割后，采用Adaptive Biotechnologies公司的ImmunoSEQ平台进行批量T细胞受体（TCR）与B细胞受体（BCR）测序（数据将另行共享）。对匹配的外周血及肿瘤标本进行单细胞RNA、TCR及BCR测序，以推断三级淋巴结构中鉴定出的T、B细胞的身份。

提交者声明，原始数据已提交至dbGap数据库（phs003672.v1.p1）