Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies.

引言：神经内分泌肿瘤（Neuroendocrine neoplasms, NENs）具有显著的免疫抑制型肿瘤免疫微环境（tumor immune microenvironments, TIMEs）。然而，神经内分泌分化（Neuroendocrine differentiation, NED）对非神经内分泌肿瘤（如胃癌（Gastric cancer, GC））的免疫学效应尚不明确。
方法：本研究在纯胃癌（Pure gastric cancer, PGC）与伴神经内分泌分化的胃癌（GC-NED）两组队列中，基于表达数据对肿瘤免疫微环境特征进行评分，并通过手术标本的连续全组织切片完成验证；同时采用多标记免疫组化染色独立评估三级淋巴结构（Tertiary lymphoid structures, TLSs）及TLS外区域。在GC-NED队列中开展肿瘤免疫微环境特征对肿瘤行为学的风险分析，并在其他器官来源的腺癌中初步探索神经内分泌分化的通用免疫学效应。
结果：基于超过11500个注释后的三级淋巴结构及2700个TLS外区域的分析结果显示，与纯胃癌组相比，伴神经内分泌分化的胃癌组具有显著更强的免疫抑制型肿瘤免疫微环境，其特征为：三级淋巴结构数量增多但处于未成熟状态，三级淋巴结构内部初始B细胞（naïve B cell）与滤泡调节性T细胞（follicular regulatory T cell）密度更高，且与三级淋巴结构成熟相关的细胞比例下调；TLS外区域内初始B细胞与调节性T细胞密度升高，且存在高比例的耗竭T细胞（exhausted T cell）。仅在伴神经内分泌分化的胃癌队列中，可观察到肿瘤PD-L1表达上调，且其与三级淋巴结构的形成及成熟过程存在显著相关性。肿瘤免疫微环境特征，尤其是与三级淋巴结构相关的特征，与肿瘤生长及侵袭行为显著相关。在结直肠、胰腺、肺及前列腺来源的腺癌中，均观察到三级淋巴结构的形成与成熟过程不同步，以及初始或调节性免疫细胞浸润增多的现象。
结论：神经内分泌分化定义了一类具有独特表型的胃癌亚型，其肿瘤免疫微环境呈现更强的免疫抑制特征，且与肿瘤行为学密切相关，提示该亚型患者可从免疫治疗中获益更多。