Supplementary Material for: Dynamic profiling of the immune tumor microenvironment in locally advanced gastric cancer treated with perioperative chemotherapy

Introduction: In locally advanced gastric cancer (GC), FLOT represents the standard perioperative regimen and combination with immunotherapy is under investigation. However, the role of immune tumor microenvironment (TME) is poorly recognized in this setting. We aimed to study TME characteristics and dynamics during FLOT.
Methods: Paired biopsy (PRE) and surgical (POST) samples of 25 patients treated with FLOT were prospectively analyzed. After collection of clinic-pathological data, NanoString analyses were performed. The primary objective of the study was to assess the changes induced by chemotherapy in POST compared to PRE samples.
Results: The unsupervised hierarchical method analysis clearly distinguished PRE and POST samples, even though some cases showed high immune gene expression at baseline. When POST samples were compared with PRE, a differential expression in hyper-expressed gene sets related to cytotoxicity, T-cell functions, complement system, tumor necrosis factor-superfamily, cell cycle and regulation was recognized. Downstaging of the primary tumor (T-regression, measured by pathologic compared to clinical T stage) was the covariate most frequently associated with these changes. Using the immune cell profiling, cases with T-regression reported a significant increase of T, CD8+ T and B-cells and a decrease in mast cells, while non-responders demonstrated an increase of T, B, cytotoxic and mast cells.
Conclusion: Our analysis shows that FLOT significantly influences immune TME of GC. While relevant modifications preferentially occur in tumors showing primary tumor regression, response to treatment seems to be associated with a specific immune profile.

引言：在局部进展期胃癌（locally advanced gastric cancer, GC）中，FLOT是标准的围手术期治疗方案，其与免疫治疗的联合应用正处于研究阶段。然而，免疫肿瘤微环境（immune tumor microenvironment, TME）在该治疗场景中的作用尚未得到充分认知。本研究旨在探究FLOT治疗期间的TME特征及其动态变化。
方法：本研究前瞻性分析了25例接受FLOT治疗患者的配对活检（PRE）与手术（POST）样本。在收集临床病理数据后，开展了NanoString分析。本研究的主要研究目标为对比POST与PRE样本，评估化疗诱导的基因表达变化。
结果：无监督分层聚类分析可清晰区分PRE与POST样本，尽管部分病例在基线时即表现出较高的免疫基因表达水平。将POST样本与PRE样本对比后，研究人员观察到与细胞毒性、T细胞功能、补体系统、肿瘤坏死因子超家族、细胞周期及其调控相关的高表达基因集存在差异表达。原发肿瘤降期（T-regression，通过病理T分期与临床T分期对比得出）是与上述变化关联最为频繁的协变量。通过免疫细胞分型分析，存在T-regression的病例中，T细胞、CD8+ T细胞及B细胞数量显著升高，肥大细胞数量降低；而无应答者则表现为T细胞、B细胞、细胞毒性细胞及肥大细胞数量升高。
结论：本研究分析表明，FLOT可显著影响胃癌（GC）的免疫肿瘤微环境。尽管相关免疫修饰多发生于出现原发肿瘤降期的肿瘤中，但治疗应答似乎与特定的免疫特征相关。