Data Sheet 1_Tertiary lymphoid structures correlate with the therapeutic efficacy and prognosis of resectable esophageal squamous cell carcinoma undergoing neoadjuvant chemoradiotherapy plus immunotherapy.docx

BackgroundTertiary lymphoid structures (TLSs) are linked to prognosis in esophageal squamous cell carcinoma (ESCC), but whether the distribution, abundance, and maturity of TLSs affect therapeutic efficacy and prognosis in ESCC treated with neoadjuvant chemoradiotherapy plus immunotherapy (NRCI) remains unclear. We explored TLS characteristics and correlated them with patient survival.
MethodsA total of 157 resectable ESCC patients treated with neoadjuvant therapy between September 2020 and May 2023 were divided into NRCI (n=49) and neoadjuvant chemoimmunotherapy (NCI, n=108) groups. Multiplex immunofluorescence (mIHC) was employed to compare the spatial distribution and cellular composition of TLSs in the NRCI (n=40) and NCI (n=40) groups. A TLSs scoring system assessed TLSs abundance and maturity across intratumoral regions (T regions), invasive margins (IM regions), and peritumoral regions (P regions). The differences in overall survival (OS) and disease-free survival (DFS) between the two groups were analyzed. Furthermore, whole-exome sequencing (WES) on 20 untreated ESCC samples examined the relationship between TLS infiltration and genetic mutations.
ResultsThe OS and DFS in the NRCI group were significantly superior to the NCI group, with a higher rate of major pathological response (MPR). MPR patients exhibited significantly longer OS and DFS, suggesting that NRCI therapy substantially enhanced patient outcomes (all P<0.05). TLSs abundance exhibited varying immune effects in different tissue regions: intratumoral and invasive margin TLSs abundance was significantly associated with longer OS, while peritumoral TLSs abundance was linked to a shorter OS (all P<0.05). Highly mature TLSs (M-TLSs) were closely associated with a better OS (all P<0.05). In the NRCI group, M-TLSs showed higher proportions of CD20+Ki-67+ B cells, CD21+ dendritic cells (DCs), CD4+Ki-67+ helper T cells (Th), and CD8+Ki-67+ cytotoxic T cells compared to the NCI group (all P<0.05), indicating that NRCI therapy enhanced antitumor immune responses.
ConclusionNRCI therapy significantly enhanced the prognosis of resectable ESCCs compared to NCI therapy. The distribution and abundance of TLSs were clearly associated with OS in ESCCs and acted as independent prognostic indicators for OS in NRCI therapy. NRCI therapy extended OS and bolstered antitumor immune responses by facilitating the proliferation and activation of M-TLSs.

背景 三级淋巴结构（Tertiary lymphoid structures, TLSs）与食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）的预后存在关联，但TLSs的分布、丰度及成熟度是否影响接受新辅助放化疗联合免疫治疗（neoadjuvant chemoradiotherapy plus immunotherapy, NRCI）的食管鳞状细胞癌患者的治疗疗效与预后，目前尚不明确。本研究探讨了TLSs的特征，并将其与患者生存情况进行关联分析。
方法 本研究纳入2020年9月至2023年5月期间接受新辅助治疗的157例可切除食管鳞状细胞癌患者，分为NRCI组（n=49）与新辅助化学免疫治疗（neoadjuvant chemoimmunotherapy, NCI，n=108）组。采用多重免疫荧光（multiplex immunofluorescence, mIHC）技术对比NRCI组（n=40）与NCI组（n=40）患者TLSs的空间分布与细胞组成。通过TLSs评分系统评估肿瘤内区域（T区域）、侵袭边缘（IM区域）及瘤周区域（P区域）的TLSs丰度与成熟度。分析两组患者的总生存期（overall survival, OS）与无病生存期（disease-free survival, DFS）差异。此外，对20例未接受治疗的食管鳞状细胞癌样本进行全外显子测序（whole-exome sequencing, WES），以探究TLSs浸润与基因突变之间的关联。
结果 NRCI组患者的总生存期与无病生存期均显著优于NCI组，且主要病理缓解（major pathological response, MPR）率更高。获得主要病理缓解的患者总生存期与无病生存期显著更长，表明NRCI治疗可显著改善患者预后（所有P<0.05）。TLSs丰度在不同组织区域呈现不同的免疫效应：肿瘤内及侵袭边缘的TLSs丰度与更长的总生存期显著相关，而瘤周TLSs丰度则与更短的总生存期相关（所有P<0.05）。高成熟度TLSs（M-TLSs）与更好的总生存期密切相关（所有P<0.05）。在NRCI组中，与NCI组相比，M-TLSs中CD20+Ki-67+ B细胞、CD21+树突状细胞（dendritic cells, DCs）、CD4+Ki-67+辅助性T细胞（Th）及CD8+Ki-67+细胞毒性T细胞的比例更高（所有P<0.05），表明NRCI治疗可增强抗肿瘤免疫应答。
结论 与NCI治疗相比，NRCI治疗可显著改善可切除食管鳞状细胞癌患者的预后。TLSs的分布与丰度与食管鳞状细胞癌患者的总生存期显著相关，并可作为NRCI治疗中总生存期的独立预后指标。NRCI治疗通过促进高成熟度TLSs的增殖与活化，延长患者总生存期并增强抗肿瘤免疫应答。