免疫检查点抑制剂与化疗联合新辅助治疗已应用于食管鳞癌（ESCC）的治疗。然而，尤其是对于老年患者，需进一步探索最佳方案，并且免疫检查点抑制剂联合化疗对ESCC演变的影响机制尚未知晓。该单臂2期临床试验入选可手术切除的（II/III/IV期无转移）ESCC患者接受纳米蛋白质结合（nab）紫杉醇治疗2个周期，口服S-1治疗2周，术前联合静脉注射托珀利麦治疗2个周期。进行手术后联合辅助治疗。主要结果为严重病理反应（MPR）。次要结果包括病理学完全缓解（pCR），总有效率（ORR），疾病控制率（D CR），无病生存（DFS），总生存（OS），Stooler吞咽困难评分改善以及日常生活能力（dADL）程度。对全外显子测序、转录组测序、PD-L1的免疫组织化学检测（IHC）、多重免疫荧光（mIF）和92种蛋白质的近距离扩展清晰图技术（PEA）等进行新辅助治疗前后组织活检和血浆的检测。从2019年11月至2021年7月，共有60名患者入选。新辅助治疗后，55（98.21％）名患者实现R0切除，27名患者（49.09％）实现MPR，16名患者（29.09％）实现pCR。PR，SD和PD患者分别为37（61.67％），21（35.00％）和2（3.33％）。治疗后，总体分期、Stooler吞咽评分和dADL得分均显著降低。11名患者（18.3％）出现≥3级AE。与PD-L1低患者相比，PD-L1高患者具有更高的PR比率。治疗过程中，具有PR的患者的肿瘤突变负荷（TMB）和肿瘤新抗原负荷（TNB）显著降低。通过分析肿瘤内异质性，证明了肿瘤内不同克隆的演化。转录组分析表明，CD4+ T淋巴细胞的浸润与临床结果相关。治疗过程中，所有患者的CD8+ T细胞和CD4+ T细胞均增加，但疲劳细胞，nTreg和iTreg在非MPR患者中明显增加。蛋白质分析显示，IFN-γ，Gal.1和LAMP3的水平可以预测临床益处。此外，CD83，TNFRSF4，TNFSF14，VEGFR2，ADA，ARG1和HO-1的表达与严重的AE有关。更重要的是，将CD4+ T细胞与IFN-γ，Gal.1或LAMP3等血浆蛋白进行整合，可以进一步区分响应者和非响应者。在该研究中，联合使用托珀利麦，nab-紫杉醇和S-1的新辅助治疗毒性较小，并在可切除的ESCC患者中显示出有希望的抗肿瘤活性。综合分析了基因组，转录组，PD-L1表达和血清蛋白的变化并与临床结果相关，这为托珀利麦联合nab-紫杉醇和S-1在ESCC患者中的作用机制提供了实质性见解。该研究由中国第13个五年计划科学技术部重大项目[资助号：2018ZX09201013]资助。Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.

Immune checkpoint inhibitors combined with chemotherapy as a neoadjuvant therapy have been applied to the treatment of esophageal squamous cell carcinoma (ESCC). However, the optimal regimen needs to be further explored, particularly for older patients, and the mechanisms by which the immune checkpoint inhibitor combined with chemotherapy modulates the evolution of ESCC are unknown.In this single-arm phase 2 trial, patients with resectable (stage II/III/IV without metastasis) ESCC were enrolled and received nanoparticle albumin-bound (nab) paclitaxel for two cycles and oral S-1 for 2 weeks, combined with intravenous toripalimab for two cycles before surgery. Combination postoperative adjuvant therapy was administered. The primary outcome was the major pathological response (MPR). Secondary outcomes included pathological complete response (pCR), overall response rate (ORR), disease control rate (DCR), disease-free survival (DFS), overall survival (OS), improvement in Stooler's dysphagia score and degree of daily living ability (dADL). Biopsies and plasma pre- and post-neoadjuvant therapy were performed using whole-exome sequencing, transcriptome sequencing, immunohistochemistry (IHC) for PD-L1, multiplex immunofluorescence (mIF) and proximity extension assay technology (PEA) for 92 proteins.From November 2019 to July 2021, 60 patients were enrolled. After neoadjuvant therapy, R0 resection was achieved in 55 (98.21%) patients. MPR was identified in 27 patients (49.09%), and 16 patients (29.09%) achieved pCR. Patients with PR, SD and PD were 37 (61.67%), 21 (35.00%) and 2 (3.33%), respectively. The overall staging, Stooler dysphagia scores and dADL were significantly decreased after treatment. 11 patients (18.3%) experienced grade ≥3 AEs. Compared to PD-L1-Low patients, PD-L1-High patients had a significantly higher ratio of PR. During therapy, the tumor mutation burden (TMB) and tumor neoantigen burden (TNB) were significantly decreased in patients with PR. Differential clonal evolution within tumors was demonstrated by analysis of intratumoral heterogeneity. Transcriptome analyses revealed that the infiltration of CD4+ T lymphocytes at baseline was associated with clinical outcome. During therapy, CD8+ T cells and CD4+ T cells were increased in all patients; however, exhausted cells, nTregs and iTregs were significantly increased in patients with non-MPR. Protein analyses revealed that the levels of IFN-γ, Gal.1 and LAMP3 can predict the clinical benefit. In addition, the expression of CD83, TNFRSF4, TNFSF14, VEGFR2, ADA, ARG1, and HO-1 was associated with serious AEs. More importantly, the integration of CD4+ T cells with plasma protein of IFN-γ, Gal.1 or LAMP3 could further distinguish responders from non-responders.In this study, neoadjuvant therapy with toripalimab, nab-paclitaxel and S-1 was less toxic and showed promising antitumor activity in patients with resectable ESCC. Changes in the genome, transcriptome, PD-L1 expression and serum proteins were comprehensively analyzed and correlated with clinical outcomes, which provides insight into the mechanism of action of toripalimab combined with nab-paclitaxel and S-1 in patients with ESCC.This study was funded by Major projects of the ministry of science and technology of the 13th five-year plan of China [grant number: 2018ZX09201013].Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.