Clinical efficacy and immune response of neoadjuvant camrelizumab plus chemotherapy in resectable locally advanced oesophageal squamous cell carcinoma: a phase 2 trial

Abstract

Background: Neoadjuvant immunotherapy is under intensive investigation for esophageal squamous cell carcinoma (ESCC). This study assesses the efficacy and immune response of neoadjuvant immunochemotherapy (nICT) in ESCC.

Methods: In this phase II trial (ChiCTR2100045722), locally advanced ESCC patients receiving nICT were enrolled. The primary endpoint was the pathological complete response (pCR) rate. Multiplexed immunofluorescence, RNA-seq and TCR-seq were conducted to explore the immune response underlying nICT.

Results: Totally 42 patients were enrolled, achieving a 27.0% pCR rate. The 1-year, 2-year DFS and OS rates were 89.2%, 64.4% and 97.3%, 89.2%, respectively. RNA-seq analysis highlighted T-cell activation as the most significantly enriched pathway. The tumour immune microenvironment (TIME) was characterised by high CD4, CD8, Foxp3, and PD-L1 levels, associating with better pathological regression (TRS0/1). TIME was categorised into immune-infiltrating, immune-tolerant, and immune-desert types. Notably, the immune-infiltrating type and tertiary lymphoid structures correlated with improved outcomes. In the context of nICT, TIM-3 negatively influenced treatment efficacy, while elevated TIGIT/PD-1 expression post-nICT correlated positively with CD8+ T cell levels. TCR-seq identified three TCR rearrangements, underscoring the specificity of T-cell responses.

Conclusions: Neoadjuvant camrelizumab plus chemotherapy is effective for locally advanced, resectable ESCC, eliciting profound immune response that closely associated with clinical outcomes.

Fig. 1. The study design.

a Flowchart for screening of eligible patients. TRS tumour regression grade. TRS0 indicates complete regression; TRS1 indicates near-complete regression; TRS2 indicates partial regression; TRS3 indicates negligible or no regression; b Trial schema. Eligible patients were treated with 2 cycle of neoadjuvant therapy of camrelizumab (200 mg, Day 1) plus nab-paclitaxel (260 mg/m², Day 1) and carboplatin (AUC = 5 mg/mL/min, Day 1), followed by surgical resection. Radiological assessment was performed at the time of baseline, and 2 weeks after 2 cycles therapy and before surgery. Tumour samples were collected at baseline and at the time of surgery. Peripheral blood samples were collected at baseline and 2 weeks after 2 cycles therapy; ESCC oesophageal squamous cell carcinoma, AUC area under the curve. c Treatment regimen in the neoadjuvant and adjuvant settings, and follow-up status per patient (n = 37); nICT neoadjuvant immunochemotherapy, ICT immunochemotherapy.

Fig. 2. Clinical efficacy.

a Waterfall plot of radiographic tumour regression (n = 37). Each bar represents one patient. The upper column shows clinical characteristics; TRS tumour regression grade, CR complete response, PR partial response, SD stable disease. b Disease-free survival for the surgical population (n = 37). c Overall survival for the surgical population (n = 37). mDFS median disease-free survival, mOS median overall survival.

Fig. 3. RNA-seq analysis of tumour specimens at baseline (n = 37).

a Differential gene expression between responders (CR/PR, n = 21) and non-responder group (SD, n = 16); The upregulated immune-related genes were marked. A cut-off of gene expression fold change of ≥1.5 and a false discovery rate (FDR) q < 0.05 was applied to select DEGs; b Typical immune-related genes in radiographic responders and non-responders; c Up-regulation of immune-related genes in patients with radiographic responders and non-responders; d The GO enrichment of RNA-seq showing the top 10 up-regulation immune-related genes in the section of biological process; e 22 types of infiltration immune cells in responders and non-responders. Responders were those having complete or partial response by RECIST 1.1, while non-responders having stable disease or disease. *p < 0.05, **p < 0.01, ***p < 0.001, asterisks (*) stand for significance levels. The statistical difference of two groups was compared through the Wilcox test.

Fig. 4. T cell infiltration and TIME types in the tumour tissue by mIF.

Expression of immune cell markers in TIME: CD4, Foxp3, CD8 and PD-L1 expression in ESCC tumour samples before (a, n = 19) and after (b, n = 31) nICT treatment; three TIME types based on the infiltration of immune cells in the tumour tissue before and after immunochemotherapy by mIF: immune infiltration (c, n = 14); immune excluded (d, n = 7); immune desert (e, n = 10)； f TIME type grouped by TRS (n = 31); g Disease-free survival in different TIME types (n = 31); h Overall survival in different TIME types (n = 31): mIF multiplexed immunofluorescence, TIME tumour immune microenvironment, TRS tumour regression stage. CD4 (green), Foxp3 (orange), CD8 (red), CD20 (rose-red) and PD-L1 (white).

Fig. 5. TLSs, TIM-3/TIGIT/PD-1 expression and TRS in tumour specimen before and after nICT.

a A presentative figure of TLSs in TIME in nICT group. TLSs tertiary lymphoid structures; CD4 (green), Foxp3 (orange), CD8 (red), CD20 (rose-red) and PD-L1 (white). b Expression of TIM-3/TIGIT/PD-1 before and after nICT treatment in a good (TRS0) and a poor (TRS3) pathological responder; compared to those with stable TIGIT/PD-1 expression (n = 9) after nICT treatment, patients with significant elevated TIGIT/PD-1 expression (n = 8) had higher expression of CD8 in tumour specimen (****p < 0.001); increased expression of TIM-3 was associated with poor efficacy before (c, n = 20) or after (d, n = 19) nICT treatment; e After nICT treatment, patients with significantly elevated TIGIT/PD-1 expression (Group A, n = 8) had higher CD8 expression in tumour specimens compared to who showed no significant change in TIGIT/PD-1 expression (Group B, n = 9); nICT neoadjuvant immunochemotherapy, TIME tumour immune microenvironment.

Fig. 6. TCR-seq analysis of 19 paired peripheral blood samples before and after nICT treatment.

a Differential expression analysis of TCR clonality before and after nICT treatment; b Differential expression analysis of TCR clonality between pathological responders (TRS0/1) and non-responders (TRS2/3). c The frequency of these shared TCRs was increased after treatment (n = 38); d Higher frequency of three shared TCRs in patients with pathological response (TRS0/1) (n = 19); sequence motif analysis of high-frequency groups of TCRs Local alignment was applied to calculate the similarity of the base (left) and amino acid (middle) of Vβ12-3Dβ1Jβ1-1 (e), Vβ12-3Dβ1Jβ2-7 (f) and Vβ12-3Dβ2Jβ2-5 (g). The question marks indicated that the sequence at that position is not conservative. The percentage of top 5 non-conserved amino acid sequences (right) in patients with high frequency of TCRs was shown.