Proteomic characterization of esophageal squamous cell carcinoma response to immunotherapy reveals potential therapeutic strategy and predictive biomarkers

Abstract

Immunotherapy is the first-line therapy for esophageal squamous cell carcinoma (ESCC), yet many patients do not respond due to drug resistance and the lack of reliable predictive markers. We collected 73 ESCC patients (including discovery cohort and validation cohort) without immune thrombocytopenia and undergoing anti-PD1 immunotherapy. Proteomic and phosphoproteomic analysis of 73 ESCC treatment-naive samples by mass spectrometry-based label-free quantification were applied to explore the potential resistant and sensitive mechanisms, and identify predictive markers of ESCC immunotherapy. Comparative analysis found the pathways related to immune and mitochondrial functions were associated with ESCC immunotherapy sensitivity; while platelet activation bioprocess showed negative correlation with CD8+ T cells and related to ESCC immunotherapy non-sensitivity. Finally, we identified 10 ESCC immunotherapy predictive biomarkers with high accuracy (≥ 0.90) to predict the immunotherapeutic response, which was validated in the independent cohort.

Keywords: Anti-PD1 immunotherapy; Esophageal squamous cell carcinoma; Immunotherapy response prediction; Platelets activation; Predictive markers; Proteomics.

Fig. 1

The association of platelets and ESCC immunotherapy response. A Overview of the workflow of proteomic profiling of ESCC immunotherapy cohort. B Heatmap of different abundance of xCell score between S and NS groups. C The representative images of immunohistochemistry (IHC) staining of CD8A and GP1BA expression in S and NS groups. The scale bar indicates 20 μm. D Spearman correlation analysis between CD8+ T-cells xCell score and platelets xCell score. P value was from two-sided Spearman correlation test. E Detection of CD8A and GP1BA in ESCC tumor tissue by multi-color IHC staining. Representative data from ESCC patients were shown. The scale bar indicates 50 or 10 μm. F Kaplan–Meier plots showing significant association of blood platelets counts with overall survival (OS) (upper) and progression-free survival (PFS) (bottom) in the MSK-IMPACT ESCC immunotherapy cohort. G The heatmap displaying the differential expression of proteins and their phosphosites involved in platelet activation, aggregation pathway between the S and NS groups. H The heatmap showing the differential expression of proteins involved in platelet activation, aggregation pathway in the IMvigor210 metastatic urothelial carcinoma immunotherapy cohort between the S and NS groups. I The Spearman correlation between the proteins involved in platelet activation, aggregation pathway and CD8+ T cells xCell score. P value was from two-sided Spearman correlation test. J Correlation between FGA protein expression and immunotherapy objective response rate in the TCGA pan-cancer cohort. P value was calculated by two-sided Spearman correlation test. K The qualification of FGA stained by IHC in the representative samples in the S and NS groups. The scale bar indicates 20 μm. L Systematic diagram summarizing the impact of the mechanism underlying ESCC patients with platelet activation is associated with immunotherapy non-sensitivity

Fig. 2

The construction and validation of predictive model for immunotherapy response. A Diagram describing a construction and validation of the predictive model for sensitive (S) and non-sensitive (NS) groups. B The heatmap displaying the 10 signatures that discriminate S and NS for ESCC immunotherapy in the discovery cohort. C Classification error matrix using logistic regression classifier of 80% training set and 20% testing set in the discovery cohort based on the 10 signatures combination. The number of samples identified is noted in each box. D ROC curves showing the predictive effect of this model in the 80% training set and 20% testing set of the discovery cohort. E Classification error matrix and ROC curve showing high sensitivity and specificity of the 10 signatures in the independent ESCC immunotherapy validation cohort