NOTCH1 Mutation and Survival Analysis of Tislelizumab in Advanced or Metastatic Esophageal Squamous Cell Carcinoma: A Biomarker Analysis From the Randomized, Phase III, RATIONALE-302 Trial

Abstract

Purpose: Although multiple agents targeting PD-1 have been approved as second-line treatment for esophageal squamous cell carcinoma (ESCC), only a fraction of patients derive long-term survival. Hence, reliable predictive biomarkers are urgently needed.

Methods: Comprehensive tumor genomic profiling and transcriptome sequencing were performed on samples from the RATIONALE-302 study. We also conducted single-cell RNA sequencing analysis on Notch1 knockdown ESCC murine models to further explore the potential molecular mechanisms underlying anti-PD-1 benefit.

Results: We identified NOTCH1 mutation as a potential predictive biomarker for longer overall survival (OS) with tislelizumab versus chemotherapy (18.4 months v 5.3 months; hazard ratio, 0.35 [95% CI, 0.17 to 0.71]). At the transcriptional level, type I IFN (IFN-I)/toll-like receptor expression signatures were positively associated with OS benefit of tislelizumab, whereas B-cell and neutrophil signatures predicted unfavorable OS. Exploratory analyses showed that the presence of NOTCH1 mutation correlated with enrichment of IFN-I signatures and reduced infiltration of B cells and neutrophils. In murine models, comparative single-cell transcriptome analyses further revealed that Notch1 deficiency facilitated a more immunologically activated tumor microenvironment which potentiated anti-PD-1 treatment.

Conclusion: Our data provide novel insights for anti-PD-1 treatment selection using NOTCH1 mutations and may provide a rationale for combination therapy in ESCC.

Trial registration: ClinicalTrials.gov NCT03430843.

FIG 1.

Study design. (A) Flowchart showing patient enrollment and sample collection workflow of the RATIONALE-302 study. Tumor tissue samples obtained from patients in the RATIONALE-302 study were tested sequentially for PD-L1 IHC, GEP, and mutation profiling. Consequently, many samples were exhausted after the first two assays, with only 48% of patients having sufficient tissue samples remaining for mutation profiling. (B) Schematic overview of the analysis workflow. GEP, gene expression profiling; ICC, investigator-chosen chemotherapy; IHC, immunohistochemistry; Tisle, tislelizumab; UMAP, uniform manifold approximation and projection.

FIG 2.

Genomic alterations associated with patient outcomes for Tisle. (A) Oncoplot depicting genomic alterations in 209 patients with ESCC in the RATIONALE-302 cohort. (B) Forest plot depicting OS improvements for Tisle versus ICC in patients with and without mutations altered in >10% of patients and patients with TMB ≥10 mut/Mb or TMB <10 mut/Mb. (C) Kaplan-Meier curve depicting OS in patients with ESCC treated with Tisle or ICC stratified by the status of NOTCH1 (mutated v WT). (D) Kaplan-Meier curve depicting OS in patients with mixed solid tumors treated with anti–PD-(L)1 monotherapy stratified by the status of NOTCH1 mutation from the MSKCC data set. Patients with annotated GOF NOTCH1 mutations and indications where NOTCH1 was reported mostly as the oncogenic activator or GOF mutations were excluded from the analysis. (E) Forest plot depicting OS in patients with ESCC treated with Tisle or ICC stratified by the combination of PD-L1 TAP score (cutoffs 1%, 5%, and 10%) and NOTCH1 mutation status. (F) Kaplan-Meier curves depicting OS in patients with ESCC treated with Tisle or ICC stratified by the combination of PD-L1 status and NOTCH1 mutation. Either positive: NOTCH1 mutant or PD-L1 TAP ≥10%; both negative: NOTCH1 WT and PD-L1 TAP<10%. ESCC, esophageal squamous cell carcinoma; GOF, gain-of-function; HR, hazard ratio; ICC, investigator-chosen chemotherapy; mOS, median OS; MSKCC, Memorial Sloan Kettering Cancer Center; MUT, mutation; NA, not applicable; NR, not reached; OS, overall survival; TAP, tumor area positivity; Tisle, tislelizumab; TMB, tumor mutational burden; WT, wild-type.

FIG 3.

Relationship between key transcriptomic markers and clinical outcomes for Tisle. (A) Heatmaps showing the expression level of representative signatures in 346 patients with ESCC in the RATIONALE-302 cohort and the association of indicated signatures with OS in each treatment arm. Color in the OS association heatmap indicates the hazard risk between high versus low within each arm, which was calculated as –log10 (P value) × (HR – 1)/|HR – 1|. Red indicates negative association; blue indicates positive association. The color of the signature name indicates the prognostic/predictive role: purple, good prognostic; green, good predictive; orange, poor prognostic; magenta, poor predictive; black, no association. (B-D) Kaplan-Meier curves depicting OS in patients with ESCC treated with Tisle or ICC stratified by the median value of indicated signatures: IFN-I, B cell, and neutrophil. *P < .1; **P < .05. DC, dendritic cell; EMT, epithelial to mesenchymal transition; ESCC, esophageal squamous cell carcinoma; HR, hazard ratio; ICC, investigator-chosen chemotherapy; IFN-I, type I IFN; mOS, median OS; NK, natural killer; OS, overall survival; Tisle, tislelizumab; TLR, toll-like receptor.

FIG 4.

TME features of NOTCH1 mutation in ESCC. (A) Volcano plot showing differentially expressed genes between NOTCH1 WT and NOTCH1-mutant subgroups. (B) Depiction of top signatures significantly enriched in NOTCH1 WT versus NOTCH1-mutant subgroups. (C) Representative images and quantification of CD66b+ neutrophils and CD20⁺ B cells in NOTCH1 WT and NOTCH1-mutant tumor tissues from an independent ESCC cohort. (D) Gene set enrichment analysis results of NOTCH1-mutant versus WT from the TCGA-SC cohort (ESCC and head and neck squamous cell carcinoma). *P < .01 by t-test. ESCC, esophageal squamous cell carcinoma; FDR, false discovery rate; GEP, gene expression profiling; MUT, mutation; NES, normalized enrichment score; SC, squamous cancer; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment; WT, wild-type.

FIG 5.

Notch1 knockdown sensitized murine ESCC tumors to anti–PD-1 by reprogramming the TME. (A) HNM007 tumor volume over time after initial treatment. Treatment of HNM007 tumors was performed with anti–PD-1 therapy (BioXCell, Cat number BE0146) or saline (control). Tumor volume was monitored for ≥10 days after treatment in tumors transfected with vehicle expressing sh-Ctrl and sh-Notch1 (n = 6 mice/group, data shown as mean ± SEM). One-way analysis of variance with multiple comparisons was used for statistical analysis. (B) Proportion of different clusters in ESCC subcutaneous tumors from WT Notch1 or Notch1 knockdown mice. (C) GSEA analysis of enriched pathways in CD8 T cells from tumors with WT Notch1 or Notch1 knockdown. (D) GSEA analysis of enriched pathways in macrophages from tumors with WT Notch1 or Notch1 knockdown. (E) Dot plot showing differentially expressed genes in macrophages from tumors with WT Notch1 or Notch1 knockdown. ESCC, esophageal squamous cell carcinoma; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MHC, major histocompatibility complex; NES, normalized enrichment score; Ro/e, ratio of observed to expected cell numbers; SEM, standard error of the mean; WT, wild-type.