An integrative analysis of genomic and transcriptional data derived from samples in a large, randomised phase III RATIONALE-302 study suggest that patients with oesophageal squamous cell cancer with inactivating NOTCH1 mutations harbour immune-activated microenvironments with higher IFN-I gene signatures and lower immune-suppressive cell infiltration.
The findings provide conceptual evidence that NOTCH1 mutation or knockdown reshaped the tumour microenvironment (TME), suggesting that targeting the NOTCH pathway may be a relevant immunotherapeutic strategy for oesophageal squamous cell cancer according to Dr. Zhihao Lu of the Department of Gastrointestinal Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute in Beijing, China and colleagues, who published the results on 3 April 2025 in the JCO.
The authors wrote in the background that only a fraction of patients with oesophageal squamous cell cancer derive long-term survival from immune checkpoint inhibitors (ICIs). Therefore, there is an urgent need to identify reliable predictive biomarkers to guide patient selection, understand response variability and resistance mechanisms, and discover novel targets for ICIs in oesophageal squamous cell cancer.
Predictive value of PD-L1 expression in oesophageal squamous cell cancer is limited because of heterogeneous and dynamic expression patterns and patient benefit regardless of PD-L1 status. Tumour mutational burden (TMB) status represents another predictive biomarker for ICI response, but its predictive role for overall survival (OS) in oesophageal squamous cell cancer has been determined only by using appropriate algorithms and cut-offs. Other biomarkers have been proposed, but most have not been prospectively validated as predictive markers or are not clinically feasible to implement in routine care.
The RATIONALE-302 study demonstrated improved OS and safety from tislelizumab versus investigator-chosen chemotherapy as second-line treatment for patients with advanced unresectable or metastatic oesophageal squamous cell cancer. The study team performed post hoc biomarker analyses using next-generation sequencing to uncover genomic and transcriptomic markers potentially linked to clinical outcomes with tislelizumab. They also explored the molecular mechanisms underlying ICI survival benefits and potential synergetic targets for ICI treatment.
Besides performing comprehensive tumour genomic profiling and transcriptome sequencing on samples from the RATIONALE-302 study, the study investigators also conducted single-cell RNA sequencing analysis on Notch1 knockdown oesophageal squamous cell cancer murine models to further explore the potential molecular mechanisms underlying anti-PD1 benefit.
The study team identified NOTCH1 mutation as a potential predictive biomarker for longer OS with tislelizumab versus chemotherapy, 18.4 versus 5.3 months (hazard ratio 0.35, 95% confidence interval 0.17 to 0.71). At the transcriptional level, IFN-I/toll-like receptor expression signatures were positively associated with OS benefit of tislelizumab, whereas B-cell and neutrophil signatures predicted unfavourable 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 TME which potentiated anti-PD1 treatment.
The authors commented that integrative analysis showed a trend of survival benefit in patients with NOTCH1-mutated tumours regardless of PD-L1 or TMB status. Because of limited sample size, these findings from retrospective analysis of RATIONALE-302 study need validation in additional data sets. Because of lack of mutation hotspots, clonality classification, and annotations for the majority of samples, further efforts are required to determine the TME regulatory function and predictive role of individual mutations.
The association between NOTCH1 mutations and improved clinical outcomes requires prospective validation in a randomised, controlled trial. The study team plans to conduct a prospective clinical trial to assess whether ICI monotherapy is sufficient for patients with oesophageal squamous cell cancer with NOTCH1 mutations.
This work was supported by BeiGene.
Reference
Lu Z, Du W, Jiao X, et al. 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. JCO; Published online 3 April 2025. DOI: https://doi.org/10.1200/JCO-24-01818