Prognostic relevance of secondary actionable fusions in MLH1-deficient dMMR/MSI-H gastrointestinal tumors treated with pembrolizumab

Background Immune checkpoint inhibitors (ICIs) are standard treatment for mismatch repair-deficient (dMMR) or microsatellite instability-high (MSI-H) gastrointestinal tumors but resistance remains relevant. Secondary actionable fusions may be enriched in MutL homolog 1 (MLH1)-deficient colorectal cancer (CRC) with mitogen-activated protein kinase wild-type biology, whereas their prevalence in non-CRC tumors and impact on ICI outcomes remain poorly defined. We evaluated secondary fusions in MLH1-deficient dMMR/MSI-H tumors treated with pembrolizumab. Materials and methods This predefined molecular subanalysis of the GAstrointestinal INestable cohort included patients with advanced dMMR/MSI-H gastrointestinal tumors with MLH1 loss treated with pembrolizumab. Molecular profiling used an RNA sequencing-based panel covering relevant fusion targets, complemented by pan-tropomyosin receptor kinase (TRK) immunohistochemistry and RET proto-oncogene (RET) FISH. Results Twenty patients were included (9 CRC and 11 non-CRC tumors). Secondary actionable fusions were detected in 4/9 CRC (44.4%; three NTRK1 , one RET) and were not detected in non-CRC tumors. Among 19 assessable patients, objective response rate was 84.2% and disease control rate 89.5%. Median overall survival (OS) was not reached in fusion-negative CRC, 43.0 months in fusion-positive CRC, and 20.2 months in non-CRC. Within CRC, OS differed by neurotrophic receptor tyrosine kinase 1 fusion status (log-rank P = 0.034), although limited by sample size. Conclusions Secondary actionable fusions may identify MLH1-deficient dMMR/MSI-H CRC with shorter long-term survival despite pembrolizumab responses. These exploratory findings support molecular profiling and warrant validation.