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Page 2 of 13 Fang et al. Cancer Drug Resist. 2025;8:42

Results: Patients harboring ROS1 mutations exhibited significantly poorer outcomes following ICI therapy, with
shorter median overall survival [OS: 5.0 vs. 11.0 months, hazard ratio (HR) = 3.22, 95%CI: 1.26-8.19, P = 0.011]
compared to ROS1 wild-type counterparts. Multivariate analysis confirmed ROS1 mutation as an independent
predictor of poor OS in ICI-treated patients (HR = 4.78, 95%CI: 1.70-13.43, P = 0.003). In contrast, ROS1 mutations
showed no prognostic significance in the treatment-naïve TCGA-HNC cohort (P = 0.26), confirming their role as a
predictive (not prognostic) biomarker for ICI response. Interestingly, despite exhibiting higher TMB and neoantigen
levels, ROS1-mutant patients showed inferior survival, underscoring the context-dependent limitations of TMB as a
predictive biomarker. Mechanistically, ROS1-mutant tumors displayed an immunosuppressive tumor
microenvironment characterized by diminished CD8 T cell infiltration, attenuated interferon-γ signaling, and
+
downregulation of immune-related genes (CXCL9, CXCL10, IFNG, PD-L1). GSEA revealed enrichment of MYC pathway
activity in ROS1-mutant tumors, which suppressed antigen presentation and T cell activation pathways.

Conclusion: ROS1 mutations drive ICI resistance in HNC by promoting an immunosuppressive TME via
MYC-mediated transcriptional reprogramming, impairing antigen presentation and T cell function. Incorporating
ROS1 status into biomarker panels may improve patient stratification and guide combinatorial therapies targeting
both immune evasion and oncogenic pathways.

INTRODUCTION
Head and neck cancer (HNC), encompassing malignancies of the oral cavity, pharynx, and larynx, is the
sixth most common cancer globally, with approximately 890,000 new cases and 450,000 deaths reported in
2022 . Despite advances in multimodal therapies, the prognosis for advanced HNC remains poor, with a
[1]
5-year survival rate below 50% for recurrent or metastatic disease .
[1,2]

Treatment options for recurrent or metastatic HNC have evolved significantly over the past decade,
improving survival outcomes . The immune checkpoint inhibitors (ICIs) pembrolizumab and nivolumab
[3,4]
are FDA-approved for cisplatin-refractory recurrent or metastatic HNC. Current national and regional
guidelines recommend first-line therapy based primarily on programmed cell death ligand 1 (PD-L1)
expression levels. Options include pembrolizumab (with or without chemotherapy) or cetuximab-based
regimens (e.g., cetuximab combined with platinum/5-FU chemotherapy) [5-7] . For second-line treatment,
nivolumab or pembrolizumab is advised, with alternatives including cetuximab (with or without
chemotherapy) or biomarker-directed therapy. Despite these advances, sustained clinical benefits are
observed in only 10%-20% of patients, highlighting the urgent need for predictive biomarkers to optimize
patient stratification .
[8,9]

Recent studies have identified tumor mutational burden (TMB), microsatellite instability (MSI), and PD-L1
expression as key determinants of ICI response; however, their predictive power in HNC remains
suboptimal. For instance, PD-L1 positivity is associated with improved ICI response in HNC, yet
approximately 80% of PD-L1-positive patients fail to achieve objective responses , highlighting the
[7]
complexity of tumor-immune interactions . Similarly, while high TMB correlates with enhanced
[10]
neoantigen presentation and ICI efficacy in melanoma and lung cancer [11,12] , its utility in HNC is limited by
molecular heterogeneity [5,8] . These observations emphasize the need for comprehensive molecular
characterization and immune profiling to integrate prognostic and predictive biomarkers into clinical
practice.

ROS1, a receptor tyrosine kinase, is implicated in the carcinogenesis of multiple cancers. ROS1 fusions are
established therapeutic targets in lung adenocarcinoma, with inhibitors such as entrectinib and taletrectinib

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