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Page 6 of 13 Fang et al. Cancer Drug Resist. 2025;8:42

ROS1-Mut tumors exhibited significantly higher TMB compared to ROS1-WT tumors (P = 0.043) [Figure
3A]. This pattern was more pronounced in the TCGA HNC cohort [Figure 3B], where ROS1-Mut tumors
showed markedly elevated TMB (P <​ 0.001). Similarly, analysis of TCGA data [Figure 3C] demonstrated that
ROS1-Mut tumors were associated with higher neoantigen levels (P <​ 0.001), suggesting enhanced
immunogenicity in ROS1-mutated cases. Despite the presumed predictive value of high TMB for ICI
response, survival analysis demonstrated that ROS1-Mut cases with TMB-H had shorter OS compared to
ROS1-WT cases [Figure 3D]. These findings highlight a critical interaction between ROS1 mutations and
TMB, where mutational burden fails to mitigate the aggressive biology of ROS1-mutant tumors in the
context of ICI therapy.

ROS1 mutation is not a prognostic factor in HNC
To evaluate the prognostic value of ROS1 mutation in HNC, survival analyses were conducted using TCGA
data. No significant differences in progression-free survival (PFS) or OS were observed between ROS1-Mut
and ROS1-WT patients (log-rank P = 0.26 for OS; Supplementary Figure 1), indicating that ROS1 mutations
do not serve as a prognostic factor in untreated HNC.

ROS1-Mut tumors exhibit a cold immune microenvironment in HNC
To investigate the molecular mechanisms underlying ROS1-mediated resistance to ICI therapy in HNC,
RNA-seq and WES data from the TCGA-HNC cohort were analyzed. Assessment of immune cell
composition revealed minimal differences between ROS1-Mut and ROS1-WT tumors [Figure 4A]. Among
22 immune cell types evaluated, only activated dendritic cells exhibited significantly higher infiltration in
ROS1-Mut tumors (P <​ 0.01), whereas regulatory T cells (Tregs) were reduced in ROS1-Mut tumors (P <​
0.05). No other immune cell types, including CD8 T cells or NK cells, showed statistically significant
+
differences. Differential expression analysis showed broad downregulation of immune-related genes in
ROS1-Mut tumors. Notably, immune checkpoint genes (CTLA4, ICOS, CD274/PD-L1), tumor necrosis
factor receptor family member CD27, IFN-γ mediating signaling pathway genes (CXCL9, CXCL10, CXCL11,
GBP1, IFNG, STAT1), and antigen-processing genes (TAP1, TAP2) were significantly reduced in ROS1-Mut
tumors [Figure 4B]. These results suggest that ROS1 mutations in HNC are associated with an
immunosuppressive microenvironment characterized by downregulation of immune-related genes and
reduced Treg infiltration, which may contribute to resistance to ICI therapy.

Pathway analysis links ROS1 mutation to immune evasion mechanisms
GSEA revealed distinct pathway activation patterns between ROS1-Mut and ROS1-WT tumors, elucidating
potential mechanisms of ICI resistance. In ROS1-Mut tumors, genes associated with the MYC signaling
pathway were significantly upregulated [Figure 5A], mediating tumor proliferation, metabolic adaptation,
and immune evasion. In contrast, immune-activating pathways including antigen receptor-mediated
signaling, T cell receptor (TCR) signaling, and cell adhesion molecules were downregulated in ROS1-Mut
tumors [Figure 5B-D]. These findings collectively highlight that ROS1 mutations drive transcriptional
reprogramming toward an immunosuppressive phenotype.

Integrative multi-omics analyses further revealed that ROS1-mutant tumors exhibit hyperactivation of the
MYC pathway, which transcriptionally represses IFN-γ signature genes (e.g., IFNG, STAT1), T cell
chemokines (e.g., CXCL9, CXCL10), and components of the antigen presentation machinery (TAP1 and
TAP2), collectively fostering an immunosuppressive TME. Despite elevated TMB and neoantigen load, these
tumors show impaired CD8 T cell infiltration and reduced responsiveness to ICI therapy. Figure 6 provides
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a schematic overview of the proposed mechanistic cascade by which ROS1 mutations drive immune evasion
and ICI resistance in HNC.

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