Identification of Metastasis-Associated Molecular Signatures and Candidate Therapeutic Targets in Head and Neck Squamous Cell Carcinoma Through Single-Cell Transcriptomic Reanalysis
Open Access DepositedHead and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with a five-year survival rate of approximately 50-60% for advanced disease 1,2. Lymph node metastasis is the dominant prognostic determinant, yet the molecular programmes governing metastatic progression remain incompletely characterised. This thesis presents a bioinformatics reanalysis of a published single-cell RNA sequencing dataset 3 comprising 29,344 cells from 23 patients, spanning primary tumours and lymph node metastases, to identify metastasis-associated molecular signatures and candidate therapeutic targets in HNSCC.Differential gene expression analysis across eight cell types identified 7,917 significant differentially expressed genes (DEGs). The malignant cell compartment yielded 1,911 significant DEGs, with metastatic cells characterised by upregulation of the immune checkpoint receptors LAG-3, TIM-3 (HAVCR2), and TIGIT, and an interferon-stimulated gene signature. Primary tumour malignant cells were characterised by upregulation of matrix metalloproteinases MMP1 and MMP2, the hypoxia marker CA9, HIF1A, and glycolytic enzymes LDHA, PKM, and HK2, with Gene Ontology enrichment confirming hypoxia response, ECM organisation, and proteolysis as the dominant primary tumour biological programmes. HPV stratification revealed that HPV-positive and HPV-negative metastatic malignant cells share only 6.5% of their upregulated metastatic gene signatures, representing a major finding of this thesis. HPV-positive metastatic cells displayed a multi-layered immune evasion programme (LAG-3, TIM-3, TIGIT, HLA-G), while HPV-negative metastatic cells showed a completely distinct signature lacking checkpoint gene upregulation 4,5. Within-patient paired analysis of four matched primary-metastatic patients validated LAG-3 upregulation across all three independent analytical approaches, establishing it as the most robustly supported candidate therapeutic target. The FDA approval of relatlimab plus nivolumab in melanoma 6 provides clinical proof-of-concept for LAG-3 blockade in solid tumours. PFKFB3, a master regulator of glycolytic flux, emerged as an additional metastasis-associated target identified specifically in the paired analysis. These findings identify LAG-3 as the primary candidate therapeutic target in metastatic HNSCC, supported by three levels of analytical evidence, and demonstrate that HPV status should be considered not only as a prognostic stratifier but as a determinant of therapeutic target selection in metastatic disease 4,7. The 6.5% signature overlap between HPV subtypes argues that HPV-positive and HPV-negative HNSCC metastasise through fundamentally distinct molecular mechanisms and should be treated as separate diseases in the context of metastasis-directed therapy.
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