Amiti Jain

Figure s6. Post-treatment changes in immune cell composition across spatial tumor compartments in responders and nonresponders

Figure s11. Carcinoma ecotype differences at baseline and after treatment in HPV+ HNSCC responders and non-responders. This figure illustrates differences in carcinoma ecotype composition before and after treatment in HPV+ HNSCC patients

Purpose:Immune checkpoint inhibitors (ICI) have demonstrated clinical benefit in head and neck squamous cell carcinoma (HNSCC); however, single-agent efficacy is limited, leaving significant unmet needs. Metformin may synergize with ICIs, offering promise to improve response rates. We leveraged multiomic data from a randomized, presurgical neoadjuvant trial (NCT03618654) evaluating a single infusion of the anti–PD-L1 ICI durvalumab with or without daily, standard dose metformin in previously untreated, nondiabetic patients with HNSCC to understand predictors of response and the effect of combination therapy.Patients and Methods:Clinical, pathologic, and correlative data were analyzed to investigate response and resistance mechanisms. We present an in-depth multiomic analysis of primary tumor specimens to study treatment response/resistance in human papillomavirus–positive HNSCC.Results:Baseline samples revealed that myofibroblastic cancer-associated fibroblast and extracellular matrix signatures were enriched in durvalumab plus metformin nonresponders, which were localized to the leading tumor edge on spatial transcriptomics. In contrast, baseline responder samples were enriched for the Langerhans-like dendritic cell (DC) state and IFN signatures. Treatment increased intratumoral CD8+ T-cell and IFN signatures and peripheral blood CCL2 levels. Responders demonstrated macrophage and DC enrichment and antigen processing and presentation upregulation. Enrichment of cell cycle–related gene sets, specifically the MYC targets V1 hallmark gene set, correlated with nonresponse.Conclusions:Early response and resistance dynamics for durvalumab plus metformin in human papillomavirus–positive HNSCC reveal baseline extracellular matrix–myofibroblastic cancer-associated fibroblast as predictive of nonresponse. In contrast, responders were distinguished by baseline enrichment in the Langerhans-like DC state and posttreatment antigen-presenting gene sets.

Figure s8: Identification and Functional Analysis of Unique Mutations in responders and non-Responders in HPV+ DM Using Whole-Exome Sequencing (WES)

Figure s2. CONSORT diagram. CONSORT diagram illustrating patient enrollment, allocation, treatment, follow-up, and analysis.

Figure s9: Integrated transcriptomic and pathway analyses reveal stress response, metabolic, and EMT-related differences in PanCK-enriched and treatment response subgroups

Figure s10. Hallmark pathway enrichment, cell cycle gene expression, and functional network analysis of differentially expressed genes in responders and nonresponders.

Figure s4. Gene set enrichment analysis (GSEA) across baseline responder vs. nonresponder comparisons and baseline vs. post-treatment analyses in the durvalumab-metformin clinical trial and other clinical trial cohorts. GSEA was performed to assess differences between baseline responders and nonresponders, as well as baseline vs. post-treatment samples