Differential Activation of Cancer-Associated Fibroblasts in HPV-Associated Head and Neck Squamous Cell Carcinoma Patients Detected Using Spatial Transcriptomics

Abstract

<h3>Purpose/Objective(s)</h3> To spatially characterize cancer-associated fibroblast (CAFs) gene expression within the tumor microenvironment (TME) of human papilloma virus (HPV)+ and HPV- head and neck squamous cell carcinoma (HNSCC). We hypothesize that CAF gene expression may differ between HPV+ and HPV- disease and that CAFs may play a role in tumor response to immune checkpoint inhibition (ICI). <h3>Materials/Methods</h3> Spatial transcriptomic analysis of tumor adjacent stroma (TAS) and leading tumor edge (LTE) was used to analyzed CAFs in samples from 3 neoadjuvant ICI trials. Patients received either Nivolumab (with tadalafil or BMS986205) or durvalumab (with metformin) as primary therapy in a window of opportunity prior to surgery. Paired pre- and post-treatment samples from 7 patients (n= 4 HPV+, 3 HPV-) were analyzed. A novel CAF-signature gene set was developed based on established literature and used for transcriptomic analysis. A "CAF score" was used for comparison of normalized transcript counts to compare expression patterns based on HPV status and response to therapy. Transcriptional regulatory patterns were evaluated by using VennPlex. <h3>Results</h3> Unsupervised clustering of both the TAS and LTE illustrates distinct CAF-related expression patterns in HPV+ and HPV- pre-tx samples. HPV+ future responders exhibited increased activation of CAF related genes, pre-treatment. Post-tx samples did not distinctly cluster based on HPV status. VennPlex analysis of the ratio of post-/pre-treatment in the TAS compartment exhibited 11 contra-regulated genes: CD9, EPCAM, ITGB1, FAP, PDPN, CLU, TGRBR1, CD59, FGF7, SLC16A1, and IL-6. The most contra-regulated of which was CD9, with a 3.4-fold change in expression levels. In the TAS, The CAF score decreased in post-tx responders, though not significantly (p= 0.52). In contrast, nonresponders exhibited a significant increase in CAF score (p= 0.04). <h3>Conclusion</h3> HPV+ HNSCC has a distinct CAF gene expression profile in both the TAS and LTE. Nonresponders upregulated CAF gene transcription in the TAS indicating a possible role for CAFs in ICI treatment resistance warranting further study.