Spatial and Transcriptomic Analysis of Perineural Invasion in Oral Cancer

Purpose: Perineural invasion (PNI), a common occurrence in oral squamous cell carcinomas, is associated with poor survival. Consequently, these tumors are treated aggressively. However, diagnostic criteria of PNI vary and its role as an independent predictor of prognosis has not been established. To address these knowledge gaps, we investigated spatial and transcriptomic profiles of PNI-positive and PNI-negative nerves. Experimental Design: Tissue sections from 142 patients were stained with S100 and cytokeratin antibodies. Nerves were identi-fied in two distinct areas: tumor bulk and margin. Nerve diameter and nerve-to-tumor distance were assessed; survival analyses were performed. Spatial transcriptomic analysis of nerves at varying distances from tumor was performed with NanoString GeoMx Digital Spatial Profiler Transcriptomic Atlas. Results: PNI is an independent predictor of poor prognosis among patients with metastasis-free lymph nodes. Patients with close nerve-tumor distance have poor outcomes even if diag-nosed as PNI negative using current criteria. Patients with large nerve(s) in the tumor bulk survive poorly, suggesting that even PNI-negative nerves facilitate tumor progression. Diagnostic criteria were supported by spatial transcriptomic analyses of > 18,000 genes; nerves in proximity to cancer exhibit stress and growth response changes that diminish with increasing nerve-tumor distance. These findings were validated in vitro and in human tissue. Conclusions: This is the first study in human cancer with high-throughput gene expression analysis in nerves with striking corre-lations between transcriptomic profile and clinical outcomes. Our work illuminates nerve-cancer interactions suggesting that cancer-induced injury modulates neuritogenesis, and supports reclassifi- cation of PNI based on nerve-tumor distance rather than current subjective criteria.

Automated summary

This study investigates the spatial and transcriptomic profiles of nerves in relation to perineural invasion (PNI) in oral squamous cell carcinomas. The findings show that PNI is an independent predictor of poor prognosis and that the distance between nerves and tumor plays a role in patient outcomes. Transcriptomic analysis reveals changes in stress and growth responses in nerves close to cancer. These findings highlight the importance of nerve-cancer interactions and suggest a need to reclassify PNI based on the distance between nerves and tumors.