Spatial and Transcriptomic Analysis of Perineural Invasion in Oral Cancer

Abstract

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 identified 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 diagnosed 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 correlations between transcriptomic profile and clinical outcomes. Our work illuminates nerve-cancer interactions suggesting that cancer-induced injury modulates neuritogenesis, and supports re-classification of PNI based on nerve-tumor distance rather than current subjective criteria.

The 3D's of Neural Phenotypes in Oral Cancer: Distance, Diameter, and Density

“…Using current criteria, detection of perineural invasion in SCC is enhanced by immunohistochemical staining with S100 or TUJ1, and cytokeratin to highlight nerves and tumor cells, respectively. [61][62][63][64] In a study on biopsy tissue from 142 patients with OSCC, detection of perineural invasion was significantly increased from 26.1% on routine hematoxylin and eosin (H&E) stained sections to 43% when H&E was used in conjunction with immunohistochemistry. [61,62] Together these studies support the use of immunohistochemistry to enhance detection of perineural invasion in OSCC.…”

“…[61][62][63][64] In a study on biopsy tissue from 142 patients with OSCC, detection of perineural invasion was significantly increased from 26.1% on routine hematoxylin and eosin (H&E) stained sections to 43% when H&E was used in conjunction with immunohistochemistry. [61,62] Together these studies support the use of immunohistochemistry to enhance detection of perineural invasion in OSCC.…”

“…These reports may not include perineural invasion in the microscopic description of the tumor; consequently, studies that use reports alone would underestimate this neural phenotype. [16,61,62] Moreover, diagnostic criteria for perineural invasion have evolved thereby impacting conclusions in studies that relied solely on archival pathology reports. Standardization of reporting criteria in pathology reports or evaluation of tissue specimens rather than review of reports could provide more accurate information for this important phenotype.…”

“…[60,73] Perineural invasion is an independent risk factor for poor disease-specific survival. [60][61][62] Studying biopsy tissue from 142 patients, Schmitd et al, [62] reported that OSCC with perineural invasion and negative lymph nodes (PNI+, N0), was associated with significantly poorer disease-specific survival, disease-free survival, and overall survival than in the absence of perineural invasion (PNI-, N0). Despite receiving adjuvant radiation, the PNI+, N0 group had a significantly higher hazard ratio than the PNI-, N0 group.…”

“…[74] However, two studies, including a large recent study showed no relationship between the number of nerves exhibiting perineural invasion and survival. [60,62] Even in patients with early-stage OSCC (Stages I and II), no survival difference was observed between patients with less than 5 or more than 5 nerves exhibiting perineural invasion. [62] This could be due to differences in treatment because early-stage lesions with multifocal perineural invasion are more likely to receive adjuvant therapy in addition to surgery, than early lesions with unifocal nerve involvement.…”