Deciphering the Worst Pattern of Invasion in Oral Cancer: Integrative Clinical, Pathological, and Multimodal...

Worst pattern of invasion-5 (WPOI-5) in oral cavity squamous cell carcinoma (OCSCC) is associated with aggressive disease behavior and worse prognosis. This study integrated clinical data analysis with multimodal nonlinear optical (MNLO) microscopy to investigate the prognostic implications and tumor microenvironment (TME) characteristics of WPOI-5 in OCSCC. Clinical data from 263 OCSCC patients were analyzed for overall survival and relapse-free survival (RFS). E-cadherin and N-cadherin expression levels were investigated in the epithelium at the tumor invasion front and within WPOI-5 regions. MNLO microscopy, combining second harmonic generation (SHG), third harmonic generation, and 2-photon fluorescence, was used to visualize and quantify structural changes in the TME associated with WPOI-5. Polarization-resolved SHG was used to investigate collagen cross-linking and remodeling. WPOI-5 was significantly associated with unfavorable overall survival and RFS in the univariate analysis and remained an independent predictor of poor RFS in multivariate analysis. Compared with the tumor invasion front, WPOI-5 exhibited reduced E-cadherin and enhanced N-cadherin expression, a hallmark of epithelial-to-mesenchymal transition, suggesting enhanced invasiveness and metastatic potential because of reduced intercellular adhesions. MNLO imaging revealed distinct TME structural organization between WPOI-5(+) and WPOI-5(-) samples, including differences in collagen fiber structure, orientation, and epithelial-to-mesenchymal transition-associated features at the tumor invasion front. Polarization-resolved SHG imaging further demonstrated increased collagen cross-linking and remodeling, as well as the perpendicular alignment of collagen fibers to the tumor boundary in WPOI-5(+) samples. This integrated approach provides preliminary evidence for the prognostic significance of WPOI-5 and offers proof-of-concept mechanistic insights into its role in OCSCC progression, offering a basis for future validation studies toward improved risk stratification and potential therapeutic targeting.