Proof-of-Concept Study: Hyperspectral Imaging for Quantification of DKK-3 Expression in Oropharyngeal Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common tumours worldwide. This study investigated the suitability of visible and near-infrared hyperspectral imaging compared to visual assessment and conventional digital image analysis for quantifying immunohistochemical staining on the example of Dickkopf-3 (DKK-3) in OSCC. A retrospective analysis of TMAs containing DKK-3 stained OSCC of 50 patients was retrieved from the archives at the Institute of Pathology, Medical University of Innsbruck. TMAs were first evaluated visually, followed by digital image analysis using QuPath (version 0.3.2, open-source software). For hyperspectral imaging, six exemplary cases were selected (three cases with strong expression and three cases with weak expression) and evaluated. The collected hyperspectral images were visualised using TIVITA (Tissue Imaging System). The resulting true-colour images and the classified HSI images were then assessed using the QuPath software. The Allred score and the H-score were used for all analyses. 97 tissue cores were used for visual and digital image analysis. No significant difference was found between the evaluations of visual and digital image analysis using the H-score (pWilcoxon = 0.278), and both H-scores correlated significantly with each other (pSpearman < 0.001). Similar results were also found using the Allred score. The kappa value was 0.67, which represents a "substantial" correlation. Finally, the H-scores and Allred scores were compared for visual, digital, and HSI imaging. No significant differences were found between the three groups concerning the H-score (pWilcoxon > 0.1). Using Cohen's Kappa, a "fair" to "moderate" correlation was observed between the three evaluations. Visible and near-infrared hyperspectral imaging (VIS-NIR-HSI) is a promising complementary tool for digital pathology workflows. This proof-of-concept study suggests that HSI offers the potential for more objective quantification of DKK-3 expression in oropharyngeal squamous cell carcinoma, particularly in cases with weak staining. However, given the small sample size and exploratory design, the findings should be regarded as hypothesis-generating. Future studies with larger, clinically annotated cohorts and standardised workflows are needed before any consideration of routine clinical application.