Combined In Situ Hybridization and Immunohistochemistry on Archival Tissues Reveals Stromal microRNA-204 as Prognostic Biomarker for Oral Squamous Cell Carcinoma

SIMPLE SUMMARY: In addition to the transformation of epithelial cells, dysfunction of stroma is crucial in carcinogenesis; cancer-associated stroma can regulate the phenotype of cancer cells and thereby influence the clinical outcome. Our study aimed to investigate the correlation of stromal miR-204 with progression of oral squamous cell carcinoma (OSCC) and assert its clinical utility. We first established a chromogen-based method that combined immunohistochemistry and in situ hybridization for exact delimitation of stroma from the tumor islands and concomitant visualization of miRs, and have developed a guide to digital miR quantification using the publicly available tool ImageJ and the licensed software Aperio ImageScope. We have then applied the method for investigating stromal miR-204 as a putative prognostic biomarker on an OSCC cohort and identified expression of miR204 in the stroma at tumor front as an independent prognostic biomarker for this disease. ABSTRACT: Micro-RNAs (miRs) are emerging as important players in carcinogenesis. Their stromal expression has been less investigated in part due to lack of methods to accurately differentiate between tumor compartments. This study aimed to establish a robust method for dual visualization of miR and protein (pan-cytokeratin) by combining chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC), and to apply it to investigate stromal expression of miR204 as a putative prognostic biomarker in oral squamous cell carcinoma (OSCC). Four different combinations of methods were tested and ImageJ and Aperio ImageScope were used to quantify miR expression. All four dual ISH-IHC methods tested were comparable to single ISH in terms of positive pixel area percentage or integrated optical density of miRs staining. Based on technical simplicity, one of the methods was chosen for further investigation of miR204 on a cohort of human papilloma virus (HPV)-negative primary OSCC (n = 169). MiR204 stromal expression at tumor front predicted recurrence-free survival (p = 0.032) and overall survival (p = 0.036). Multivariate Cox regression further confirmed it as an independent prognostic biomarker in OSCC. This study provides a methodological platform for integrative biomarker studies based on simultaneous detection and quantification of miRs and/or protein and reveals stromal miR204 as a prognostic biomarker in OSCC.