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

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.

Profiling and Functional Analysis of microRNA Deregulation in Cancer-Associated Fibroblasts in Oral Squamous Cell Carcinoma Depicts an Anti-Invasive Role of microRNA-204 via Regulation of Their Motility

“…Reduced expression of miR-204 in cancers, likening or leading to aggressiveness and metastasis of cancer cells, was previously reported. We have previously investigated miR204 expression and its correlation with clinical-pathological parameters and survival in a cohort of 169 HPV-negative OSCC patient cohort [ 36 ]. Data from that cohort showed a correlation between the expression of miR-204 in tumor center and the degree of differentiation, namely higher miR-204 expression in well-differentiated OSCC lesions.…”

“…Lower expression of miR-204 has been observed in oral premalignant lesions [ 32 ] and OSCC tumors compared with normal tissues [ 33 , 34 ], and the anti-tumor effect of miR-204 has also been demonstrated in vitro in OSCC cells, but not in OSCC-derived CAFs [ 35 ]. In a recent study on a cohort of 169 patients with human papilloma virus (HPV)-negative primary OSCC, we showed that presence of miR-204 in the stroma at the tumor front predicted better overall survival and recurrence free survival [ 36 ]. Nevertheless, none of these previous studies investigated the dysregulation of miR-204 in CAFs and its impact on the behavior of OSCC cells.…”

Diagnostic and Prognostic Value of MicroRNAs in Metastasis and Recurrence of Head and Neck Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis

“…In this review, we systematically analyzed 1,093 HNSCC samples from 10 studies (22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and identified a panel of 21 microRNAs related to poor RFS in HNSCC patients for the first time. Besides, we investigated the diagnostic accuracy of microRNAs for (14).…”

Progression Risk Score Estimation Based on Immunostaining Data in Oral Cancer Using Unsupervised Hierarchical Clustering Analysis: A Retrospective Study in Taiwan

“…S h = P PLK1 − 2.46 + P PhosphoMet − 3.08 + P SGK2 − 3.06 + P SHC1 − 3.21 (4) According to Equation (2), the S l can be computed using Equation (5). The harmonic means of PLK1, PhosphoMet, SGK2, and SHC1 in the low-risk group were 1.76 (L PLK1 ), 2.14 (L PhosphoMet ), 2.38 (L SGK2 ), and 2.76 (L SHC1 ), respectively.…”

“…In oral cancer, well-known clinicopathologic factors, such as tumor size, stage, nodal status, the positivity of margin, lymphovascular invasion, perineural invasion, and extranodal extension, are widely regarded as having potential for risk stratification for further therapeutic strategies [3,4]. Some studies have evaluated chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC) biomarkers from cancerous tissues or databases, such as stromal microRNA-204 and RFC4, to assess the feasibility of prognostic prediction [5,6]. Recent studies have started to emphasize personalized biomarker-driven therapeutic strategies to guide treatments in refractory advanced cancer, including basket trials and umbrella trials [7,8].…”