{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T04:02:46Z","timestamp":1790308966674,"version":"4.1.0"},"reference-count":45,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2021,3,15]],"date-time":"2021-03-15T00:00:00Z","timestamp":1615766400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002551","name":"Norwegian Research Council","doi-asserted-by":"publisher","award":["22325"],"award-info":[{"award-number":["22325"]}],"id":[{"id":"10.13039\/501100002551","id-type":"DOI","asserted-by":"publisher"}]},{"name":"The Western Norway Regional Health Authority","award":["912260\/2019"],"award-info":[{"award-number":["912260\/2019"]}]},{"name":"The Norwegian Centre for International Cooperation in Education","award":["CPEA-LT-2016\/10106"],"award-info":[{"award-number":["CPEA-LT-2016\/10106"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/cancers13061307","type":"journal-article","created":{"date-parts":[[2021,3,15]],"date-time":"2021-03-15T11:38:58Z","timestamp":1615808338000},"page":"1307","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Combined In Situ Hybridization and Immunohistochemistry on Archival Tissues Reveals Stromal microRNA-204 as Prognostic Biomarker for Oral Squamous Cell Carcinoma"],"prefix":"10.3390","volume":"13","author":[{"given":"Saroj","family":"Rajthala","sequence":"first","affiliation":[{"name":"Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Harsh","family":"Dongre","sequence":"additional","affiliation":[{"name":"Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7185-0844","authenticated-orcid":false,"given":"Himalaya","family":"Parajuli","sequence":"additional","affiliation":[{"name":"Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anjie","family":"Min","sequence":"additional","affiliation":[{"name":"Department of Oral Maxillofacial Surgery, Xiangya Hospital, Central South University, Changsha 410083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisabeth Sivy","family":"Nginamau","sequence":"additional","affiliation":[{"name":"Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Department of Pathology, Haukeland University Hospital, N-5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arild","family":"Kvalheim","sequence":"additional","affiliation":[{"name":"Oral Surgery Private Referral Practice \u201cTannteam\u201d, N-5221 Nesttun, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stein","family":"Lybak","sequence":"additional","affiliation":[{"name":"Head and Neck Clinic, Haukeland University Hospital, N-5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dipak","family":"Sapkota","sequence":"additional","affiliation":[{"name":"Institute of Oral Biology, Faculty of Dentistry, University of Oslo, N-0316 Oslo, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne Christine","family":"Johannessen","sequence":"additional","affiliation":[{"name":"Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Department of Pathology, Haukeland University Hospital, N-5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7673-0358","authenticated-orcid":false,"given":"Daniela Elena","family":"Costea","sequence":"additional","affiliation":[{"name":"Gade Laboratory for Pathology, Department of Clinical Medicine, Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Centre for Cancer Biomarkers (CCBIO), Faculty of Medicine, University of Bergen, N-5020 Bergen, Norway"},{"name":"Department of Pathology, Haukeland University Hospital, N-5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1038\/nrc.2016.73","article-title":"The biology and function of fibroblasts in cancer","volume":"16","author":"Kalluri","year":"2016","journal-title":"Nat. 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