Research Article In Search of TGCT Biomarkers: A Comprehensive In Silico and Histopathological Analysis Dora Raos , 1,2 Jure Krasic , 1,2,3 Silvija Masic , 4 Irena Abramovic , 1,2 Marijana Coric , 3,5 Bozo Kruslin , 2,3,4 Ana Katusic Bojanac , 1,3 Floriana Bulic-Jakus , 1,3 Davor Jezek , 3,6 Monika Ulamec , 2,3,4,7 and Nino Sincic 1,2,3 1 Department of Medical Biology, University of Zagreb School of Medicine, Šalata 3, 10000 Zagreb, Croatia 2 Scientific Group for Research on Epigenetic Biomarkers, University of Zagreb School of Medicine, Šalata 3, 10000 Zagreb, Croatia 3 Scientific Centre of Excellence for Reproductive and Regenerative Medicine, University of Zagreb School of Medicine, Šalata 3, 10000 Zagreb, Croatia 4 Ljudevit Jurak Clinical Department of Pathology and Cytology, Sestre Milosrdnice University Hospital Center, Vinogradska Cesta 29, 10000 Zagreb, Croatia 5 Department of Pathology and Cytology, University Hospital Centre Zagreb, Kišpatićeva Ulica 12, 10000 Zagreb, Croatia 6 Department of Histology and Embryology, University of Zagreb School of Medicine, Šalata 3, 10000 Zagreb, Croatia 7 Department of Pathology, University of Zagreb School of Dental Medicine and School of Medicine, Gundulićeva Ulica 5, 10000 Zagreb, Croatia Correspondence should be addressed to Nino Sincic; nino.sincic@mef.hr Received 21 April 2020; Revised 10 July 2020; Accepted 31 July 2020; Published 6 November 2020 Academic Editor: Kishore Chaudhry Copyright © 2020 Dora Raos et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Testicular germ cell tumors (TGCTs) are ever more affecting the young male population. Germ cell neoplasia in situ (GCNIS) is the origin of TGCTs, namely, seminomas (SE) and a heterogeneous group of nonseminomas (NS) comprising embryonal carcinoma, teratoma, yolk sac tumor, and choriocarcinoma. Response to the treatment and prognosis, especially of NS, depend on precise diagnosis with a necessity for discovery of new biomarkers. We aimed to perform comprehensive in silico analysis at the DNA, RNA, and protein levels of six prospective (HOXA9, MGMT, CFC1, PRSS21, RASSF1A, and MAGEC2) and six known TGCT biomarkers (OCT4, SOX17, SOX2, SALL4, NANOG, and KIT) and assess its congruence with histopathological analysis in all forms of TGCTs. Cancer Hallmarks Analytics Tool, the Search Tool for the Retrieval of Interacting Genes/Proteins database, and UALCAN, an interactive web resource for analyzing cancer OMICS data, were used. In 108 TGCT and 48 tumor-free testicular samples, the immunoreactivity score (IRS) was calculated. SE showed higher frequency in DNA alteration, while DNA methylation was significantly higher for all prospective biomarkers in NS. In GCNIS, we assessed the clinical positivity of RASSF1 and PRSS21 in 52% and 62% of samples, respectively, in contrast to low or nil positivity in healthy seminiferous tubules, TGTCs as a group, SE, NS, or all NS components. Although present in approximately 80% of healthy seminiferous tubules (HT) and GCNIS, HOXA9 was diagnostically positive in 64% of TGCTs, while it was positive in 82% of NS versus 29% of SE. Results at the DNA, mRNA, and protein levels on putative and already known biomarkers were included in the suggested panels that may prove to be important for better diagnostics of various forms of TGCTs. 1. Introduction Testicular germ cell tumors (TGCTs) represent the most common malignancy among males between 15-45 years of age in Caucasian populations [1]. Although they make up around 1% of all newly diagnosed neoplasms [2], according to GLOBOCAN 2018, TGCT incidence will increase by the year 2040 to a yearly incidence of 85,635 new cases world- wide [3]. Mortality is stable in most high-resource countries, with the curative rate of TGCT being above 95% due to advances in treatment strategies and surgical techniques [4]. Still, some populations like Croatian are experiencing a Hindawi Disease Markers Volume 2020, Article ID 8841880, 18 pages https://doi.org/10.1155/2020/8841880