m⁶A RNA modification and its writer/reader VIRMA/YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance.

BACKGROUND:Covalent RNA modifications, such as N-6-methyladenosine (m⁶A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m⁶A dynamics depends on specific enzymes whose deregulation may also impact in tumorigenesis. Herein, we assessed the differential abundance of m⁶A, its writer VIRMA and its reader YTHDF3, in testicular germ cell tumors (TGCTs), looking for clinicopathological correlates. METHODS:In silico analysis of TCGA data disclosed altered expression of VIRMA (52%) and YTHDF3 (48%), prompting subsequent validation. Formalin-fixed paraffin-embedded tissues from 122 TGCTs (2005-2016) were selected. RNA extraction, cDNA synthesis and real-time qPCR (Taqman assays) for VIRMA and YTHDF3 were performed, as well as immunohistochemistry for VIRMA, YTHDF3 and m⁶A, for staining intensity assessment. Associations between categorical variables were assessed using Chi square and Fisher's exact test. Distribution of continuous variables between groups was compared using the nonparametric Mann-Whitney and Kruskal-Wallis tests. Biomarker performance was assessed through receiver operating characteristics (ROC) curve construction and a cut-off was established by Youden's index method. Statistical significance was set at p < 0.05. RESULTS:In our cohort, VIRMA and YTHDF3 mRNA expression levels differed among TGCT subtypes, with Seminomas (SEs) depicting higher levels than Non-Seminomatous tumors (NSTs) (p < 0.01 for both). A positive correlation was found between VIRMA and YTHDF3 expression levels. VIRMA discriminated SEs from NSTs with AUC = 0.85 (Sensitivity 77.3%, Specificity 81.1%, PPV 71.6%, NPV 85.3%, Accuracy 79.7%). Immunohistochemistry paralleled transcript findings, as patients with strong m⁶A immunostaining intensity depicted significantly higher VIRMA mRNA expression levels and stronger VIRMA immunoexpression intensity (p < 0.001 and p < 0.01, respectively). CONCLUSION:Abundance of m⁶A and expression of VIRMA/YTHDF3 were different among TGCT subtypes, with higher levels in SEs, suggesting a contribution to SE phenotype maintenance. VIRMA and YTHDF3 might cooperate in m⁶A establishment in TGCTs, and their transcript levels accurately discriminate between SEs and NSTs, constituting novel candidate biomarkers for patient management.

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