Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors

Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5(m)C) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5(m)C erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5(m)C levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5(m)C to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5(m)C was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5(m)C in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5(m)C staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5(m)C erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.