Abstract 4939: Testicular Germ Cell Tumors: Could Aberrant Promoter Methylation differentiate Seminomas from Non-Seminomas
Authors: Mariana Brait, Leonel Maldonado, Shahnaz Begum, Myriam Loyo, Danielle Wehle, Fabio F. Tavora, Cynthia Lebron, Jeanne Kowalski, Zhe Zhang, George J. Netto, David Sidransky, Mohammad O. Hoque
Journal: Cancer Research (2010)

Abstract Methylation associated silencing inactivates known or putative tumor suppressor genes and represent one of the hits needed for cancer development. It remains important to identify cancer specific methylation in relatively less studied cancer like testicular cancer to understand the biology of this disease and develop new molecular markers. Testicular germ cell tumors (GCT) represent 95% of the testicular cancers and are histopathologically subdivided mainly as seminomas or non-seminomas. The molecular mechanisms behind these two subtypes that present morphological and clinical differences are yet to be established. Our aim was to study by candidate gene approach, methylation frequency of cancer related genes in different histological types of GCT and normal progenitor cells. We selected 15 genes (AIM1, ARF, CCNA1, MGMT, hMLH1, PGP9.5, S100A2, APC, RARβ, ERα, ERβ, MCAM, VGF, FKBP4, SSBP2) and analyzed them by quantitative fluorogenic real-time methylation specific PCR (QMSP) in 43 seminomas, 14 non seminomas, and 23 testicular normal tissues. The presence or absence of methylation was evaluated for an association with cancer using cross tabulations and Fisher's exact tests. Multivariate logistic regression was done comparing patients characteristics and gene methylation values. Observed methylation frequencies on the seminoma samples samples (total of 43) were: AIM1 9.3%, ARF 11.6%, CCNA1 7%, MCAM 51.2%, MGMT 2.3%, MLH1 27.9%, PGP9.5 44.2%, S100 11.6%, SSBP2 32.6%, APC 11.6%, RAR β 2.3%, VGF 16.3%, ERα 32.6%, ERβ 34.9% and FKBP4 4.7%. On the normal testicular tissues from archived autopsy samples (total of 23), the frequencies observed were AIM1 26.1%, ARF 21.7%, CCNA1 0%, MCAM 34.8%, MGMT 0%, MLH1 4.3%, PGP9.5 8.7%, S100 60.9%, SSBP2 73.9%, APC 43.5%, RARβ and VGF 0%, ERα 87%, ERβ 34.8% and FKBP4 0%. Considering APC and MCAM, 76.7% of the seminomas showed methylation, whereas only 21.7% of the normal tissues (p= 0.00002). Multivariate logistic regression was performed and these 2 genes remained statistically significant. On the non seminomas (total of 24): AIM1 21.4%, ARF 7.1%, CCNA1 14.3%, MCAM 100%, MGMT 50%, MLH1 71.4%, PGP9.5 14.3%, S100 64.2%, SSBP2 71.4%, APC 78.6%, RAR β 21.4%, VGF 50%, ERα and ERβ 64.3%, and FKBP4 35.7%. Considering APC, MLH1 and ERβ, 85.7% of the non-seminomas showed methylation, whereas only 17.4% of the normal tissues (p= 0.00008). APC is the only gene included in both combinations of genes, but we can still notice a higher frequency of methylation in non-seminomas. Our results indicate that the methylation profile of cancer associated genes in GCT correlates with histological types and also show cancer specific pattern. These epigenetic alterations have potential of being molecular markers for this cancer type and also distinguish between the two main subtypes of GCT. Further analyses in a larger testicular GCT cohort are needed to elucidate their role in GCT development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4939.