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Introduction and Objective: Alterations in methylation of cytosine residues at the 5 position in CpG dinucleotides (5mC) are a hallmark of cancer. While specific genes can be silenced by acquiring somatic 5mC in their promoter regions, global hypomethylation of the remainder of the genome has been observed in many somatic malignancies. Studies on global 5mC (GDM) status in germ cell tumors (GCT) are lacking. We evaluated GDM status and DNA methyl transferase 1 (DMT) expression in seminomatous (Sm-GCT) and non-seminomatous (NS-GCT) germ cell tumors.

Methods: A total of 37 GCT including 16 Sm-GCT, 19 NS-GCT and 2 mixed GCTx were examined. Paraffin sections were immunostained using monoclonal antibodies for 5mC and DMT. Distribution and intensity of nuclear staining were assessed for each marker as a percentage of cells staining in four ( 0, 1+,2+ and 3+) intensity categories. 5mC and DMT scores ( intensity x percentage) were calculated for each tumor (score range: 0-300). Tumors with a 5mC score 150 were considered of low GDM status. The same cut-off was used for low DMT expression status. 5mC DMT staining intensity was also assessed in associated intratubular germ cell neoplasm (IGCNU).

Results: 5mC: The median 5mC score for the entire GCT cohort was 290. 5mC content was markedly lower in Sm-GCT (median 120) compared to NS-GCT (median 300). 14/18 (78%) Sm-GCT had 5mC score that was lower than cohort median and 11/18 (61%) tumors were of low GDM status. This compares to 2/21 (9.5%) of NS-GCT with 5mC score less than median and 1/21 (5%) with low GDM status; p=0.000. Low GDM status was also encountered in the Sm-GCT area of one of the two mixed GCT but not in corresponding NS-GCT component. 5mC staining was negative or weak intensity in 11/12 IGCNU studied.

DMT: No significant difference in DMT expression was detected between Sm-GCT and NS-GCT. DMT score was low in 4/18 Sm-GCT (range: 100-300) and in 5/21 NS-GCT (range: 40-300); p=0.8. DMT staining was of moderate (2+) to marked (3+) intensity in 8 of the 12 studied IGCNU. No statistically significant correlation was present between low GDM and low DMT expression (p=0.5).

Conclusions: Seminonas have markedly lower levels of global genomic cytosine methylation than non-seminomatous GCT. The difference in methylation levels could not be ascribed to differences in DMT expression; thus lack of maintenance methylation is unlikely to explain the low 5mC in Sm-GCT.. Additional studies to explore potential diagnostic or prognostic utility of our findings are warranted.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA