172 Vladusic T et al. / Tumor suppressor genes in testicular germ cell tumors TABLE 2. A) Observed loss of heterozygosity (LOH) and B) distribution of observed LOH of CDKN2A and RB1 genes in testicular germ cell tumors Patient no. 20 25 31 34 Tumor Seminoma, Σ 18 Nonseminoma, Σ 22 A) observed LOH CDKN2A B) distribution of observed LOH LOH LOH CDKN2A 0% (0/16) 11% (2/18) RB1 LOH LOH NI I RB1 0% (0/15) 10.5% (2/19) I = informative (heterozygous); NI = not informative (homozygous) Numbers in parentheses: the number of tumors demonstrating LOH over the number of informative tumors. gene in only one of 29 TGCTs with a yolk sac tumor component, using seven different markers. These observations indicate that CDKN2A gene inactiva- tion might be an important mechanism leading to cell deregulation in TGCTs. Despite of promoter methylation and muta- tions being the most common ways of inactivat- ing CDKN2A in TGCTs, various studies detected LOH at the position of the CDKN2A gene, varying from as low as 5.5% to as high as 42%. The LOHs of CDKN2A were reported mostly in nonsemi- nomas.5,19 Genomic region containing CDKN2A (9p21) is reported to be the most commonly dele- ted region early in the development of nonsemino- mas, which may be implicated in their ability to di- fferentiate into various types, for various markers located within this region.20 In our study only nonseminomas demonstrated LOH (Table 2). Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, one sample also having an embryonal carcinoma component. Furthermore, one nonsemi- noma with the LOH of CDKN2A demonstrated LOH of TP53 gene, and the other showed LOH of the CDH1 gene.21 The RB1 gene is often deleted or mutated to an inactive form in a variety of human tumors. Cells of embryonal testes and intratubular germ cell neoplasia (ITGCN) show no expression of pRB, whereas it is expressed in healthy testes during spermatogenesis. The lack of pRB in most TGCTs may, therefore, reflect its deregulation by normal mechanisms in testicular germ cells. However, the lack of pRB may facilitate the transition of those cells to tumor cells of ITGCN and thus contribute to molecular pathogenesis of TGCTs.7,12 Lowered Radiol Oncol 2010; 44(3): 168-173. levels of pRB mRNA compared with normal tes- tis did not reflect a grossly altered structure of the DNA coding regions, but instead relates to a potentially reversible transcriptional modulation through the promoter methylation. The pRB ap- pears to be differentially expressed according to the differentiation status of the tumor, more dif- ferentiated cells of teratocarcinoma show positive immunohistochemical staining, less differentiated forms of TGCT such as embryonal carcinoma are stained negatively.12,22,23 In contrast, deletions of RB1 gene are, along with its mutations, also reported as one of the most com- mon alterations of the RB pathway. Various studies revealed deletions of the RB1 gene region in tes- ticular cancer.5 For example, Peng et al.24 used short variable number of tandem repeats in RB1 introns 16 and 20, and found LOH in 5% of seminomas and 28% of nonseminomas analyzed within 93% of informative TGCT cases. The location of the RB1 gene is reported to be one of the most common- ly involved in allelic imbalance within TGCTs.4 The exact alterations of the RB1 in various forms of TGCTs needs to be further elucidated in more detail. Studies also revealed a different pattern of LOH in different histological types of nonsemino- mas for markers located within the genomic region containing the RB1 gene (13q14), varying from 0% in yolk sac tumor component to 50% in choriocar- cinoma.25 In our study, LOH of the RB1 gene was found in nonseminomas with an embryonal carcinoma component, and both nonseminomas with LOH of RB1 also demonstrated LOH of the TP53 gene.21 Interestingly, the amount of embryonal carcinoma component in TGCT, along with vascular invasion, has been proved so far to be the only clinically val- id prognostic factor for the development of stage II metastatic testicular cancer.26 LOH of CDKN2A, RB1, TP53 and CDH1 in TGCTs may increase their tumorigenic potential by the increased proliferation capacity due to RB1 loss and decreased rate of apoptosis due to TP53 altera- tion.19,21,27 It has been shown that TP53 is abundant but inactive in cells of TGCTs. In healthy testes such reversibly inactivated TP53 may play a role in switching between proliferation and apoptosis in cells undergone meiosis.27 It was reported that, in cells that sustained lesion in the RB pathway, there was a strong selection for the loss or inactivation of wild type TP53. Alterations of RB1 are often seen together with alterations of TP53 in variety of dif- ferent cancers.6,10,15 It is possible that the inactiva- tion of both RB1 and TP53 genes in a cell produces