Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours

Abstract

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase-polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM.

Figure 1

Hierarchical and K-means clustering of genes expressed specifically in testicular SEM or N-SEM. Results from the microarray analysis were filtered to show genes whose expression would discriminate between the two overt tumour types. This list was then subjected to hierarchical and K-means clustering (K was set to 4 according to the number of groups filtered for) using Euclidian distance measures. The clustering was carried out using the Genesis software (Sturn et al, 2002). Results on the CIS samples are from Almstrup et al (2004).

Figure 2

Verification of the microarray data by RT–PCR. RT–PCR primers for selected genes were used on a panel of different testicular tissue samples and cell lines. Gene-specific primers were designed preferentially to span intron–exon boundaries. The genes were divided according to the microarray analysis into: (A) Genes preferentially overexpressed in SEMs and (B) genes preferentially overexpressed in N-SEM. At the lower part of the figure, expression of ACTB is shown as a control of the RT–PCR protocol. Sample abbreviations can be found in Table 1, except for: M=100 bp marker, H₂O=control, NT2=NT2 cell line days 0–15 after RA treatment, 2102Ep=2102Ep cell line days 0–10 after RA treatment.

Figure 3

Identification of the expressing cell types by ISH. In situ hybridisation was performed with antisense and control sense (inserted images) RNA probes. Expression is shown for three genes: KIT (A–D), HLXB9 (E–H), and the IMAGE clone number 260215 (I–M). KIT was highly expressed in CIS cells (A) and in SEM (C), and present in components of teratoma, for example, keratinised intestine-like epithelium as shown here (B) and embryonal carcinoma (D). HLXB9 expression was high in CIS cells (E+F) and SEM (G); HLXB9 was present in embryonal carcinoma tumour components (H) and it was detected in spermatocytes, spermatids and Leydig cells in normal testicular tissue (E). IMAGE clone 260215 was present in spermatocytes, spermatids and Sertoli cells in normal testicular tissue (I); the expression in Sertoli cells was confirmed in a tubule with Sertoli cell-only pattern (J). Additionally, the transcript was highly expressed in CIS cells (K) and present in SEM (L) and components of embryonal carcinoma (M). Scale bar represents 50 μm.