Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin-based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo-sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo-resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26-27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo- resistance to cisplatin-based treatment in TGCTs and other cancers.

3742 Testicular germ cell tumour (TGCT) is the commonest cancer in young men, and its incidence has increased in recent years. Around 90% of TGCT patients are cured due to its exquisite sensitivity to cisplatin-based chemotherapy, placing testicular cancer as an ideal model for the study of chemo-resistance that occurs in most human cancers. Our previous study using three pairs of cisplatin-sensitive and their derivative resistant TGCT cell lines revealed consistent expression changes of some chromosomal regions across all three pairs of cell lines, comparing the parental lines against their resistant derivatives. However, no consistent genomic alterations were found. Therefore, we further analysed the expression changes in these three pairs of cell lines using Affymetrix microarray analysis. Expression differences between untreated cells, and after 24-hour exposure to cisplatin at the EC50 concentration of the resistant cell line in each pair, were investigated using the Affymetrix gene expression microarray profiling approach (Human Genome U133 plus 2.0 arrays). Genes potentially involved in the mechanisms of TGCT cisplatin-resistance were identified using GeneSpring v7.2 software. With a 2-fold expression difference as a cut-off value, no consistent gene expression changes were found between the untreated sensitive and resistant lines. However, 7 genes were up-regulated in the resistant lines when a 1.5-fold cut-off value was used. Following cisplatin exposure, consistent 2-fold expression changes were found across all three cell line pairs. Interestingly, 71 of these 2-fold genes, including 32 up-regulated and 39 down-regulated, showed consistent expression changes in the three treated sensitive, but not the resistant cell lines, compared to the untreated cells. We have validated the expression changes of five selected genes in the three pairs of cell lines using quantitative real-time PCR and we are currently also evaluating the significance of these candidate cisplatin resistant genes in clinical samples. This study demonstrates that gene expression changes are associated with cisplatin resistance, and that the immediate response to drug exposure differs between sensitive and resistant cells. These differentially expressed genes have potential to be used both as prognostic markers for stratified treatment and as targets to re-sensitise tumour cells to cisplatin.

The genotype of a tumor determines its biology and clinical behavior. The genetic alterations associated with the unique embryonal morphology of nonseminomatous subtypes of testicular germ cell tumors remain to be established. Using single nucleotide polymorphism microarray analysis, we found in all of the 15 nonseminomas analyzed, large-scale chromosomal homozygosities, most of which were not associated with relative chromosome loss. This unusual genotype, distinguishing nonseminoma from seminomas and other human tumors, may be associated with the special embryonal development morphologic transition of this malignancy. Based on these genetic data, we hypothesized a new potential origin of nonseminomas through sperm fusion. Nonrandom involvement of certain chromosomes also suggests that genes on these chromosome regions may play an important role in nonseminoma development.