Development Of Testicular Germ Cell Tumors Research Articles

Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines

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.

Genes, chromosomes and the development of testicular germ cell tumors of adolescents and adults

Testicular germ cell tumors (TGCTs) of adults and adolescents are thought to be derived from primordial germ cells or gonocytes. TGCTs develop postpuberty from precursor lesions known as intratubular germ cell neoplasia undifferentiated. The tumors can be divided into two groups based on their histology and clinical behavior; seminomas resemble primordial germ cells or gonocytes and nonseminomas resemble embryonic or extraembryonic tissues at various stages of differentiation. The most undifferentiated form of nonseminoma, embryonal carcinoma, resembles embryonic stem cells in terms of morphology and expression profiling, both mRNAs and microRNAs. Evidence supports both environmental factors and genetic predisposition underlying the development of TGCTs. Various models of development have been proposed and are discussed. In TGCTs, gain of material from the short arm of chromosome 12 is invariable: genes from this region include the proto-oncogene KRAS, which has activating mutations in approximately 10% of tumors or is frequently overexpressed. A number of different approaches to increase the understanding of the development and progression of TGCTs have highlighted the involvement of KIT, RAS/RAF/MAPK, STAT, and PI3K/AKT signaling. We review the role of these signaling pathways in this process and the potential influence of environmental factors in the development of TGCTs.

Mutations of c- kit gene in bilateral testicular germ cell tumours in Japan

About 10–40% of testicular germ cell tumours (TGCTs) have been reported to have activating c- kit gene mutations. A European study group has reported that most bilateral TGCTs from European patients have c- kit mutations at codon 816, although few unilateral cases harbour the mutations. This implies that the presence of a c- kit mutation in a unilateral TGCT predicts the development of TGCT in the contralateral testis (bilateral disease). However, since little is known about on c- kit gene mutational frequencies in bilateral TGCTs from patients of Asian origin, we examined 12 bilateral TGCTs from seven Japanese patients, along with 39 unilateral TGCTs from Japanese patients, for the presence of c- kit mutations. We analyzed c- kit exons 11 and 17 by PCR followed by direct sequencing, and also analyzed the hotspot mutations at codon 816 by loop-hybrid mobility shift assay, a sensitive PCR-based method. We found c- kit mutations in seven of 39 (18%) unilateral TGCTs: two of the seven mutations were in exon 11 and the others, including four point mutations at codon 816, were in exon 17. No mutations, however, were observed in bilateral TGCTs. Thus, the presence of c- kit gene mutations in TGCTs may not be associated with bilateral diseases, at least in Japan.

Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: Report of 220 tumors and review of literature

Mutations in the KIT gene occur in approximately 8% of all testicular germ cell tumors (TGCT) and KIT is the most frequently mutated known cancer gene. One report has shown that 93% of patients with bilateral disease have a mutation at codon 816 of the KIT gene. Importantly, this suggests that the identification of a mutation in KIT is predictive of the development of a contralateral TGCT. We investigated the frequency and type of mutations in KIT in a series of 220 tumors from 211 patients with TGCTs and extragonadal germ cell tumors. In 170 patients with unilateral TGCT and no additional germ cell tumour, we identified one exon 11 mutation in a patient with unilateral TGCT and eight activating KIT mutations in exon 17 (9/175, 5.1%). In 32 patients with bilateral TGCT, one patient had an activating KIT mutation in exon 17 (3.1%). The incidence of activating KIT mutations in sporadic TGCT vs. familial TGCT was not significantly different. All mutations were identified in seminomas. Three extragonadal primary germ cell tumors were examined and in one tumor an activating KIT mutation was demonstrated in the pineal germinoma. Interestingly, this mutation was also seen in the patient's testicular seminoma. We find no evidence for an increased frequency of KIT mutations in bilateral TGCT.

KIT and RAS signalling pathways in testicular germ cell tumours: new data and a review of the literature

Testicular germ cell tumours (TGCTs) are the leading cause of cancer deaths in young male Caucasians. Identifying changes in DNA copy number can pinpoint genes involved in tumour development. We defined the smallest overlapping regions of imbalance in TGCTs using array comparative genomic hybridization analysis. Novel regions, or regions which refined those previously reported, were identified. The expression profile of genes from 12p, which is invariably gained in TGCTs, and amplicons defined at 12p11.2-12.1 and 4q12, suggest KRAS and KIT involvement in TGCT and seminoma development, respectively. Amplification of these genes was not found in intratubular germ cell neoplasia adjacent to invasive disease showing these changes, suggesting their involvement in tumour progression. Activating mutations of RAS genes (KRAS or NRAS) and overexpression of KRAS were mutually exclusive events. These, correlations between the expression levels of KIT, KRAS and GRB7 (which encodes an adapter molecule known to interact with the KIT tyrosine kinase receptor) and other reported evidence reviewed here, are consistent with a role for activation of KIT and RAS signalling in TGCT development. In order to assess a role for KIT in seminomas, we modulated the level of KIT expression in TCam-2, a seminoma cell line. The likely seminomatous origin of this cell line was supported by demonstrating KIT and OCT3/4 overexpression and gain of 12p material. Reducing the expression of KIT in TCam-2 through RNA inhibition resulted in decreased cell viability. Further understanding of KIT and RAS signalling in TGCTs may lead to novel therapeutic approaches for these tumours.

What leads from dead-end?

The 129 mouse strain develops congenital testicular germ cell tumors (TGCTs) at a low frequency. TGCTs in mice resemble the testicular tumors (teratomas) that occur in human infants. The genes that cause these tumors in 129 have not been identified. The defect at the Ter locus increases TGCT incidence such that 94% of 129-Ter/Ter males develop TGCTs. The primary effect of the Ter mutation is progressive loss of primordial germ cells (PGCs) during embryonic development. This results in sterility in adult Ter/Ter mice on all mouse strain backgrounds. However, on the 129 background, Ter causes tumor development in addition to sterility. Therefore, Ter acts as a modifier of 129-derived TGCT susceptibility genes. Ter was identified to be a mutation that inactivates the Dead-end1 (Dnd1) gene. In this perspective, I discuss the possible areas of future investigations to elucidate the mechanism of TGCT development due to Dnd1 inactivation.

Novel epigenetically deregulated genes in testicular cancer include homeobox genes and SCGB3A1 (HIN‐1)

Testicular germ cell tumours (TGCTs) are classified into two main histological subgroups: seminomas and non-seminomas. The latter comprise several subtypes: embryonal carcinomas, yolk sac tumours, choriocarcinomas, and teratomas. These embryonal and extra-embryonal-like differentiation lineages represent a caricature of early normal development, and inactivation of gene expression through promoter hypermethylation may therefore be of particular importance in germ cell tumourigenesis. The promoter methylation status of ten candidate genes-CDH13, DLX6, EMX2, HOXA9, HOXB5, MSX1, MSX2, RASSF1A, RUNX3, and SCGB3A1 (alias HIN-1)-was assessed by methylation-specific PCR in seven intratubular germ cell neoplasias and 55 primary TGCTs. Furthermore, by a discovery-based global approach, comparing cDNA microarray expression profiles of two germ cell tumour cell lines before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine, a gene list of potentially epigenetic targets was identified, from which CGGBP1, CGRRF1, SMARCC2, SORBS1, and XPA were analysed further. Overall, the non-seminomas were significantly more often methylated than were seminomas (p < 0.001). The three most frequently methylated genes among this subtype were SCGB3A1 (54%), RASSF1A (29%), and HOXA9 (26%). CDH13 and HOXB5 were methylated at low frequencies (10-15%), and EMX2, MSX1, RUNX3, SORBS1, and XPA only rarely (<10%). In conclusion, this study has identified several novel epigenetically deregulated target genes in TGCT development, including homeobox genes and SCGB3A1, suggesting that epigenetic inactivation of key genes in normal development also has an important role in TGCTs.

Lack of association between the incidence of testicular germ cell tumors and Y‐chromosome haplogroups in the Japanese population

Despite being relatively uncommon, testicular germ cell tumors (TGCT) are the most common malignant disease in young men. Epidemiological studies concerning patients with testicular cancer indicate that the most of them have poor semen quality or testicular dysgenesis. However, many studies have shown that the Y chromosome harbors many candidate genes responsible for spermatogenesis process and development and maintenance of the germ cells. The Y chromosome is thought to have a relationship with the formation and progression of TGCT. To verify this relationship, we investigated if there is any correlation between the Y chromosome structural variations presented as different haplogroups and the occurrence of TGCT in the Japanese population. Using combined haplogroups based on typing of three Y chromosome polymorphic binary markers, we analyzed 68 TGCT derived from Japanese patients together with randomly selected 104 unrelated healthy Japanese matched male controls who were confirmed as residents of the same geographic area. Our findings showed a lack of association between the incidence of TGCT and the different Y- chromosome haplogroups in Japanese population. We concluded that there are no significant variations in males from different Y chromosome lineages regarding their susceptibility or resistance for developing TGCT. The previously hypothesized role of the Y chromosome in the development of TGCT is still uncertain and needs further verification.

Association of XRCC1 gene polymorphisms with the susceptibility and chromosomal aberration of testicular germ cell tumors

It is known that many genomic and genetic alterations caused by aging or environmental factors are responsible for cancer development and progression. XRCC1 is involved in the repair of DNA single-strand breaks formed by exposure to ionizing radiation and alkylating agents. The objective of this study was to investigate the association of genomic alterations and the susceptibility of testicular germ cell tumors with XRCC1 polymorphisms. Two polymorphisms of XRCC1, Arg194Trp and Arg399Gln, were genotyped in 83 patients with testicular germ cell tumors (TGCT) and 87 male controls. Allelic imbalances (AI) were evaluated using 4 microsatellite markers in a subgroup of 50 patients. Patients with at least one Gln allele of the Arg399Gln polymorphism had an increased risk of TGCT than those with the Arg/Arg genotype (aOR=1.775, 95% CI=1.045-3.016, P=0.034). Furthermore, the increased risk associated with the Gln allele against the Arg homozygote was more strongly observed in patients with pure seminoma (aOR=2.242, 95% CI=1.149-4.374, P=0.018) or with metastasis (aOR=2.481, 95% CI=1.267-4.862, P=0.008). In the Arg194Trp polymorphism, there was no significant difference in the genotype distribution between TGCT patients and the controls. In AI analysis, the frequency of AI was significantly higher in tumors with at least one Gln allele than those with the Arg/Arg genotype in D13S317 (P=0.010) and in a combination of 4 markers (0.51+/-0.32 vs 0.32+/-28, P=0.028). Our results suggest that the Gln allele of the XRCC1 Arg399Gln polymorphism may genetically modify the development and progression of TGCT through genomic instability.

Activating Mutations and/or Expression Levels of Tyrosine Kinase Receptors GRB7, RAS, and BRAF in Testicular Germ Cell Tumors

Amplification and/or overexpression of genes encoding tyrosine kinase receptors KIT and ERBB2 have been reported in testicular germ cell tumors (TGCTs). These receptors can bind the adaptor molecule GRB7 encoded by a gene adjacent to ERBB2 at 17q12, a region also frequently gained in TGCTs. GRB7 binding may be involved in the activation of RAS signaling and KRAS2 maps to 12p, which is constitutively gained in TGCT and lies within a minimum overlapping region of amplification at 12pl1.2–12.1, a region we have previously defined. RAS proteins activate BRAF, and activating mutations of genes encoding these proteins have been described in various tumors. Here we determine the relationships between expression levels and activating mutations of these genes in a series of 65 primary TGCTs and 4 TCGT cell lines. High levels of expression and activating mutations in RAS were mutually exclusive events, and activating mutations in RAS were only identified in the seminoma subtype. Mutations in BRAF were not identified. Increased ERBB2 expression was associated with differentiated nonseminoma histology excised from lymph nodes postchemotherapy. Mutation, elevated expression, and correlations between expression levels of KRAS2, GRB7, and KIT are consistent with their involvement in the development of TGCTs.

Sequence analysis of the protein kinase gene family in human testicular germ‐cell tumors of adolescents and adults

The protein kinase gene family is the most frequently mutated in human cancer. Previous work has documented activating mutations in the KIT receptor tyrosine kinase in testicular germ-cell tumors (TGCT). To investigate further the potential role of mutated protein kinases in the development of TGCT and to characterize the prevalence and patterns of point mutations in these tumors, we have sequenced the coding exons and splice junctions of the annotated protein kinase family of 518 genes in a series of seven seminomas and six nonseminomas. Our results show a remarkably low mutation frequency, with only a single somatic point mutation, a K277E mutation in the STK10 gene, being identified in a total of more than 15 megabases of sequence analyzed. Sequencing of STK10 in an additional 40 TGCTs revealed no further mutations. Comparative genomic hybridization and LOH analysis using SNP arrays demonstrated that the 13 TGCTs mutationally screened through the 518 protein kinase genes were uniformly aneuploid with consistent chromosomal gains on 12p, 8q, 7, and X and losses on 13q, 18q, 11q, and 4q. Our results do not provide evidence for a mutated protein kinase implicated in the development of TGCT other than KIT. Moreover, they demonstrate that the general prevalence of point mutations in TGCT is low, in contrast to the high frequency of copy number changes.

The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours

In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.

Absence of constitutional Y chromosome AZF deletions in patients with testicular germ cell tumors

To investigate the frequency of azoospermia factor (AZF) deletions in Dutch patients with testicular germ cell tumors (TGCTs). Reduced fertility is associated with TGCTs and reduced fertility and TGCTs might share genetic risk factors according to the testicular dysgenesis hypothesis. Up to 8% of infertility and reduced fertility in the general male population can be explained by the presence of constitutional deletions of part of the long arm of the Y chromosome (Yq11), referred to as the AZF region. In 112 patients with TGCT, screening for constitutional deletions in the AZF region was performed by multiplex polymerase chain reaction analysis in DNA extracted from peripheral blood lymphocytes. A set of 24 primer pairs, of which 20 primer pairs are homologous to previously identified and mapped sequenced tag sites within the AZF region were used. No deletions in the Yq11 region were detected in any of the 112 patients. Large Y chromosome microdeletions in the AZF region are not a major contributor to the development of TGCT and TGCT-associated reduced fertility.

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2-p12.1.

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

Genetic control of susceptibility to spontaneous testicular germ cell tumors in mice.

Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men. TGCT is a polygenic trait and genes that control susceptibility for TGCT development have not yet been identified. The 129/Sv inbred strain of mice is an important experimental model to study the genetics and development of TGCTs. We review several novel approaches that were developed to study the susceptibility of TGCTs in the 129/Sv mouse model and its application in humans. These approaches showed that several spontaneous and engineered mutations interact with 129/Sv-derived susceptibility genes to enhance or suppress susceptibility; two of these mutations (Ter and Trp53) revealed novel linkages for susceptibility genes in sensitized polygenic trait analysis. Linkage analysis with a chromosome substitution strains suggests that as many as 100 genes control susceptibility. Bilateral TGCTs result from the coincidental occurrence of unilateral tumors. These results highlight the important contributions that this mouse model can make to studies of TGCT susceptibility in humans.

The testicular germ cell tumour genome.

Human testicular germ cell tumour (TGCT) of adolescents and young adults develop from precursor lesions called carcinoma in situ (CIS), which is believed to originate from diploid primordial germ cells during foetal life. CIS is initiated by an aneuploidisation event accompanied by extensive chromosome instability. The further transformation of CIS into invasive TGCT (seminomas and nonseminomas) is associated with increased copy number of chromosome arm 12p, most often seen as isochromosome 12p. Despite the morphological distinctions between seminomatous and nonseminomatous TGCTs, they have many of the same regional genomic disruptions, although frequencies may vary. However, the two histological subtypes have quite distinct epigenomes, which is further evident from their different gene expression patterns. CIS develops from cells with erased parental imprinting, and the seminoma genome is under-methylated compared to that of the nonseminoma genome. High throughput microarray technologies have already pinpointed several genes important to TGCT, and will further unravel secrets of how specific genes and pathways are regulated and deregulated throughout the different stages of TGCT tumourigenesis. In addition to acquiring new insights into the molecular mechanisms of TGCT development, understanding the TGCT genome will also provide clues to the genetics of human embryonic development and of chemotherapy response, as TGCT is a good model system to both.

Allelic losses in carcinoma in situ and testicular germ cell tumours of adolescents and adults: evidence suggestive of the linear progression model.

Testicular germ cell tumours (TGCTs) may arise through a process of multi-step carcinogenesis, and loss of heterozygosity (LOH) at specific loci is likely to be an important early event, although this has not been studied in detail. In order to explore the pathogenetic relationships among TGCTs, we investigated the genetic changes in testicular tumours that exhibit a disease continuum through the precursor carcinoma in situ (CIS) to either seminoma (SE) and/or non-seminomatous germ cell tumour (NSGCT). Universal amplification has been performed on 87 TGCT specimens and 36 samples of CIS cells microdissected from single paraffin-embedded tumour sections from 40 patients, including multiple specimens of CIS and TGCT cells of varied histology microdissected from 24 individual patients. Seventy-seven microsatellite markers were used to assay these samples for LOH at candidate regions selected from the literature, mapping to 3q, 5q, 9p, 11p, 11q, 12q, 17p and 18q. Construction of deletion maps for each of these regions identified common sites of deletion at 3q27–q28, 5q31, 5q34–q35, 9p22–p21 and 12q22, which correlate with allelic losses we have also observed in the precursor CIS cells. Evidence for allelic loss at 3q27–q28 was observed in all of the embryonal carcinoma samples analysed. We conclude that inactivation of gene(s) within these regions are likely to be early events in the development and progression of TGCTs. These results also provide molecular evidence in support of the hypothesis that SE is an intermediate stage of development within a single neoplastic pathway of progression from CIS precursor cells to NSGCT. © 2000 Cancer Research Campaign

Comparative genomic hybridization of microdissected samples from different stages in the development of a seminoma and a non-seminoma.

Human testicular germ cell tumours (TGCTs) of adolescents and adults, both seminomas and non-seminomas, originate from intratubular germ cell neoplasia (IGCN). Comparative genomic hybridization (CGH) was applied to microdissected samples from different stages of the development of a seminoma and a mixed non-seminoma, including IGCN of both. The different stages of the seminoma development, namely IGCN, intratubular and invasive seminoma, showed a very similar pattern of chromosomal imbalances, including gains of parts of 7, 8, 12,14, and X, and losses of parts of 3, 4, 5, 10, 11, 12q, 16, 18, 22, and Y. A more heterogeneous pattern was found for the non-seminoma. Some aberrations were present only in IGCN, or in IGCN and in all invasive components (gains of parts of 1q, 17, 19p, 20q, and 22, and losses of parts of 4, 5, 9p, 13, and 18q), while others were present in a less consistent pattern. These are the first reported CGH data from different stages in the development of TGCTs. Although only two cases were studied, the results suggest that particular numerical changes of (parts of) chromosomes are involved in the early development and progression of this cancer.

Loss of heterozygosity, differentiation, and clonality in microdissected male germ cell tumours.

Testicular germ cell tumours (TGCTs) are heterogeneous neoplasms with different histological patterns and malignant potential. The aim of this study was to determine whether the main TGCT subtypes (seminoma, embryonal carcinoma, yolk sac tumour, choriocarcinoma, and mature teratoma) are distinguished by their loss of heterozygosity (LOH) patterns and whether LOH typing can help to distinguish between clonal and multifocal development of different components in mixed TGCTs. In 76 tumours analysed for allelic losses at 25 chromosomal loci, different LOH patterns were found in distinct histological subtypes. A region around D18S543 frequently lost in yolk sac tumours could harbour one or more tumour suppressor genes. In 20 microdissected mixed tumours, losses of identical alleles in different histological components in 11 of 20 cases (over 50 per cent) were found, which is in favour of current histogenetic models of clonal TGCT development. Clonal losses were most often found at D13S317 (6 of 20 tumours). Two classes of allelic losses may therefore occur during TGCT development: clonal losses which are involved in early transformational events and others related to TGCT differentiation along different lines.

Persistence of parvovirus B19 DNA in testis of patients with testicular germ cell tumours.

Germ cell tumours (GCT) of the testis are the most common malignant tumours occurring in young adults. In view of the young age of patients, the increasing incidence of GCT and the overexpression of wild-type p53 observed in a majority of tumours, the possibility of the involvement of a virus in the development of this cancer was considered. Testicular GCT were analysed for the presence of cytomegalovirus and Epstein-Barr virus (EBV), which are known to cause overexpression of wild-type p53 protein, and parvovirus B19. The testicular tissue of 39 patients with testicular GCT and 12 patients with healthy testicular tissues was tested for presence of viral DNA by PCR. Neither cytomegalovirus nor EBV DNAs were detected in the 39 tumours analysed, but parvovirus B19 DNA sequences were demonstrated in the testicular tissue of 85% (33/39 cases) of patients with GCT. The sera of 16 of the 39 patients with GCT were tested for the presence of parvovirus B19 IgM and IgG. B19-specific IgG was detected in the sera of 11 patients (69%). Only one case was positive for parvovirus B19 IgM, which was also shown to have B19 genome sequences in the serum by PCR, indicating that in a majority of cases an acute B19 infection can be excluded as being the source of the B19 DNA sequences in the testis. B19 DNA could not be detected in normal testicular tissue and thus parvovirus B19 could play a role, direct or indirect, in the development of testicular GCT or have tropism for the tumour cells.