GENETIC CONTROL OF SUSCEPTIBILITY TO TESTICULAR CANCER
Human germ cell tumours comprise a heterogeneous group of neoplasms. Testicular germ cell tumors can be divided into three groups (infantile/prepubertal, adolescent/young adult and spermatocytic seminoma), each with its own constellation of clinical histology, molecular and clinical features. They originate from germ cells at different stages of development.
Testicular germ cell tumors (TGCTs) are the most common cancer in males aged 20-35 year. TGCTs affect 1 in 500 men and the incidence varies among geographical areas.
TGCTs often present clinically as a painless swelling or lump. The tumor may cause mild discomfort because of its weight, but rarely pain. Most testicular cancer cases are unilateral and predominately affect the right testis. Bilateral tumors are rare at diagnosis, but in two percent of patients with unilateral TGCT, a metachronous new tumor will develop in the remaining testis.
TGCTs are often malignant and metastasize readily if not treated early. The major route of metastasis is lymphatic, the lumbar and mediastinal nodes being commonly involved. Right testicular tumors usually metastasize to nodes between the aorta and the inferior vena cava and left testicular tumors metastasize to nodes lateral to the aorta.
Intratubular germ cell neoplasia, unclassified type (IGCNU) is the precursor to these invasive tumors. Several factors have been associated with their pathogenesis, including cryptorchidism, elevated estrogens in utero and gonadal dysgenesis.
Studies on epidemiology, histology, clinical behaviour, and chromosomal constitution of these tumours support the concept of distinct entities derived from germ cells but each with a different pathogenesis. Either the teratomas of the infantile testis show no chromosomal aberrations, or display a pattern of over- and under-representation of (parts of) chromosomes as detected in the yolk sac tumours of the infantile testis. In contrast, the seminomas and nonseminomas reveal a consistent pattern of losses and gains, that is, chromosomes 11, 13 and 18, and 7, 8 and X, respectively, that is different from that found in the infantile testis teratomas and yolk sac tumours. The most consistent structural chromosomal abnormality is an isochromosome 12p. Tumours lacking i(12p) have other structural abnormalities of 12p, among them amplification of 12p11.2-p12.1. The pathogenetically relevant genes on 12p11.2-p12.1 are probably on a fragment of about 1.7 mb. Gain of 12p sequences may be related to invasive growth. Gain of chromosome 9 is the only consistent chromosomal anomaly of spermatocytic seminomas. Infantile teratomas and spermatocytic seminomas are benign tumours. Infantile yolk sac tumour is a malignant germ cell tumour. Seminomas and nonseminomas are malignant, and the most common cancer in young Caucasian males. The cure rate of seminomas and non-seminomas with radio- and chemotherapy is over 90%, which is higher than that of any other solid cancer in adults. In addition, the precursor lesions of these tumours can be treated readily, justifying efforts to develop means for early diagnosis.
To date, a TGCT susceptibility gene has not been identified in humans. Linkage studies have been difficult because of the rarity of multigenerational pedigrees with several affected individuals, sterility resulting from chemotherapies, and the complexity of genetic control. Mapping studies of families collected by the International Testicular Cancer Linkage Consortium revealed weak linkages on chromosomes 3, 4, 5, 11, 12 and 18 (BISHOP 1998; LEAHY et al. 1995).
The increased risk of TGCT between fathers and sons is less than the risk between brothers which suggests that there may be X-linked inheritance. In fact, linkage studies extended to the X chromosome demonstrated the first significant linkage on Xq27 (RAPLEY et al. 2000). Interestingly, the LOD score increased when linkage analysis were performed only on families with at least one bilateral tumor case (RAPLEY et al. 2000). This data suggest that the locus on Xq27 to be linked to predisposition to bilateral tumors.
Tumorigenesis appears to be very complicated and requires two stages. The first stage is the formation of carcinoma in situ (CIS). CIS cells have been recognized as the precursor cells of all types of TGCTs, except spermatocytoma (RAJPERT-DE MEYTS et al. 1996). CIS cells can act as pluripotent stem cells, and give rise to non-seminomas, or they can lose stem cell potential with age and give rise to more limited cell and tissue types .The second stage of tumorigenesis is a somatic event that triggers the CIS to develop into TGCTs. It is not evident how or when CIS arises, but the CIS resembles the embryonal carcinoma (EC) cells found in the early stage of mouse TGCTs.
It has been proposed that TGCTs develop because some primordial germ cells (PGS) fail to enter mitotic G1 arrest and continue to divide for several days giving rise to pluripotent stem cells called embryonal carcinoma (EC) cells that become disorganized and form tumors of various cells and tissues (Stevens and Mackensen 1961). Therefore, cell cycle regulation is likely to play a major role in TGCT tumorigenesis.
An alternative hypothesis is germ cell tumors may arise from germ cells that do not migrate properly (STALLOCK et al. 2003). During embryogenesis, a significant number of PGCs fail to migrate correctly to the genital ridges. These PGCs usually die in ectopic locations. Studies have shown that MGF-KIT interaction is necessary for germ cell survival. Mgf stimulates PGCs to survive or proliferate in culture (DOLCI et al. 1991; MATSUI et al. 1991). Bax has also been shown to be required for germ cell death during and after migration of germ cells that do not reach the genital ridge (RUCKER et al. 2000). W locus mutations include large deletions, rearrangements and point mutations which affect the amount of c-Kit protein expressed and the level of kinase activity (DUBREUIL et al. 1990). The ligand for the c-Kit receptor is mast cell growth factor (Mgf) or Kit ligand (KL) or Steel factor (SLF), which is encoded at the Sl locus (ANDERSON et al. 1990; COPELAND et al. 1990). Mgf is produced as a membrane bound growth factor that undergoes proteolytic cleavage to generate a soluble form. The Sl locus is highly mutable and many induced (X-irradiation) and spontaneous mutations have been described
It can be argued against the hypothesis that germ cell tumors arise from germ cells that do not migrate properly because it may be possible that germ cells that migrated away from the genital ridges may require specific growth factors which are not present at the ectopic locations and the lack of proper environment and signals will most likely cause PGCs to undergo cell death. In addition, if PGCs that do not migrate properly do survive and transform into tumors, these tumors would most probably not be TGCTs since the PGCs never arrived at the genital ridges and instead migrated to other ectopic locations. Therefore, it is necessary to determine the function of all the different molecular controls of PGC development with respect to TGCT development.
The consistency of 12p-over-representation in all histological subtypes of TGCTs, including their preinvasive stage, suggests that gain of one or more genes on 12p(amplifications corresponding to 12p11.1-p12.1) is crucial in the development of this cancer. So far, studies aimed at the identification of the relevant gene(s) were based on the 'candidate-gene approach'. Using bicolour-FISH, physical mapping, and semi-quantitative polymerase chain reactions, the size of the shortest region of overlap of amplification (SROA) was estimated to be between 1750-3000 kb. In addition, we mapped a number of genes in and around this region. While fourteen known genes could be excluded as candidates based on their location outside this region, we demonstrate that KRAS2, JAW1 and SOX5 genes are localized within the SROA. While KRAS2 and JAW1 map to the proximal border of the SROA, SOX5 maps centrally in the SROA. KRAS2 and JAW1 are expressed in all TGCTs, whereas one 12p amplicon-positive TGCT lacks expression of SOX5. The critical region of 12p over-represented in TGCTs is less than 8% of the total length of the short arm of chromosome 12.
Three known genes map within the newly determined shortest region of overlap of amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to the telomeric region of the SROA, DAD-R is the first gene at the centromeric region within the 12p amplicon. Although all three genes are amplified to the same level within the SROA, expression of DAD-R is significantly up-regulated in seminomas with the restricted 12p amplification compared with seminomas without this amplicon. DAD-R is also highly expressed in nonseminomas of various histologies and derived cell lines, both lacking such amplification. This finding is of particular interest because seminomas with the restricted 12p amplification and nonseminomas are manifested clinically in the third decade of life and show a low degree of apoptosis. In contrast, seminomas lacking a restricted 12p amplification, showing significantly lower levels of DAD-R with pronounced apoptosis, manifest clinically in the fourth decade of life. A low level of DAD-R expression is also observed in normal testicular parenchyma and in parenchyma containing the precursor cells of this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression in seminomas and nonseminomas correlates with invasive growth and a reduced level of apoptosis associated with an earlier clinical presentation. These data implicate DAD-R as a candidate gene responsible in part for the pathological effects resulting from gain of 12p sequences in TGCTs.
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.
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 12p11.2-12.1, 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.
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.