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.     

c-KIT is frequently mutated in bilateral germ cell tumours and down-regulated during progression from intratubular germ cell neoplasia to seminoma

Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men; 5% of these patients develop a second TGCT in the contralateral testis. The pathogenesis of TGCT is closely linked to primordial germ cells (PGCs) or gonocytes. The receptor tyrosine kinase (c-KIT) is necessary for migration and survival of PGCs and is expressed in intratubular neoplastic germ cells (IGCNUs) and seminomas. We studied the frequency of c-KIT exon 11 and 17 mutations in 155 unilateral (108 seminomas and 47 non-seminomas) and 22 bilateral (18 seminomas, two embryonal carcinomas, two IGCNU) cases. While no mutations were detected in exon 11, the mutation frequency in exon 17 was significantly higher in bilateral (14/22, 63.6%) compared to unilateral TGCT (10/155, 6.4%) (p < 0.001). Different activating mutations (Y823D, D816V, D816H and N822K) were detected in bilateral TGCT. Y823D mutation was identical in both testes in three cases and quantitative pyrosequencing showed that up to 76% of the cells analysed in tumour samples carried this mutation. One bilateral synchronous seminoma revealed a S821F mutation in one testis and a Y823D mutation contralaterally. To study the role of c-KIT in TGCT progression, we compared its expression in 41 seminomas and adjacent IGCNUs. Immunohistochemical analysis revealed that c-KIT expression was significantly reduced in seminomas compared to IGCNUs (p < 0.006) and that there were no significant changes in c-KIT mRNA copy numbers in progressed compared to low-stage seminomas. In summary, our study shows that patients with c-KIT mutations are more prone to develop a bilateral TGCT and suggests that in a portion of bilateral TGCTs, c-KIT mutations occur early during embryonal development, prior to the arrival of PGCs at the genital ridge. Furthermore, our findings show that c-KIT down-regulation occurs during the progression of IGCNU to seminoma.     

Analysis of the DND1 gene in men with sporadic and familial testicular germ cell tumors

A base substitution in the mouse Dnd1 gene resulting in a truncated Dnd protein has been shown to be responsible for germ cell loss and the development of testicular germ cell tumors (TGCT) in the 129 strain of mice. We investigated the human orthologue of this gene in 263 patients (165 with a family history of TGCT and 98 without) and found a rare heterozygous variant, p. Glu86Ala, in a single case. This variant was not present in control chromosomes (0/4,132). Analysis of the variant in an additional 842 index TGCT cases (269 with a family history of TGCT and 573 without) did not reveal any additional instances. The variant, p. Glu86Ala, is within a known functional domain of DND1 and is highly conserved through evolution. Although the variant may be a rare polymorphism, a change at such a highly conserved residue is characteristic of a disease-causing variant. Whether it is disease-causing or not, mutations in DND1 make, at most, a very small contribution to TGCT susceptibility in adults and adolescents.     

Localisation of susceptibility genes for familial testicular germ cell tumour

Approximately 1700 men in the United Kingdom develop testicular germ cell tumours (TGCT) per year. Among the known risk factors a family history of disease remains one of the strongest (1, 2). Two-percent of TGCT cases report another affected family member. Epidemiological studies have shown that there is an eight to ten fold increase in relative risk of TGCT to brothers of patients and a fourfold increased risk to fathers and sons (2-5). This relative risk is considerably higher than for most other common cancers, which rarely exceeds four and strongly suggests that genes may play an important role in TGCT. Linkage analysis of the set of families compatible with X-linkage (i.e. no male to male transmission) provided the first statistically significant evidence for a TGCT predisposition locus (6). The gene called TGCT1 is located at Xq27 and seems to be associated with a risk of bilateral disease and undescended testis. However TGCT1 does not account for all TGCT pedigrees and additional susceptibility genes must exist. Our group has now genotyped 179 TGCT pedigrees and identified additional genomic regions that might also harbour TGCT susceptibility genes. This paper reviews the current data for the region at Xq27 and presents evidence for several other possible candidate regions.     

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.     

Multipoint methylation analysis indicates a distinctive epigenetic phenotype among testicular germ cell tumors and testicular malignant lymphomas

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. Although a growing number of genes showing hypermethylation is being reported in human cancer, methylation profiles of tumor-related genes in testicular neoplasms have not been well elucidated. This study was designed to show the methylation profiles of multiple CpG islands in testicular germ cell tumors (TGCTs) in comparison with those in testicular malignant lymphomas. We studied the methylation status of E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 by use of TGCT tissues and testicular malignant lymphoma tissues (25 primary TGCT tissues and three primary testicular lymphoma tissues). Methylation was not observed in E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 in any of the TGCT tissues. In contrast, all three (100%) of the testicular lymphoma tissues demonstrated hypermethylation of E-cadherin, RASSF1A, and RARB, but not CDKN2B, CDKN2A, BRCA1, RB1, VHL, and GSTP1. These data demonstrate that a distinctive epigenetic phenotype underlies the TGCTs and testicular lymphomas at the CpG sites of E-cadherin, RASSF1A, and RARB; a distinctive epigenetic phenotype was not observed among seminomatous TGCTs and non-seminomatous TGCTs at the CpG sites examined.     

Trans-generational epistasis between Dnd1Ter and other modifier genes controls susceptibility to testicular germ cell tumors

The genetic basis for susceptibility to testicular germ cell tumors (TGCTs) has been remarkably elusive. Although TGCTs are the most common cancer in young men and have an unusually strong familial risk, only one low-frequency susceptibility gene has been identified for this highly multigenic trait. In tests to determine whether pairs of genetic variants act epistatically to modulate susceptibility in the 129/Sv mouse model of spontaneous TGCTs, we discovered an unusual mode of inheritance that involved interactions between different genes in different generations. Any of six genetic variants, in either the female or male parent interacted with the Dnd1(Ter) mutation in male offspring to significantly increase both the frequency of affected Ter/+ males and the proportion of bilateral cases. Trans-generational epistasis is a novel mode of epigenetic inheritance that could account for the difficulty of finding TGCT susceptibility genes in humans and might represent a mechanism for transmitting information about genetic and environmental conditions from parents to offspring through the germline.     

Chromosomes, genes, and development of testicular germ cell tumors

A literature review found 265 articles on testicular germ cell tumors (TGCTs) detailing the copy number of chromosomal regions and expression of 245 genes. An initial precursor stage, intratubular germ cell neoplasia (IGCN), is characterized by triploidization and an upregulation of KIT, ALPP, CCDN2, and ZNF354A, and a downregulation of CDKN2D. TGCT regularly have a series of chromosomal aberrations: a decrease in copy number at 4q21 approximately qter and 5q14 approximately qter; an increase at 7p21 approximately pter, 7q21 approximately q33, and 8q12 approximately q23 (especially high increase in seminoma); a decrease at 11p11 approximately p15 and 11q14 approximately q24; an increase at 12p11 approximately pter; a decrease at 13q14 approximately q31; an increase of 17q11 approximately q21 (only for nonseminoma); a decrease of 18q12 approximately qter; and an increase at 21q21 approximately qter, 22q11 approximately qter (only for seminoma), and Xq. Macroscopically overt TGCT is associated with a characteristic series of abnormalities in the retinoblastoma pathway including upregulation of cyclin D2 and p27 and downregulation of RB1 and the cyclin-dependent kinase inhibitors p16, p18, p19, and p21. TGCT thus has a synergistic pattern in gene expressions of the retinoblastoma pathway that is rare in other malignancies.     

Testicular cancer susceptibility in the 129.MOLF-Chr19 mouse strain: additive effects,gene interactions and epigenetic modifications

Testicular germ cell tumors (TGCTs) are the most common solid cancers affecting young men. Although the evidence for genetic predisposition to TGCTs in humans is compelling, the genetic control of susceptibility is poorly understood. The 129S1/SvImJ (129/Sv) inbred strain of mice is an excellent model for studying TGCT susceptibility. We previously reported a new mouse strain, the 129.MOLF-Chr19 chromosome substitution strain, which develops spontaneous TGCTs at a high frequency (70-80%) as compared with the much lower rate in the 129/Sv strain (5%). To characterize the genetic control of TGCT susceptibility, we created a panel of single- and double-congenic strains derived from 129.MOLF-Chr19. The frequency of TGCTs in these strains suggests that several genes with additive and epistatic effects located at distinct sites on chromosome 19 control susceptibility. However, an alternative interpretation involving epigenesis is based on a striking correlation between TGCT frequency and the length of the MOLF-derived congenic segment, regardless of their chromosomal location on Chr 19 in each congenic strain. We also show that bilateral TGCT cases result from the coincidental co-occurrence of unilateral TGCTs rather than from the action of distinct genes that control susceptibility to bilateral versus unilateral TGCT cases. Finally, we propose that these TGCTs result from disrupted testicular and spermatogenic developmental programs.     

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.     

COMMENTS

Approximately 1700 men in the United Kingdom develop testicular germ cell tumours (TGCT) per year. Among the known risk factors a family history of disease remains one of the strongest (1, 2). Two-percent of TGCT cases report another affected family member. Epidemiological studies have shown that there is an eight to ten fold increase in relative risk of TGCT to brothers of patients and a fourfold increased risk to fathers and sons (2-5). This relative risk is considerably higher than for most other common cancers, which rarely exceeds four and strongly suggests that genes may play an important role in TGCT. Linkage analysis of the set of families compatible with X-linkage (i.e. no male to male transmission) provided the first statistically significant evidence for a TGCT predisposition locus (6). The gene called TGCT1 is located at Xq27 and seems to be associated with a risk of bilateral disease and undescended testis. However TGCT1 does not account for all TGCT pedigrees and additional susceptibility genes must exist. Our group has now genotyped 179 TGCT pedigrees and identified additional genomic regions that might also harbour TGCT susceptibility genes. This paper reviews the current data for the region at Xq27 and presents evidence for several other possible candidate regions.     

Novel c-KIT germline mutation in a family with gastrointestinal stromal tumors and cutaneous hyperpigmentation

Mutations in the c-KIT gene have been identified in many sporadic and familial cases of gastrointestinal stromal tumor (GIST). We report a familial case of GIST with cutaneous hyperpigmentation associated with a novel germline mutation in the c-KIT gene. Screening for mutations in exon 11 of the c-KIT gene in genomic DNA from tumors and peripheral blood of the members of a family with GISTs was undertaken by direct genomic sequencing. Tumors from GIST patients were analyzed histologically and immunohistochemically. Clinical examination of GIST patients was also performed to detect other systemic diseases associated with c-KIT mutations. Histological study showed that the tumors were GISTs expressing CD34 and c-KIT protein. This GIST-hyperpigmentation disease was associated in the family with a germline mutation in the c-KIT gene. The mutation is a duplication of the sequence CAACTT located in exon 11 of the c-KIT gene, which introduces two extra glutamine and leucine residues in the encoding protein between positions 576 and 577. This Spanish family was affected with GISTs and cutaneous hyperpigmentation associated with a novel germline mutation Leu576_Pro577insGlnLeu in the juxtamembrane domain of the c-KIT receptor. These types of mutation in the c-KIT gene activate the tyrosine kinase activity of the c-KIT receptor and induce constitutive signaling leading to GISTs, in some cases associated with cutaneous hyperpigmentation.     

Clinical characteristics of metachronous bilateral testicular tumors in the chemotherapeutic era.

We wanted to present the results of our experience with bilateral testis tumor and to suggest the effects of chemotherapy in suppressing the development of second primary testicular tumors. Between 1978 and 1997, 2,345 patients were treated for testicular tumor at The University of Texas M. D. Anderson Cancer Center. Of these, 2,107 had germ cell cancers. There were 22 (0.94%) cases of bilateral testicular tumor in the overall patient population and 21 (1.0%) cases among patients with germ cell cancer. We reviewed the medical records to determine the incidence of the histological subtype, the incidence of metachronous versus synchronous formation of contralateral tumors, and tumor stage in this patient population. We also examined the effect of chemotherapy in treating the first tumor and preventing the occurrence of a second tumor. Finally, we compared the effect of ultrasonography, serum tumor marker elevation, and physical examination in detecting second tumors. Only one contralateral germ cell tumor developed synchronously; all others developed metachronously. Fifty percent of first tumors were seminomas, compared to 55% of second tumors. The histologic concordance rate for first and second tumors was 35%. Tumor stage was higher among first tumors than second tumors. The majority of second tumors in patients who received chemotherapy for first malignancies tended to be metachronous seminomas. Ultrasonography detected 6 of 21 (28.6%) contralateral tumors before they were evident by physical examination or serum tumor marker elevation. Seminomas were more prevalent among patients with bilateral germ cell disease than patients with unilateral disease. Chemotherapy, when used as treatment for first tumors, may have some effect in preventing the development of nonseminomatous germ cell tumors in the contralateral testicle. Close follow-up of the contralateral testis with ultrasonography is essential for early detection of second tumors. The outcome for patients with bilateral testicular germ cell cancer is excellent, secondary to early detection.     

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.     

Frequency, phenotype, and genotype of minute gastrointestinal stromal tumors in the stomach: an autopsy study

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the human digestive tract. Up to 85% of these tumors show somatic gain-of-function mutation of the receptor tyrosine kinase c-KIT gene. A recent study has shown a high frequency (22.5%) of minute gastrointestinal stromal tumors in stomachs examined during routine autopsies. The aims of our study were to confirm the previously reported incidence of gastric gastrointestinal stromal tumors in routine autopsies and to investigate their molecular alterations. Gastrointestinal stromal tumors were collected prospectively from 578 autopsies over an 18-month period. After recording the size and location of each lesion, representative tissue samples were processed for hematoxylin and eosin staining and immunohistochemically stained for CD117 and CD34. Microdissected DNA from all identified gastrointestinal stromal tumors was studied for c-KIT and platelet-derived growth factor receptor α mutations. We identified 17 gastrointestinal stromal tumors in 578 consecutive autopsies (2.9%) located in the gastric body (47%) and fundus (47%). One tumor location was not recorded. All tumors were immunohistochemically positive for CD117 and CD34. DNA analysis showed c-KIT mutations in 11 cases. One platelet-derived growth factor receptor α mutation was found. The incidence of gastric minute gastrointestinal stromal tumors (2.9%) is higher than the reported clinical incidence. All are benign tumors, and most, including minute tumors, contain c-KIT mutations. This finding highlights the fact that c-KIT mutations are an early event in the evolution of gastrointestinal stromal tumors but are not sufficient per se for clinically relevant disease.     

Genome-wide linkage screen for testicular germ cell tumour susceptibility loci

A family history of disease is a strong risk factor for testicular germ cell tumour (TGCT). In order to identify the location of putative TGCT susceptibility gene(s) we conducted a linkage search in 237 pedigrees with two or more cases of TGCT. One hundred and seventy-nine pedigrees were evaluated genome-wide with an average inter-marker distance of 10 cM. An additional 58 pedigrees were used to more intensively investigate several genomic regions of interest. Genetic linkage analysis was performed with the ALLEGRO software using two model-based parametric analyses and a non-parametric analysis. Six genomic regions on chromosomes 2p23, 3p12, 3q26, 12p13-q21, 18q21-q23 and Xq27 showed heterogeneity LOD (HLOD) scores of greater than 1, with a maximum HLOD of 1.94 at 3q26. Genome-wide simulation studies indicate that the observed number of HLOD peaks greater than one does not differ significantly from that expected by chance. A TGCT locus at Xq27 has been previously reported. Of the 237 pedigrees examined in this study, 66 were previously unstudied at Xq27, no evidence for linkage to this region was observed in this new pedigree set. Overall, the results indicate that no single major locus can account for the majority of the familial aggregation of TGCT, and suggests that multiple susceptibility loci with weak effects contribute to the disease.     

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.     

A point mutation,C to T,in exon 8 of the porphobilinogen deaminase gene in a Japanese family with acute intermittent porphyria

Summary Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a deficiency of porphobilinogen deaminase (PBGD). To date, only two mutations have been reported in Japanese patients. We report here another mutation of the gene in a Japanese patient. Analysis of the PCR amplified DNA fragments of the gene by direct-sequencing method revealed the gene abnormality responsible for the disease. The mutation found was a point mutation, C to T, in exon 8 of the gene at position 346 of the housekeeping cDNA from the translation codon ATG. This mutation resulted in an Arg¹¹⁶ to Trp substitution. Four carriers in the family were successfully diagnosed by detecting the mutation using restriction analysis of PCR products.