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.     

Altered expression of p27Kip1‐interacting cell‐cycle regulators in the adult testicular germ cell tumors: potential role in tumor development and histological progression

We examined the potential role of cell‐cycle dysregulation in the development and histological progression of adult testicular germ cell tumors (TGCTs). Expressions of p27^Kip1‐interacting cell‐cycle regulators (down‐regulation of p27^Kip1 and overexpression of Skp2, Cks1, cyclin A, and cyclin E) and Ki‐67 labeling index (LI) were immunohistochemically examined in histological components of 50 intratubular germ cell neoplasms, unclassified (IGCNUs); 74 seminomas; and 25 embryonal carcinomas, identified from 88 patients. Altered expression of p27^Kip1, Skp2, Cks1, cyclin A, and cyclin E was observed in 20%, 12%, 16%, 10%, and 24% of IGCNUs; 26%, 36%, 27%, 89%, and 23% of seminomas; and 48%, 68%, 56%, 100%, and 60% of embryonal carcinomas, respectively. A significant difference in the frequency of Skp2 and cyclin A overexpression was observed between IGCNUs and seminomas. Significantly more frequent alterations of Skp2, Cks1, and cyclin E and p27^Kip1 were detected in embryonal carcinomas than in seminomas. Alterations of all cell‐cycle regulators were significantly more frequent in embryonal carcinomas than in IGCNUs. The mean Ki‐67 LI significantly increased from IGCNU (21.2%) through seminoma (34.7%) to embryonal carcinoma (54.2%). These results suggest that alterations of the p27^Kip1‐interacting cell‐cycle regulators are common in TGCTs and may be involved in their histological progression.     

Fatty acid synthase overexpression in adult testicular germ cell tumors: potential role in the progression of non-seminomatous germ cell tumors

Overexpression of fatty acid synthase (FASN), which is a key enzyme responsible for the endogenous synthesis of fatty acids, and its association with multistep progression have been demonstrated in various human malignant tumors. We aimed to clarify the potential role of FASN overexpression in the development and progression of adult testicular germ cell tumors (TGCTs). From the primary sites of a cohort of 113 TGCT cases, we obtained 221 histological components: 53 intratubular germ cell neoplasias, unclassified (IGCNUs), 84 seminomas, 32 embryonal carcinomas, seven choriocarcinomas, 21 yolk sac tumors, and 24 teratomas. Samples were analyzed for overexpression of FASN by immunohistochemistry. Intensities of immunoreactivity and the fraction of positive cells were classified into each four categories (intensity, 0 to 3; fraction, 0–10 %?=?1, 11–50 %?=?2, 51–80 %?=?3, and >80 %?=?4). The overall score was determined by multiplication of both scores and overall scores greater than 6 were considered FASN overexpression. On a component basis, FASN overexpression was detected in 8 % of seminomas but not in IGCNUs (0 %) and was detected frequently in non-seminomatous germ cell tumors (NSGCTs) (88 % of embryonal carcinomas, all choriocarcinomas, 81 % of yolk sac tumors, and 54 % of teratomas). There were no cases of a mixed tumor (i.e., a tumor with multiple histological components) that overexpressed FASN in seminoma components but not in co-existing NSGCT components, suggesting sequential progression. Our immunohistochemical data suggest that FASN overexpression occurs as a late event during the progression from IGCNUs/seminomas to NSGCTs.     

Frequent Fas gene mutations in testicular germ cell tumors

The Fas (Apo-1/CD95)/Fas ligand (L) system is involved in cell death signaling, and has been suggested to be important for the regulation of germ cell apoptosis in the testis. Mutations of the Fas gene may result in accumulation of germ cells and thus might contribute to testicular carcinogenesis. The open reading frame of Fas cDNA was examined in 24 cases of testicular germ cell tumors (TGCTs), comprised of 19 pure histological type (15 seminomas, 3 embryonal carcinomas, 1 immature teratoma) and 5 mixed-type tumors. Mutations of the Fas gene were found in nine (37.5%) of these cases. Each lesion with a homogeneous histological picture was selectively microdissected using a laser capture microdissection method: samples consisted of 18 lesions from seminomas, 7 embryonal carcinomas, 4 immature teratomas, 2 choriocarcinomas, and 1 from a yolk sac tumor. Microdissected genomic DNA was examined to determine which mutations were derived from which kind of histological lesion. Eleven mutations were detected in 10 TGCT lesions from nine cases, but none were found in benign lesions. All were point mutations, and eight missense mutations occurred in exon 9 encoding the core protein of the death domain essential for apoptotic signal transduction. Three were silent mutations. Mutations were found in the seminoma (27.8%) and embryonal carcinoma lesions (62.5%), but none were found in the one yolk sac tumor, two choriocarcinomas, or four immature teratoma lesions. Each seminoma and embryonal carcinoma lesion found in the same case had a different type of Fas mutation from the others. Mouse T-cell lymphoma cells transfected with missense mutated genes were resistant to apoptosis induced by anti-Fas antibody, indicating these to be loss-of-function mutations. These findings suggested a role of Fas gene mutations in the pathogenesis of TGCTs.     

KIT mutations are common in testicular seminomas

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT.     

Detection of c-kit exons 11- and 17-activating mutations in testicular seminomas by high-resolution melting amplicon analysis.

A subgroup of testicular seminomas has been reported to contain activating mutations in KIT, the transmembrane tyrosine kinase receptor encoded by the c-kit gene. Most mutations are in exon 17, although exon 11-activating mutations have recently been described. For patients refractory to standard therapeutic protocols for seminoma, the presence of c-kit-activating mutations in some of these neoplasms might suggest an alternative therapy with KIT targeting drugs. We used the novel mutation scanning technique, high-resolution melting amplicon analysis, to screen a series of 22 testicular seminomas for c-kit-activating mutations. Four cases (18%) had exon 17-activating mutations and these included D816Y, D816V, Y823N and one case that contained both D816E and D820H. A single case (5%) had an exon 11-activating mutation. Interestingly, the exon 11-activating mutation was L576P, the same mutation that characterizes the rare c-kit mutation-positive cases of malignant melanoma. Fluorescence in situ hybridization (FISH) for c-kit suggested that most seminomas are probably polysomic for c-kit and there was not a significant difference in c-kit FISH characteristics between the mutation-positive and mutation-negative cases. The use of high-resolution melting amplicon analysis as a screening technique will allow for the rapid identification of patients with testicular seminomas whose tumors contain c-kit-activating mutations. This could benefit patients whose tumors are refractory to standard therapeutic protocols.     

N-cadherin expression in malignant germ cell tumours of the testis

Background

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18–35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth.

Methods

Tumour tissues were acquired from 113 male patients and investigated by immunohistochemistry, as were the three TGCT cell lines NCCIT, NTERA-2 and Tcam2. A monoclonal antibody against N-cadherin was used.

Results

Tumour-free testis and intratubular germ cell neoplasias (unclassified) (IGCNU) strongly expressed N-cadherin within the cytoplasm. In all seminomas investigated, N-cadherin expression displayed a membrane-bound location. In addition, the teratomas and yolk sac tumours investigated also differentially expressed N-cadherin. In contrast, no N-cadherin could be detected in any of the embryonal carcinomas and chorionic carcinomas examined. This expression pattern was also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma.

Conclusions

N-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT.

     

The association between intratubular seminoma and invasive germ cell tumors

Intratubular seminoma (ITS) has been defined as the complete filling of the seminiferous tubules with seminoma cells with no Sertoli cells present. This contrasts with intratubular germ cell neoplasia, unclassified (IGCNU), where the malignant germ cells are interspersed by Sertoli cells. We aimed to determine the relationship between these 2 entities and the association between ITS and invasive classic seminomas. We therefore examined the morphology and immunochemistry of ITS and IGCNU adjacent to germ cell tumors to differentiate the patterns, frequency, and distribution of these lesions. We found that ITS was seen in equal frequency adjacent to seminomas as it was to nonseminomas. The presence of ITS in non-seminomatous germ cell tumors suggests that it is a true in situ lesion rather than representative of intratubular spread of an existing seminoma. However, because it is not specifically associated with seminoma, we suggest that it is not useful to discriminate this lesion from IGCNU and that it merely represents an advanced form of IGCNU on the way to invasive malignancy.     

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. Copyright © 1999 John Wiley & Sons, Ltd.     

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.     

Human testicular (non)seminomatous germ cell tumours: the clinical implications of recent pathobiological insights

Human germ cell tumours (GCTs) comprise several types of neoplasias with different pathogeneses and clinical behaviours. A classification into five subtypes has been proposed. Here, the so‐called type II testicular GCTs (TGCTs), ie the seminomas and non‐seminomas, will be reviewed with emphasis on pathogenesis and clinical implications. Various risk factors have been identified that define subpopulations of men who are amenable to early diagnosis. TGCTs are omnipotent, able to generate all differentiation lineages, both embryonic and extra‐embryonic, as well as the germ cell lineage itself. The precursor lesion, composed of primordial germ cells/gonocytes, is referred to as carcinoma in situ of the testis (CIS) and gonadoblastoma of the dysgenetic gonad. These pre‐malignant cells retain embryonic characteristics, which probably explains the unique responsiveness of the derived tumours to DNA‐damaging agents. Development of CIS and gonadoblastoma is crucially dependent on the micro‐environment created by Sertoli cells in the testis, and granulosa cells in the dysgenetic gonad. OCT3/4 has high sensitivity and specificity for CIS/gonadoblastoma, seminoma, and embryonal carcinoma, and is useful for the detection of CIS cells in semen, thus a promising tool for non‐invasive screening. Overdiagnosis of CIS due to germ cell maturation delay can be avoided using immunohistochemical detection of stem cell factor (SCF). Immunohistochemistry is helpful in making the distinction between seminoma and embryonal carcinoma, especially SOX17 and SOX2. The different non‐seminomatous histological elements can be recognized using various markers, such as AFP and hCG, while others need confirmation. The value of micro‐satellite instability as well as BRAF mutations in predicting treatment resistance needs validation in prospective trials. The availability of representative cell lines, both for seminoma and for embryonal carcinoma, allows mechanistic studies into the initiation and progression of this disease. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Molekulare Pathologie der testikulären Keimzelltumoren: ein Update

Germ cell tumors (GCT) comprise a heterogeneous group of benign and malignant tumors. Based on their different biological characteristics and their origin, five types of GCT are classified. Among these, malignant seminomatous and non-seminomatous GCT in males and females are designated as type II GCT. They occur most frequently as malignant testicular GCTs. Many characteristics of type II GCT can be linked to embryonic stem cells. Intratubular germ cell neoplasia, unclassified (IGCNU) is the precursor of type II GCT and derives from undifferentiated germ cells, gonocytes, which persist in the newborn testis and escape the normal differentiation process. It is suggested that Exon-17-activated mutations of the receptor tyrosine kinase, c-KIT, occur early in germ cell development and that gonocytes with an activated c-KIT receptor are restricted in their differentiation, thereby escaping normal development. New diagnostic markers for neoplastic germ cells, including OCT3/4 and AP-2gamma, are specifically detected in IGCNU, seminomas and embryonal carcinomas and are helpful in the differentiation of type II GCT from other malignant tumors.     

Loss of heterozygosity of selected tumor suppressor genes in human testicular germ cell tumors

Human testicular germ cell tumors (TGCTs) are histologically heterogenous neoplasms with a variable malignant potential. Two main groups of germ cell tumors occur in men: seminomas and nonseminomas. In the present study, a set of four tumor suppressor genes was investigated in testicular cancers. CDH1, APC, p53, and nm23-H1 genes were tested for loss of heterozygosity (LOH). Thirty-eight testicular germ cell tumors (17 seminomas and 21 nonseminomas) were analyzed by PCR using restriction fragment length polymorphism or the dinucleotide/tetranucleotide repeat polymorphism method. An allelic loss of p53 at exon 4 was detected in five nonseminomas, whereas LOH of p53 at intron 6 occurred in one of the seminoma and two of the nonseminoma samples. Allelic losses of the APC gene were present in three seminomas and one nonseminoma, whereas one seminoma and three nonseminomas showed LOH of CDH1. The analysis of allelic losses showed no common structural genetic alterations in tumor tissues, although a different pattern of LOH was observed between the two main histological groups of TGCTs.     

Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is approximately 8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.     

Immunohistochemical staining of germ cell tumors and intratubular malignant germ cells of the testis using antibody to placental alkaline phosphatase and a monoclonal anti-seminoma antibody.

Antibody to placental alkaline phosphatase (PLAP) has been previously used in immunoperoxidase (IMP) staining studies of germ cell tumors and intratubular malignant germ cells (ITMGC) of the testis, the latter believed to be the precursor of these tumors. In this study, we compared staining by IMP using monoclonal antibody (mAb) and polyclonal antibody to PLAP with that seen using a mAb, M2A, which was previously shown to react with testicular seminomas and ITMGC. Antibody to PLAP and M2A reacted with different cellular components, as assessed by IMP staining of placenta and prepubertal testis and by Western blotting of seminoma lysates. Antibody to PLAP stained pure seminomas (seven of seven), pure embryonal carcinomas (four of four), and the seminoma (three of three) and embryonal carcinoma (six of six) components of mixed testicular germ cell tumors. M2A stained pure seminomas (26 of 26) and the seminoma component (three of three) of the mixed tumors, but failed to stain pure embryonal carcinomas (zero of four) or the embryonal carcinoma component (zero of five) of the mixed tumors. Both antibody to PLAP and M2A stained ITMGC of the testis. Since M2A stained seminomas and ITMGC but not embryonal carcinomas, seminomas would appear to be more closely related to ITMGC than embryonal carcinomas. This result has led us to speculate on the histogenesis of testicular germ cell tumors.     

The epigenome of testicular germ cell tumors

Gene expression is tightly regulated in normal cells, and epigenetic changes disturbing this regulation are a common mechanism in the development of cancer. Testicular germ cell tumor (TGCT) is the most common malignancy among young males and can be classified into two main histological subgroups: seminomas, which are basically devoid of DNA methylation, and nonseminomas, which in general have methylation levels comparable with other tumor tissues, as shown by restriction landmark genome scanning (RLGS). In general, DNA methylation seems to increase with differentiation, and among the nonseminomas, the pluripotent and undifferentiated embryonal carcinomas harbor the lowest levels of DNA promoter hypermethylation, whereas the well-differentiated teratomas display the highest. In this regard, TGCTs resemble the early embryogenesis. So far, only a limited number of tumor suppressor genes have been shown to be inactivated by DNA promoter hypermethylation in more than a minor percentage of TGCTs, including MGMT, SCGB3A1, RASSF1A, HIC1, and PRSS21. In addition, imprinting defects, DNA hypomethylation of testis/cancer associated genes, and the presence of unmethylated XIST are frequent in TGCTs. Aberrant DNA methylation has the potential to improve current diagnostics by noninvasive testing and might also serve as a prognostic marker for treatment response.     

Pathogenesis of adult testicular germ cell tumors. A cytogenetic model

In essence, two models exist of the pathogenetic relationship between seminomas and nonseminomatous germ cell tumors (NSGCTs). In the first model, the histogenesis of seminomas is assumed to diverge from that of the other testicular germ cell tumors (TGCTs) at an early stage. The neoplastic pathway of seminomas and NSGCTs is different, with limited or no crossover. The second model suggests that seminomas and NSGCTs have a common origin with a single neoplastic pathway on which seminomas are an intermediate stage in development of NSGCTs. Our data on the cytogenetics and ploidy of seminomas, combined tumors, and NSGCTs lend support to the model of pathogenesis of seminomas and NSGCTs in which all TGCTs (with the possible exception of spermatocytic seminoma and infantile yolk sac tumor) have a single origin and neoplastic pathway, with seminomas representing an intermediate stage in development of NSGCT components, as opposed to the model in which seminomas and NSGCTs develop separately. The progression of TGCTs probably proceeds from high to lower numbers of chromosomes and is therefore accompanied by a net loss of chromosomal material. This decrease will be the end result of loss of specific chromosomes, gain of some other chromosomes (or part of chromosomes), and development of structural abnormalities.