Invasive Testicular Germ Cell Tumors Research Articles

Molecular Pathogenesis of Testicular Germ Cell Tumors

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men and their incidence has been increasing over the past decades. Several factors may combine and play a role in the TGCT etiology, including environmental factors and genetic predispositions at multiple genomic loci that affect both testicular germ cells and stromal cells, and their interactions within the testicular microenvironment. The pathogenesis of TGCT starts prenatally with primordial germ cell arrest, and further proceeds postnatally, giving rise to in situ germ cell neoplasia and, finally, to invasive TGCT with the characteristic 12p chromosome amplification. Apart from the genes localized here, further molecular mechanisms have been linked to TGCT pathogenesis, such as the activation of the KIT/KITL signaling pathway, and aberrations in genes involved in DNA reparation, regulation of cellular differentiation, proliferation, and survival. Despite the relatively good prognosis and known etiopathogenesis of these tumors, neither targeted therapy nor molecular prognostic/predictive factors have yet been implemented in the management of TGCT, because there is not enough information about the molecular pathways or molecules involved in TGCT development that could be used for patient stratification and treatment. Current high-throughput technologies, such as next generation sequencing at the exome or transcriptome level could provide this missing information on genetic predispositions and other factors influencing the clinical course of the disease and treatment response in TGCT. In this review, we summarize the main molecular characteristics of TGCT and the probable mechanisms participating in tumor initiation and progression.Key words: testicular germ cell tumors - signaling pathways - molecular aberrations - predictive factors - prognostic factors The work was supported by the Czech Ministry of Education, Youth and Sports NPU I nr.LO 1604. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 20. 3. 2017Accepted: 23. 7. 2017.

Malignant testicular germ cell tumors in postpubertal individuals with androgen insensitivity: prevalence, pathology and relevance of single nucleotide polymorphism-based susceptibility profiling.

What is the prevalence of malignant testicular germ cell tumors (TGCT) and its precursors, (pre-) germ cell neoplasia in situ (GCNIS), in late teenagers and adults who have androgen insensitivity syndrome (AIS) and the impact of an individual's genetic susceptibility to development of TGCT? No GCNIS or TGCT was diagnosed, but pre-GCNIS was identified in 14 and 10% of complete and partial AIS patients, respectively, and was associated with a higher genetic susceptibility score (GSS), with special attention for KITLG (rs995030) and ATFZIP (rs2900333). Many adult women with AIS decline prophylactic gonadectomy, while data regarding the incidence, pathophysiology and outcomes of TGCT in postpubertal individuals with AIS are lacking. The relevance of genetic factors, such as single nucleotide polymorphisms (SNPs), in predisposing AIS individuals to TGCT is unknown. This multicenter collaborative study on prophylactically removed gonadal tissue was conducted in a pathology lab specialized in germ cell tumor biology. Material from 52 postpubertal individuals with molecularly confirmed AIS (97 gonadal samples) was included; the median age at surgery was 17.5 (14-54) years. Immunohistochemical studies and high-throughput profiling of 14 TGCT-associated SNPs were performed. The main outcome measures were the prevalence of pre-GCNIS, GCNIS and TGCT, and its correlation with a GSS, developed based on the results of recent genome-wide association studies. The earliest recognizable change preceding GCNIS, referred to as pre-GCNIS, was present in 14% of individuals with complete and 10% of those with partial AIS at a median age of 16 years. No GCNIS or invasive TGCT were found. The median GSS was significantly greater for those with, compared to those without, pre-GCNIS (P = 0.01), with an overlap between groups. Our data suggest important roles for risk alleles G at KITLG (rs995030) and C at ATFZIP (rs2900333), among the 14 studied TGCT-associated SNPs. N/A. A limited number of cases were included. Our data suggest that the prevalence of pre-GCNIS in individuals with AIS beyond puberty is around 15%. Genetic susceptibility likely contributes to pre-GCNIS development in AIS but factors related to malignant progression remain unclear. Although data in older patients remain scarce, malignant progression appears to be a rare event, although the natural history of the premalignant lesion remains unknown. Therefore, the practice of routine prophylactic gonadectomy in adults with AIS appears questionable and the patient's preference, after having been fully informed, should be decisive in this matter. This study was supported by research grants from the Research Foundation Flanders (FWO) (to M.C.), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq G0D6713N) (to B.B.M. and M.C.) and the European Society for Pediatric Endocrinology (ESPE), granted by Novo Nordisk AB (to J.K.). There are no competing interests.

Is a CIS phenotype apparent in children with Disorders of Sex Development? Milder testicular dysgenesis is associated with a higher risk of malignancy.

All malignant testicular germ cell tumors (TGCT) of adult men are preceded by an in situ stage (CIS) of protracted evolution. The adult CIS is well characterized, but there is debate on the phenotype of infantile CIS, its distinction from delayed maturation of germ cells and prognostic potential. A large series of 43 patients with Disorders of Sex Development (DSD) and dysgenetic testes (90% ranging from neonates to 12years, mean age 4.7years), was studied by quantifying dysgenetic features, degree of germ cell abnormalities/atypia (GCA), expression of OCT 3/4 (a pluripotency-undifferentiation marker), germ cell ploidy and evolution to CIS and invasive TGCT. Findings were compared with those of normal testes. The type of gonads present defined three groups of patients: bilateral testes (BT-DSD, n=21), one testis and one streak gonad (CT-DSD, C for combined, n=13), and ovarian-testicular combinations (OT-DSD, n=9). There were 5 boys with infantile CIS, bilateral in 3 (total of 8 infantile CIS) and two patients with adult CIS, bilateral in one (total of 3 adult CIS). Two patients had bilateral seminomas one at 12-17 and the other at 23years. Histological dysgenesis was significantly higher in CT-DSD (p<0.05), that had only 1 CIS. The highest frequency of GCA was in BT-DSD (p<0.05), which coincided with a total of 11CIS+Seminomas. In all patients, aneuploidy was significantly higher (63%) than diploidy (p<0.02), and GCA were more frequent in aneuploid than in diploid samples (p<0.02). All CIS and TGCT were OCT 3/4 positive. Finally, there was a significant association between the triad Aneuploidy+GCA+OCT 3/4 positivity and the incidence of CIS (Fisher Exact test p<0.002, relative risk 7.0). The degree of testicular dysgenesis (derived from abnormal organization of Sertoli cells in fetal testicular cords) is inversely related to the incidence of CIS. Our data demonstrate that the combined use of OCT 3/4 expression, quantification of germ cell abnormalities-atypia and ploidy in dysgenetic testes can satisfactorily identify infantile CIS with high risk of malignant evolution and set it aside from delayed germ cell maturation with lower or nil neoplastic potential.

Vitamin D and male reproduction

Vitamin D is a versatile signalling molecule with a well-established role in the regulation of calcium homeostasis and bone health. The spectrum of vitamin D target organs has expanded and the reproductive role of vitamin D is highlighted by expression of the vitamin D receptor (VDR) and enzymes that metabolize vitamin D in testis, male reproductive tract and human spermatozoa. The expression levels of VDR and CYP24A1 in human spermatozoa serve as positive predictive markers of semen quality, and VDR mediates a nongenomic increase in intracellular calcium concentration that induces sperm motility. Interestingly, functional animal models show that vitamin D is important for estrogen signalling and sperm motility, while cross-sectional studies support the positive association between serum 25-hydroxyvitamin D level and sperm motility in both fertile and infertile men. Expression of VDR and enzymes that metabolize vitamin D in fetal testis indicates a yet unknown role during development, which may be extrapolated from invasive testicular germ cell tumours where 1α,25-dihydroxyvitamin D induces a mesodermal differentiation of the pluripotent testicular cancer cells. Taken together, vitamin D signalling has a positive effect on semen quality, increases estrogen responsiveness and differentiates germ cell tumours. Future studies are needed to determine when 1α,25-dihydroxyvitamin D acts in a paracrine manner and whether systemic changes, which are subject to pharmacological modulation, could influence male reproductive function.

The downside of ‘inappropriate messaging’: new insight into the development of testicular germ cell tumours in young men?

How invasive testicular germ cell tumours (TGCTs) develop from precursor carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/IGCNU) cells, and only after puberty, is unknown. In the current issue of The Journal of Pathology, Jørgensen and colleagues have compared the protein expression profile of CIS before and after puberty and in pre-invasive versus invasive TGCT and show that the mitosis-meiosis controller DMRT1 switches off in CIS cells postpubertally and is associated with invasiveness. They also show that CIS cells express a 'confusing' mix of pro- and anti-meiotic proteins; this may predispose CIS cells to accumulate extra chromosomal material which ultimately leads to tumourigenesis.

Dysregulation of the mitosis–meiosis switch in testicular carcinoma in situ

Testicular germ cell tumours (TGCT) of young adults arise from the intratubular precursor, carcinoma in situ (CIS). CIS cells are thought to be developmentally arrested and transformed fetal germ cells that survive through childhood and gain invasive capacity after puberty. Given that germ cell neoplasms arise frequently in undervirilized and dysgenetic gonads and the striking physiological difference between meiotic entry in ovaries (fetal life) versus testes (at puberty), this study aimed to investigate whether errors in regulation of meiosis may be implicated in the pathogenesis of CIS or its invasive progression to TGCT. The main focus was on a key sex differentiation and meiosis regulator, DMRT1, which has also been linked to TGCT risk in recent genetic association studies. Expression patterns of DMRT1 and other meiosis regulators (SCP3, DMC1, STRA8, CYP26B1, NANOS2, NANOS3) were investigated in pre- and post-pubertal CIS samples and TGCT by quantitative RT-PCR and immunohistochemistry. The results demonstrated that meiosis markers and meiosis inhibitors were simultaneously expressed in CIS cells, in both pre- and post-pubertal testis samples. DMRT1 was present in a restricted subset of CIS cells, which was relatively greater in pre-pubertal (27%) compared to adult (2.6%) samples. In contrast to the majority of CIS cells, DMRT1-positive CIS cells in adult testes were not proliferating. DMRT1 and most of the other meiosis regulators were absent or expressed at low levels in invasive TGCT, except in spermatocytic seminoma (not derived from CIS). In conclusion, this study indicates that meiosis signalling is dysregulated in CIS cells and that a key regulator of the mitosis-meiosis switch, DMRT1, is expressed in 'early-stage' CIS cells but is down-regulated with further invasive transformation. Whether this mixed meiosis signalling in CIS cells is caused by insufficient virilization of the fetal somatic niche or a partial post-pubertal maturation remains uncertain and requires further study.

Vitamin D Metabolism and Effects on Pluripotency Genes and Cell Differentiation in Testicular Germ Cell Tumors In Vitro and In Vivo

Testicular germ cell tumors (TGCTs) are classified as either seminomas or nonseminomas. Both tumors originate from carcinoma in situ (CIS) cells, which are derived from transformed fetal gonocytes. CIS, seminoma, and the undifferentiated embryonal carcinoma (EC) retain an embryonic phenotype and express pluripotency factors (NANOG/OCT4). Vitamin D (VD) is metabolized in the testes, and here, we examined VD metabolism in TGCT differentiation and pluripotency regulation. We established that the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human fetal germ cells, CIS, and invasive TGCTs. VD metabolism diminished markedly during the malignant transformation from CIS to EC but was reestablished in differentiated components of nonseminomas, distinguished by coexpression of mesodermal markers and loss of OCT4. Subsequent in vitro studies confirmed that 1,25(OH)2D3 (active VD) downregulated NANOG and OCT4 through genomic VDR activation in EC-derived NTera2 cells and, to a lesser extent, in seminoma-derived TCam-2 cells, and up-regulated brachyury, SNAI1, osteocalcin, osteopontin, and fibroblast growth factor 23. To test for a possible therapeutic effect in vivo, NTera2 cells were xenografted into nude mice and treated with 1,25(OH)2D3, which induced down-regulation of pluripotency factors but caused no significant reduction of tumor growth. During NTera2 tumor formation, down-regulation of VDR was observed, resulting in limited responsiveness to cholecalciferol and 1,25(OH)2D3 treatment in vivo. These novel findings show that VD metabolism is involved in the mesodermal transition during differentiation of cancer cells with embryonic stem cell characteristics, which points to a function for VD during early embryonic development and possibly in the pathogenesis of TGCTs.

AZFa protein DDX3Y is differentially expressed in human male germ cells during development and in testicular tumours: new evidence for phenotypic plasticity of germ cells

DDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1-S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes. DDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry. Germ cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function. The fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.

Overexpression of SOX5 by a Novel Chromosome Translocation Involving the IGH Locus In a Follicular Lymphoma without BCL2 Expression

AbstractAbstract 4453The majority of follicular lymphoma (FL) cases are characterized by upregulation of BCL2 expression in tumor cells as results of a specific oncogenic event, t(14;18)(q32;q21), or other molecular mechanisms including amplification of the BCL2 gene. Approximately 10% of FL cases are lack of t(14;18)(q32;q21) and BCL2 expression. Molecular pathogenesis of BCL2 negative-FL has not been clarified.We found a case of BCL2-negative FL with a chromosome abnormalities including t(12;14)(p12;q32). Translocations involving 14q32 are frequently seen in B-lymphoid neoplasms, and breakpoints are often clustered within the IGH gene locus. Chromosome translocations involving the IGH locus represent the molecular mechanism of activation of a number of proto-oncogenes in B-cell neoplasms. In the present case, we hypothesized that as a result of the translocation a target gene on 12p12 is upregulated by juxtaposition to an enhancer region within the IGH gene and may play an important role in lymphoma development. Therefore, we attempted to determine a breakpoint of t(12;14)(p12;q32) by using a long-distance inverse PCR (LDI-PCR). Genomic DNA isolated from a lymph node sample was digested with restriction enzymes. Fragmented DNA was ligated to form circular DNA, and subjected to LDI-PCR. LDI-PCR was carried out using primer sets complementary to regions within the IGH locus. PCR products were subcloned into pGEM-T vector, and sequenced. The sequences were analyzed by a BLAST computational search. We determined the sequence around a breakpoint, in which we found the IGH gene J region on 14q32, as expected, and intron 1 of the SRY-related high-morbidity-group (HMG) box 5 (SOX5) gene on 12p12. In the rearranged chromosome region, an enhancer region within the IGH gene is located to up-stream of the exon 2 of the SOX5 gene. Coding region of the SOX5 genes spans from exons 5 to 20 so that all of coding exons of the SOX5 gene are retained on the rearranged chromosome. We examined SOX5 expression by immunohistochemistry using anti-SOX5 rabbit polyclonal antibody. SOX5 is overexpressed in nuclei of tumor cells in neoplastic follicle.SOX5 is a member of SOX family transcriptional factors which are characterized by the HMG box DNA binding domain. SOX5 is involved in the regulation of embryonic development and in the determination of the cell fate. In addition, SOX5 may play roles in the pathogenesis of some types of human cancers. In invasive testicular germ cell tumors, in which gain of short arm of chromosome 12 is frequently found, the SOX5 gene amplification is observed. In nasopharyngeal carcinoma, SOX5 overexpression is associated with poor clinical outcome, and SOX5 works as a transcriptional repressor for SPARC which is a putative tumor suppressor gene. These findings suggest that the SOX5 gene is a kind of proto-oncogene. Currently, Storlazzi et al. reported that SOX5 expression is upregulated by promoter swapping with the P2RY8 gene, as a result of t(X;12)(p22;p12) in primary splenic FL without BCL2 expression. Their report and our present result suggest that SOX5 overexpression play a role in BCL2-negative FL development. Further studies are necessary to clarify a molecular biological role of SOX5 overexpression in the pathogenesis of BCL2-negative FL. Disclosures:No relevant conflicts of interest to declare.

Intratubular germ cell neoplasms of the testis and bilateral testicular tumors: Clinical significance and management options

Objectives:Intratubular germ cell neoplasia (ITGCN) is the precursor lesion for invasive testicular germ cell tumors (TGCTs) of adolescents and young adults. The rising incidence of these tumors has prompted a rigorous investigation of the etiology, diagnosis and management of ITGCN. Bilateral testicular cancer is closely linked with ITGCN, as patients with unilateral testicular cancer are at the highest risk for a future malignancy in the contralateral testicle.Methods:A literature review directed at ITGCN and bilateral testis cancer was performed using the Medline/PubMed database. Our review focused on the pathogenesis, risk factors, diagnosis and treatment regimens utilized.Results:Major advances have been made in the understanding of ITGCN over the past 30 years. There is evidence that TGCTs arise from ITGCN, ITGCN is closely related to fetal gonocytes, and that events in pre- and perinatal period may result in abnormal persistence of fetal gonocytes leading to ITGCN and subsequent TGCT. Controversy exists regarding the need to biopsy men at increased risk of TGCT, as well as the best approach to managing patients with known ITGCN. Bilateral testicular cancer has excellent outcomes in the current era of platinum-based chemotherapy.Conclusion:The optimal management of patients at risk for ITGCN and future TGCT is still a matter of debate. Individualization of management, including biopsy and treatment, should be based on risk factors for TGCT, compliance with potential surveillance, and patient preferences particularly with regard to fertility.

Deregulation of EZH2 expression in human spermatogenic disorders and testicular germ cell tumors

Enhancer of Zeste 2 (EZH2) is an epigenetic transcriptional repressor involved in cell cycle control and cell fate decisions. Since these processes play key roles during intact spermatogenesis, deregulation of EZH2 expression may contribute to the development and progression of benign and malignant testicular diseases. The objective of this study was to investigate the expression profile of EZH2 in testicular germ cell tumors (TGCT) and spermatogenic defects. Real-time RT-PCR was applied to quantify the m-RNA expression of EZH2 in 64 seminomas 36 non-seminomas, 4 carcinomas in situ (CIS), 40 samples harboring impaired spermatogenesis and 24 normal testicular reference biopsies. EZH2 was expressed in 99% of TGCT samples and in all biopsies with intact spermatogenesis. Its expression levels were highest in normal testicular tissue, and continuously decreased with malignant transformation to CIS and further progression to invasive TGCT (P<0.001). EZH2 tumor levels were not related to the histological TGCT subtype or clinical tumor stage. Comparison of distinct stages of spermatogenic failure revealed an inverse association of EZH2 levels to the severity of the spermatogenic defect (P<0.001). Our data strongly suggest that in TGCT EZH2 does not exert its often assumed oncogenic properties during malignant transformation and progression. High EZH2 levels in normal testicular tissue and the inverse association of its expression levels with the severity of spermatogenic failure point to its potential value as a molecular marker for spermatogenic defects and may indicate an important physiological role of EZH2 during intact spermatogenesis.

Evaluation of testicular biopsies for carcinoma in situ: immunohistochemistry is mandatory

Carcinoma in situ (CIS) is the common precursor of all type II testicular germ cell tumors (TGCTs), i.e. seminomas and non-seminomas, which can be diagnosed using a surgical biopsy. The objective of this study was to investigate the additional value of immunohistochemistry for the diagnosis of CIS in assessing testicular biopsies taken in the context of infertility. A series of 21 infertile patients were retrieved from the Dutch pathological database (PALGA), being diagnosed with an invasive TGCT, while a matched previously obtained testicular biopsy was diagnosed as non-malignant. From 20 patients, both the invasive tumors as well as the biopsies were revised using morphology and immunohistochemistry for OCT3/4, placental-like alkaline phosphatase and c-KIT, all known established markers for CIS. The presence of CIS or invasive malignancies was scored. There are no interventions. Morphological criteria alone allowed an experienced pathologist in TGCTs to diagnose CIS in five and an invasive tumor in two cases (total n = 7, 35%). Application of immunohistochemistry resulted in the identification of an additional four cases of CIS (total n = 11, 55%, additional value of 20%). The initial correct diagnosis of CIS could have prevented a second gonadectomy in four patients (20%). This study, for the first time, really shows that time of progression from CIS to seminoma is longer than to non-seminoma. Our study demonstrates that immunohistochemistry should be performed for the diagnosis of CIS of the testis on single biopsies obtained because of infertility, resulting in an extra diagnostic yield of at least 20%. Application of this protocol will allow early diagnosis, and therefore prevent any adverse anti-cancer treatment sequelae including gonadectomy, and requiring life long androgen supplementation in some patients.

Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors

Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5mC) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5mC erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5mC levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5mC to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5mC was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5mC in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5mC staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5mC erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.

Association of Intratubular Seminoma and Intratubular Embryonal Carcinoma With Invasive Testicular Germ Cell Tumors

The classification of intratubular germ cell neoplasia of the testis includes an unclassified type (IGCNU), in addition to various other intratubular lesions that show specific forms of differentiation, such as intratubular seminoma and intratubular embryonal carcinoma. Although IGCNU is recognized as a precursor lesion for testicular germ cell tumors, the relationship between differentiated types of intratubular germ cell neoplasia and invasive germ cell tumors of the testis is not well established. The aim of the present study was to examine the association between invasive testicular germ cell tumors and intratubular neoplastic lesions, with particular emphasis on differentiated types of intratubular germ cell neoplasia. The seminiferous tubules adjacent to 42 testicular germ cell tumors were evaluated for the presence of various forms of intratubular germ cell neoplasia. IGCNU was observed in 37 (88%) of 42 cases, whereas intratubular seminoma and intratubular embryonal carcinoma were seen in 19% and 7% of the cases, respectively. Intratubular seminoma was associated primarily with seminomas or mixed germ cell tumors with a seminomatous component, but was also present in a case of a nonseminomatous germ cell tumor. Intratubular embryonal carcinoma was associated exclusively with nonseminomatous germ cell tumors. All cases of intratubular embryonal carcinoma were identified morphologically and exhibited histologic features corresponding to traditional definitions of this lesion. No examples of intratubular embryonal carcinoma as defined by CD30 expression alone in the absence of an intratubular proliferation were observed. The presence of intratubular seminoma in a nonseminomatous germ cell tumor suggests that it is a true preinvasive lesion rather than a manifestation of intratubular spread of an established invasive seminoma. The low incidence of intratubular embryonal carcinoma supports the theory that most nonseminomatous germ cell tumors evolve initially as seminomas, rather than directly from a differentiated intratubular neoplastic lesion.

Diagnostic value of OCT3/4 for pre‐invasive and invasive testicular germ cell tumours

Human testicular germ cell tumours of adolescents and adults (TGCTs), the seminomatous and non-seminomatous germ cell tumours, show morphological and biological similarities to normal embryonic development, presumably determined by their supposed cell of origin, the primordial germ cell/gonocyte. Based on this knowledge, OCT3/4, also known as POU5F1, was recently defined as a diagnostic marker for these tumour types. In the adult testis, positive immunohistochemistry for OCT3/4 is an absolute indicator for the presence of the TGCT precursor carcinoma in situ/intratubular germ cell neoplasia undifferentiated (CIS/ITGCNU), seminoma, and/or embryonal carcinoma. Several studies have confirmed this observation, using the same polyclonal antibody. The present study demonstrates the usefulness of OCT3/4 immunohistochemistry in a diagnostic setting of a consecutively collected series of more than 200 testicular tumours and over 80 testicular biopsies. Moreover, it is shown that a monoclonal antibody directed against OCT3/4 is as informative as the polyclonal antibody, both in immunohistochemistry and in western blot analysis. The antibodies are robust and applicable with different methods of pretreatment and storage of tissue. This allows routine application of this diagnostic marker.

Fluorescence in situ hybridization analysis of chromosome 12p in paraffin-embedded tissue is useful for establishing germ cell origin of metastatic tumors

The over-representation of chromosome 12p sequences is crucial for the development of invasive testicular germ cell tumors. Testicular cancer patients may have metastatic tumors of diverse histologic types, including adenocarcinoma, undifferentiated carcinoma, sarcoma, or other malignancies that lack features of germ cell tumors. We sought to investigate the possible germ cell origin of such tumors using interphase fluorescence in situ hybridization. In all, 10 metastatic malignant somatic-type tumors from patients with histories of testicular cancer, as well as one malignant somatic-type tumor from a patient with primary mediastinal germ cell tumor were studied and included: adenocarcinoma (five cases), poorly differentiated carcinoma (one), sarcoma (four), and neuroendocrine carcinoma (one). The tumors were analyzed using fluorescence in situ hybridization using 12p spectrum green and 12 centromeric spectrum orange probes in paraffin sections. The patients ranged in age from 27 to 55 years (mean, 43). Colon and lung cancers from patients without germ cell tumors were used as controls. Adequate signals were observed in all tumors. Gain of chromosome 12p was seen in six tumors. None of the control tumors showed 12p amplification. Fluorescence in situ hybridization for 12p amplification in routinely processed surgical specimens is a useful adjuvant diagnostic tool in confirming the germ cell origin of metastatic tumors having the histologic appearance of somatic-type neoplasms.

12p-amplicon structure analysis in testicular germ cell tumors of adolescents and adults by array CGH.

All invasive testicular germ cell tumors of adolescents and adults (TGCTs), that is, seminomas and nonseminomas, show gain of 12p sequences, mostly as isochromosomes. Although several candidate genes have been suggested, the relevant gene(s) have not been identified yet. About 10% of testicular seminomas, however, show a more restricted amplification of the 12p11.2-p12.1 region, in which the various amplicons show an apparent overlap, allowing for the shortest region of amplification overlap approach, aiming at the identification of pathogenetically relevant sequences residing in this region. Here we report on a high-resolution 12p-amplicon architecture analysis using microarray-based comparative genomic hybridization, the results of which were subsequently confirmed by fluorescent in situ hybridization studies. The 12p-specific microarray contained 63 positionally selected BAC clones, which are more or less evenly distributed over the short arm of chromosome 12 (average spacing: less than 500 Kb), including 20 clones within the region of amplification. Out of a series of 17 seminomas, seven seminomas showed amplification of the whole amplicon region, of which three showed a dip in T/R value in the center of the amplified area. A more complex amplification pattern was found in the other 10 seminomas: three showed predominant amplification at the centromeric border; one mainly at the telomeric border; six showed a balanced amplification of both the centromeric and telomeric regions. The only nonseminoma investigated showed a structure in which the centromeric border was only amplified. These data support a mechanistic model in which at least two 12p genes, situated at the border regions of the amplicon, are positional candidates capable of actively supporting tumor progression in TGCTs.

Deregulation of the RB pathway in human testicular germ cell tumours.

Deregulation of the RB pathway is shared by most human malignancies. Components upstream of the retinoblastoma tumour suppressor (pRB), namely the INK4 family of cyclin-dependent kinase (CDK) inhibitors, the D-type cyclins, their partner kinases CDK4/CDK6, and pRB as their critical substrate, are differentially targeted in diverse types of cancer. An 'unorthodox' spectrum of defects within this cascade occurs in testicular germ cell tumours (TGCTs), including silencing of pRB transcription, overexpression of cyclin D2, and loss of p18INK4c. To improve understanding of the role of this pathway in spermatogenesis, and its subversion in TGCTs, we examined immunohistochemical expression patterns of CDK4, p16INK4a, p15INK4b, and pRB, and established an in situ assay for cyclin D-mediated phosphorylation of serine795, a phosphorylation event critical for neutralization of pRB's growth-restraining ability. pRB was expressed throughout adult spermatogenesis and was detectable in teratomas, but was absent or grossly reduced in carcinoma in situ (CIS) and most seminomas and embryonal carcinomas. Unexpectedly, we also found that pRB was absent from fetal human gonocytes, the candidate target cell for all types of TGCTs. Thus, rather than a tumorigenesis-promoting loss of pRB, the lack of pRB in TGCTs likely reflects its developmental control. Widespread expression of p15INK4b, found in normal testes, was preserved in TGCTs. In contrast, p16INK4a was lost or reduced in large subsets of TGCTs. CDK4 was expressed in normal spermatogonia, CIS, and invasive TGCTs, as was serine795-phosphorylated pRB. Our data on expression of pRB support the plausible origin of TGCTs from fetal gonocytes, and the serine795 phosphorylation demonstrates that the cyclin D-dependent kinases are active, and neutralize pRB in spermatogonia and in those TGCTs that express pRB. We hope that this study will inspire further immunohistochemical applications of phosphospecific antibodies in pathology, and examination of the RB pathway defects in relation to curability of TGCTs.

Role of gain of 12p in germ cell tumour development.

Within the human testis, three entities of germ cell tumours are distinguished: the teratomas and yolk sac tumors of newborn and infants, the seminomas and nonseminomas of adolescents and young adults, referred to as testicular germ cell tumours (TGCT), and the spermatocytic seminomas. Characteristic chromosomal anomalies have been reported for each group, supporting their distinct pathogenesis. TGCT are the most common cancer in young adult men. The initiating pathogenetic event of these tumours occurs during embryonal development, affecting a primordial germ cell or gonocyte. Despite this intra-uterine initiation, the tumour will only be clinically manifest after puberty, with carcinoma in situ (IS) as the precursor. All invasive TGCT, both seminomas and nonseminomas, as well as CIS cells are aneuploid. The only consistent (structural) chromosomal abnormalities in invasive TGCT are gains of the short arm of chromosome 12, mostly due to isochromosome (i(12p)) formation. This suggests that an increase in copy number of a gene(s) on 12p is associated with the development of a clinically manifest TGCT. Despite the numerous (positional) candidate gene approaches that have been undertaken thus far, identification of a causative gene(s) has been hampered by the fact that most 12p gains involve rather large genomic intervals, containing unmanageable numbers of candidate genes. Several years ago, we initiated a search for 12p candidate genes using TGCT with a restricted 12p-amplification, cytogenetically identified as 12p11.2-p12.1. This approach is mainly based on identification of candidate genes mapped within the shortest region of overlap of amplification (SROA). In this review, data will be presented, which support the model that gain of 12p-sequences is associated with suppression of apoptosis and Sertoli cell-independence of CIS cells. So far, DAD-R is one of the most likely candidate genes involved in this process, possibly via N-glycosylation. Preliminary results on high through-put DNA- and cDNA array analyses of 12p-sequences will be presented.

Overrepresentation of the short arm of chromosome 12 is related to invasive growth of human testicular seminomas and nonseminomas.

Overrepresentation of 12p-sequences, mostly due to isochromosome formation, is the only consistent chromosomal alteration found in invasive testicular germ cell tumors of adolescents and young adults (TGCTs), both seminomas and the various histological elements of nonseminomas. The biological role of extra 12p in the pathogenesis of this cancer is unclear, and it is also unknown so far, whether it is an early event, i.e., already present in carcinoma in situ, or related to invasive growth. Using comparative genomic hybridization (CGH) with DOP-PCR amplified DNA isolated from micro-dissected tumor cells, and double fluorescent in situ hybridization (FISH) on frozen tissue sections, we investigated the presence of overrepresentation of 12p sequences in different development stages of four seminomas and seven nonseminomas, in total 17 invasive components, in addition to the carcinoma in situ of each. CGH demonstrated relative gain of 12p-sequences in all invasive components except one, confirmed by FISH in most samples. In contrast, no gain was found in the carcinoma in situ samples by any of the methods. These findings show that overrepresentation of 12p is not an early event in the development of TGCTs, but relates to invasive growth.