Gillis, A.J.

The seminoma cell line TCam-2 is sensitive to HDAC inhibitor depsipeptide but tolerates various other chemotherapeutic drugs and loss of NANOG expression (Article)

2011-12-01T00:00:00Z

Seminomas and embryonal carcinomas (EC) are both type II germ cell tumor (GCT) entities and develop from the same precursor lesion (carcinoma-in situ, CIS). However, they show significant differences in growth behavior, differentiation potential, and gene expression. Although ECs are prone to differentiate into all three germ layers and give rise to the non-seminomatous GCT entities teratoma, choriocarcinoma, and yolk-sac tumor, differentiation of seminomas to these entities is only rarely observed. This might reflect the ability of seminomas to actively inhibit differentiation processes evoked by environmental cues. Also, it is not known why CIS gives rise to seminoma in some patients and to non-seminoma in the others. Here, we treated the seminoma-like cell line TCam-2 with the HDAC-inhibitor Depsipeptide, the global demethylating agent 5-aza-2'-deocycytidine, all-trans retinoic acid and the monaminooxidase inhibitor Tranylcipromine and also used knock down approaches to reduce expression of the pluripotency marker NANOG and/or the inhibitor of primordial germ cell differentiation TFAP2C. We found that TCam-2 cells induce apoptosis when treated with Depsipeptide (>10 nM) but are resistant to treatments with 5-aza-2'-deocycytidine, all-trans retinoic acid and Tranylcipromine, highlighting Depsi as a treatment option for seminomas. We show that TCam-2 cells up-regulate endoderm- and throphectoderm-associated genes after down-regulation of NANOG expression; however, morphologically no indications of differentiation could be found. Instead, we observed up-regulation of OCT3/4 and SOX17 in TCam-2-NANOG knockdown and speculate that this compensates for the loss of the NANOG protein. Hence, NANOG is not a primary target gene responsible for the inhibition of differentiation in seminomas.

Specific detection of OCT3/4 isoform A/B/B1 expression in solid (germ cell) tumours and cell lines: Confirmation of OCT3/4 specificity for germ cell tumours (Article)

2011-09-06T00:00:00Z

Background: OCT3/4 (POU5F1) is an established diagnostic immunohistochemical marker for specific histological variants of human malignant germ cell tumours (GCTs), including the seminomatous types and the stem cell component of non-seminomas, known as embryonal carcinoma. OCT3/4 is crucial for the regulation of pluripotency and the self-renewal of normal embryonic stem-and germ cells. Detection of expression of this transcription factor is complicated by the existence of multiple pseudogenes and isoforms. Various claims have been made about OCT3/4 expression in non-GCTs, possibly related to using nonspecific detection methods. False-positive findings undermine the applicability of OCT3/4 as a specific diagnostic tool in a clinical setting. In addition, false-positive findings could result in misinterpretation of pluripotency regulation in solid somatic cancers and their stem cells. Of the three identified isoforms-OCT4A, OCT4B and OCT4B1-only OCT4A proved to regulate pluripotency. Up until now, no convincing nuclear OCT4A protein expression has been shown in somatic cancers or tissues. Methods: This study investigates expression of the various OCT3/4 isoforms in GCTs (both differentiated and undifferentiated) and somatic (non-germ cell) cancers, including representative cell lines and xenografts. Results: Using specific methods, OCT4A and OCT4B1 are shown to be preferentially expressed in undifferentiated GCTs. The OCT4B variant shows no difference in expression between GCTs (either differentiated or undifferentiated) and somatic cancers. In spite of the presence of OCT4A mRNA in somatic cancer-derived cell lines, no OCT3/4 protein is detected. Significant positive correlations between all isoforms of OCT3/4 were identified in both tumours with and without a known stem cell component, possibly indicating synergistic roles of these isoforms. Conclusion: This study confirms that OCT4A protein only appears in seminomatous GCTs, embryonal carcinoma and representative cell lines. Furthermore, it emphasises that in order to correctly assess the presence of functional OCT3/4, both isoform specific mRNA and protein detection are required.

Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance (Article)

2011-08-01T00:00:00Z

Human type II germ cell tumours (GCTs) originate from an embryonic germ cell, either as a primordial germ cell or gonocyte. This start determines the biological as well as clinical characteristics of this type of cancer, amongst others their totipotency as well as their overall (exceptional) sensitivity to DNA damaging agents. The histology of the precursor lesion, either carcinoma in situ or gonadoblastoma, depends on the level of testicularization (i.e. testis formation) of the gonad. The impact of either intrinsic (genetic) - and environmental factors involved in the pathogenesis is demonstrated by disorders of sex development as well as testicular dysgenesis syndrome as risk factors, including cryptorchidism, hypospadias and disturbed fertility as parameters. This knowledge allows identification of individuals at risk for development of this type of cancer, being a population of interest for screening. Factors known to regulate pluripotency during embryogenesis are proven to be of diagnostic value for type II GCTs, including OCT3/4, even applicable for non-invasive screening. In addition, presence of stem cell factor, also known as KITLG, allows distinction between delayed matured germ cells and the earliest stages of malignant transformation. This is of special interest because of the identified association between development of type II GCTs of the testis and a limited number of single nucleotide polymorphisms, including some likely related to KITL. Transition from the precursor lesion to an invasive cancer is associated with gain of the short arm of chromosome 12, in which multiple genes might be involved, including KRAS2 and possibly NANOG (pseudogenes). While most precursor lesions will progress to an invasive cancer, only a limited number of cancers will develop treatment resistance. Putative explanatory mechanisms are identified, including presence of microsatellite instability, BRAF mutations, apoptosis suppression and p21 sub-cellular localization. It remains to be investigated how these different pathways integrate to each other and how informative they are at the patient-individual level. Further understanding will allow development of more targeted treatment, which will benefit quality of life of these young cancer patients. © 2011 The Authors. International Journal of Andrology

TGF-β1, EGF and FGF4 synergistically induce differentiation of the seminoma cell line TCam-2 into a cell type resembling mixed non-seminoma (Article)

2011-08-01T00:00:00Z

Malignant germ-cell tumours arise from a neoplastic precursor, the carcinoma in situ, and develop into seminomas and/or non-seminomas (embryonal carcinomas, teratomas, yolk-sac tumours and choriocarcinomas). Based on histological and clinical findings, it has been postulated that seminomas can eventually transform into non-seminomas. Here, we used the cell line TCam-2 as model for seminomas and interrogated their differentiation potential. We demonstrate that TCam-2 cells are able to differentiate into mixed non-seminomatous lineages after supplementing the media with TGF-β1, EGF and FGF4. On a molecular level, the differentiation is initiated by repression of BMP/SMAD signalling. As a consequence, BLIMP1, a molecule known to inhibit the differentiation of murine primordial germ cells, is down-regulated and differentiation-inhibiting histone modifications are lost. The appearance of multinucleated giant cells and the expression of marker genes indicate that cells differentiate predominantly into extra-embryonic choriocarcinoma-like cells. This is most likely due to the presence of components of the Hippo pathway, TEAD4 and YAP1. These molecules have been described to trigger extra-embryonic fate determination in the murine system. This study supports the model that seminomas indeed have an intrinsic ability to transform into a non-seminoma. In addition, the data suggest that the transformation does not require an additional mutation, but can be triggered by changes in the tumour microenvironment. © 2011 The Authors. International Journal of Andrology

Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis (Article)

2011-08-01T00:00:00Z

OCT3/4, NANOG, SOX2 and, most recently, LIN28 have been identified as key regulators of pluripotency in mammalian embryonic and induced stem cells, and are proven to be crucial for generation of the mouse germ-cell lineage. These factors are a hallmark of certain histological types of germ-cell tumours (GCTs). Here, we report novel information on the temporal and spatial expression pattern of LIN28 during normal human male germ-cell development as well as various types of GCTs. To investigate LIN28 expression, immunohistochemical analyses and quantitative proximity ligation assay-based TaqMan protein assays were applied on snap-frozen and formalin-fixed, paraffin-embedded samples as well as representative cell lines. LIN28 was found in primordial germ cells, gonocytes and pre-spermatogonia, in contrast to OCT3/4 and NANOG, which were found only in the first two stages. LIN28 was also found in all precursor lesions (carcinoma in situ and gonadoblastoma) of type II GCTs, as well as the invasive components seminoma and the non-seminomatous elements embryonal carcinoma and yolk sac tumour. Choriocarcinoma showed a heterogeneous pattern, while teratomas and spermatocytic seminomas (type III GCTs) were negative. This expression pattern suggests that LIN28 is associated with malignant behaviour of type II GCTs. Cell line experiments involving siRNA knockdown of LIN28, OCT3/4 and SOX2 showed that LIN28 plays a role in the maintenance of the undifferentiated state of both seminoma and embryonal carcinoma, closely linked to, and likely upstream of OCT3/4 and NANOG. In conclusion, LIN28 regulates the differentiation status of seminoma and embryonal carcinoma and is likely to play a related role in normal human germ-cell development. © 2011 The Authors. International Journal of Andrology

Diagnosis of testicular carcinoma in situ'(intratubular and microinvasive)' seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections (Article)

2011-02-01T00:00:00Z

Aims: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for the presence of invasive germ cell tumour components, and the precursor carcinoma in situ (CIS). Currently, diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mainly unavoidable. The aim was to develop an intraoperative assessment technique using alkaline phosphatase as a marker. Methods and results: A total of 4093 snap-frozen samples and matched paraffin-embedded tissue of 1500 patients were included. Besides standard haematoxylin and eosin (H&E) staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15min) was applied on frozen sections, while H&E and immunohistochemistry for detection of OCT3/4, α-fetoprotein, human chorionic gonadotrophin (hCG) and cytokeratin was performed on the paraffin-embedded slides. Endothelial cells served as control for the dAP test. Positive staining was found in all CIS (n=965), seminoma (n=1035) and embryonal carcinoma (n=584), either intratubular, microinvasive or invasive. Differentiated non-seminomas (n=1238) showed variable staining. No staining was identified in spermatocytic seminomas (n=5), testicular lymphomas (n=42), testicular rhabdomyosarcomas (n=7), Leydig cell tumours (n=31), Sertoli-cell-only nodules (n=4), (epi) dermoid cyst (n=16), normal testicular parenchyma (n=116), testicular torsion (n=32) and inflammation of the epididymis (n=19). The dAP test results matched H&E-stained parallel sections, as well paraffin-embedded tissue analysis, including immunohistochemistry. Conclusions: The dAP test is an informative, reproducible and easy tool to diagnose CIS, (intratubular and microinvasive) seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.

Critical function of AP-2gamma/TCFAP2C in mouse embryonic germ cell maintenance (Article)

2010-05-18T00:00:00Z

Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdml activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation.

Stem cell factor as a novel diagnostic marker for early malignant germ cells (Article)

2008-09-01T00:00:00Z

Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low-dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c-KIT, PLAP, AP-2γ, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c-KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT-derived cell lines, specifically in cases of CIS and GB. Both membrane-bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay.

A Pure Triphasic Testicular Wilms Tumor of Primordial Germ Cell Origin (Article)

2008-07-01T00:00:00Z

Further characterization of the first seminoma cell line TCam-2 (Article)

2008-03-01T00:00:00Z

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

High-throughput microRNAome analysis in human germ cell tumours (Article)

2007-11-01T00:00:00Z

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow intersample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. Copyright

Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines (Article)

2007-10-01T00:00:00Z

Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the UI33Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity.

JKT-1 is not a human seminoma cell line (Article)

2007-08-01T00:00:00Z

The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors (Article)

2003-01-01T00:00:00Z

Human germ cell tumors (GCTs) may have variable histology and clinical behavior, depending on factors such as sex of the patient, age at clinical diagnosis, and anatomical site of the tumor. Some types of GCT, i.e., the seminomas/germinomas/dysgerminomas and embryonal carcinomas (the stem cell component of nonseminomas), have pluripotent potential, which is demonstrated by their capacity to differentiate into somatic and/or extraembryonic elements. Although embryonal carcinoma cells are intrinsically pluripotent, seminoma/germinoma/dysgerminoma cells, as well as their precursor carcinoma in situ/gonadoblastoma cells, have the phenotype of early germ cells that can be activated to pluripotency. The other types of GCT (teratomas and yolk sac tumors of infants and newborn, dermoid cyst of the ovary, and spermatocytic seminoma of elderly) are composed of (fully) differentiated tissues and lack the appearance of undifferentiated and pluripotent stem cells. OCT3/4, a transcription factor also known as OTF3 and POU5F1, is involved in regulation of pluripotency during normal development and is detectable in embryonic stem and germ cells. We analyzed the presence of POU5F1 in GCT and other tumor types using immunohistochemistry. The protein was consistently detected in carcinoma in situ/gonadoblastoma, seminomas/germinoma/dysgerminoma, and embryonal carcinoma but not in the various types of differentiated nonseminomas. Multitumor tissue microarray analysis covering >100 different tumor categories and 3600 individual cancers verified that POU5F1 expression is specific for particular subtypes of GCT of adults. No protein was observed in GCT of newborn and infants, spermatocytic seminomas, and the various tumors of nongerm cell origin. In addition, no difference in staining pattern was found in chemosensitive and chemoresistant GCT of adults. These results indicate preservation of the link between POU5F1 and pluripotency, as reported during normal development, after malignant transformation. Therefore, POU5F1 immunohistochemistry is an informative diagnostic tool for pluripotent GCT and offers new insights into the histological heterogeneity of this cancer.

Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors (Article)

2003-01-01T00:00:00Z

Testicular germ-cell tumors (TGCTs) of adolescents and adults originate from intratubular germ cell neoplasia (ITGCN), which is composed of the malignant counterparts of embryonal germ cells. ITGCN cells are characterized, among others, by the presence of stem cell factor receptor c-KIT. Once established, ITGCN will always progress to invasiveness. Approximately 2.5-5% of patients with a TGCT will develop bilateral disease and require complete castration, resulting in infertility, a need for lifelong androgen replacement, and psychological stress. To date, the only way to predict a contralateral tumor is surgical biopsy of the contralateral testis to demonstrate ITGCN. We did a retrospective study of 224 unilateral and 61 proven bilateral TGCTs (from 46 patients, in three independently collected series in Europe) for the presence of activating c-KIT codon 816 mutations. A c-KIT codon 816 mutation was found in three unilateral TGCT (1.3%), and in 57 bilateral TGCTs (93%; P < 0.0001). In the two wild-type bilateral tumors for which ITGCN was available, the preinvasive cells contained the mutation. The mutations were somatic in origin and identical in both tumors. We conclude that somatic activating codon 816 c-KIT mutations are associated with development of bilateral TGCT. Detection of c-KIT codon 816 mutations in unilateral TGCT identifies patients at risk for bilateral disease. These patients may undergo tailored treatment to prevent the development of bilateral disease, with retention of testicular hormonal function.

Coamplification of DAD-R, SOX5, and EKI1 in human testicular seminomas, with specific overexpression of DAD-R, correlates with reduced levels of apoptosis and earlier clinical manifestation (Article)

2002-01-01T00:00:00Z

Seminomas and nonseminomas represent the invasive stages of testicular (TGCTs) of adolescents and adults. Although TGCTs are characterized by extra copies of the short arm of chromosome 12, the genetic basis for gain of 12p in the pathogenesis of this cancer is not yet understood. We have demonstrated that gain of 12p is related to invasive growth and that amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates with reduced apoptosis in the tumors. Here we show that three known genes map within the newly determined shortest region of overlap of amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to the telomeric region of the SROA, DAD-R is the first gene at the centromeric region within the 12p amplicon. Although all three genes are amplified to the same level within the SROA, expression of DAD-R is significantly up-regulated in seminomas with the restricted 12p amplification compared with seminomas without this amplicon. DAD-R is also highly expressed in nonseminomas of various histologies and derived cell lines, both lacking such amplification. This finding is of particular interest because seminomas with the restricted 12p amplification and nonseminomas are manifested clinically in the third decade of life and show a low degree of apoptosis. In contrast, seminomas lacking a restricted 12p amplification, showing significantly lower levels of DAD-R with pronounced apoptosis, manifest clinically in the fourth decade of life. A low level of DAD-R expression is also observed in normal testicular parenchyma and in parenchyma containing the precursor cells of this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression in seminomas and nonseminomas correlates with invasive growth and a reduced level of apoptosis associated with an earlier clinical presentation. These data implicate DAD-R as a candidate gene responsible in part for the pathological effects resulting from gain of 12p sequences in TGCTs. In addition, our results also imply differences in expression regulation of DAD-R between seminomas and nonseminomas.

Microsatellite instability of germ cell tumors is associated with resistance to systemic treatment (Article)

2002-01-01T00:00:00Z

Systemic cisplatin-based chemotherapy cures > or =90% of patients with metastatic germ cell tumors (GCTs). The biological basis of this exquisite chemo-sensitivity and the resistant phenotype encountered in 10-15% of patients with GCT is yet unclear. A defective mismatch repair pathway leading to microsatellite instability (MSI) has been related to resistance to cytotoxic drugs. We investigated 100 unselected GCTs and 11 clinically defined chemotherapy-resistant GCTs for MSI using 8 mono- or dinucleotide markers and the presence of the mismatch repair factors MLH1, MSH2, and MSH6 by immunohistochemistry. The resistant tumors, both chemo-naive (n = 8) and pretreated (n = 3), showed a significantly higher incidence of MSI compared with the unselected series (45 versus 6% in at least one locus and 36 versus 0% in > or =2 of 8 loci, both P < or = 0.001). In 5 of all 11 unstable tumors, MSI correlated with immunohistochemical findings. This study demonstrates for the first time a positive correlation between MSI and treatment resistance in GCT.

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

2000-01-01T00:00:00Z

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.

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas (Article)

1999-01-01T00:00:00Z

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.