Genomic and Expression Profiling of Human Spermatocytic Seminomas: Primary Spermatocyte As Tumorigenic Precursor and DMRT1 As Candidate Chromosome 9 Gene

Research Article

Genomic and Expression Profiling of Human Spermatocytic Seminomas: Primary Spermatocyte as Tumorigenic Precursor and DMRT1 as Candidate Chromosome 9 Gene

Leendert H.J. Looijenga,1 Remko Hersmus,1 Ad J.M. Gillis,1 Rolph Pfundt,4 Hans J. Stoop,1 Ruud J.H.L.M. van Gurp,1 Joris Veltman,1 H. Berna Beverloo,2 Ellen van Drunen,2 Ad Geurts van Kessel,4 Renee Reijo Pera,5 Dominik T. Schneider,6 Brenda Summersgill,7 Janet Shipley,7 Alan McIntyre,7 Peter van der Spek,3 Eric Schoenmakers,4 and J. Wolter Oosterhuis1

1Department of Pathology, Josephine Nefkens Institute; Departments of 2Clinical Genetics and 3Bioinformatics, Erasmus Medical Center/ University Medical Center, Rotterdam, the Netherlands; 4Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; 5Howard Hughes Medical Institute, Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts; 6Clinic of Paediatric Oncology, Haematology and Immunology, Heinrich-Heine University, Du¨sseldorf, Germany; 7Molecular Cytogenetics, Section of Molecular Carcinogenesis, The Institute of Cancer Research, Sutton, Surrey, United Kingdom

Abstract histochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for Spermatocytic seminomas are solid tumors found solely in the involvement in the development of spermatocytic seminomas. testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and (Cancer Res 2006; 66(1): 290-302) numerical chromosomal changes through conventional karyotyping, spectral karyotyping, and array comparative Introduction genomic hybridization using a 32 K genomic tiling-path Spermatocytic seminomas are benign testicular tumors that resolution BAC platform (confirmed by in situ hybridization). exceptionally may progress to sarcoma (1–4). It is becoming Our panel of five spermatocytic seminomas showed a specific increasingly clear that these tumors are not a variant of (classic) pattern of chromosomal imbalances, mainly numerical in seminoma (see ref. 5 for review). On the other hand, the histology nature (range, 3-24 per tumor). Gain of chromosome 9 was the of dysgerminomas of the ovary is virtually undistinguishable from only consistent anomaly, which in one case also involved seminomas of the testis (see ref. 6 for review). These types of germ amplification of the 9p21.3-pter region. Parallel chromosome cell tumors are thought to originate from a primordial germ cell or level expression profiling as well as microarray expression gonocyte, whereas the spermatocytic seminomas are suggested to analyses (Affymetrix U133 plus 2.0) was also done. Unsuper- originate from a more mature germ cell, likely a spermatogonium/ vised cluster analysis showed that a profile containing spermatocyte. This is based on morphologic characteristics, as well transcriptional data on 373 genes (difference of z3.0-fold) is as expression of markers, including placental-like alkaline phos- suitable for distinguishing these tumors from seminomas/ phatase (PLAP; ref. 7), xeroderma pigmentosa protein A (XPA), dysgerminomas. The diagnostic markers SSX2-4 and POU5F1 synaptonemal complex protein 1 (SYCP1), and the synovial (OCT3/OCT4), previously identified by us, were among the top sarcoma protein on the X chromosome (SSX2-SSX4; ref. 8). discriminatory genes, thereby validating the experimental Moreover, this has been supported by analysis of CHEK2, P53, set-up. In addition, novel discriminatory markers suitable for p19INK4Ad (CDKN2D), the cancer testis antigen (CTA) MAGEA4 diagnostic purposes were identified, including Deleted in (9), and transcription factor POU5F1 (OCT3/OCT4), the latter Azospermia (DAZ). Although the seminomas/dysgerminomas related to pluripotency (10). The seminomas and spermatocytic were characterized by expression of stem cell–specific genes seminomas differ also in their pattern of genomic imprinting (11); (e.g., POU5F1, PROM1/CD133,andZFP42), spermatocytic the seminomas show an erased pattern of genomic imprinting (12), seminomas expressed multiple cancer testis antigens, includ- whereas spermatocytic seminomas show a more paternal pattern ing TSP50 and CTCFL (BORIS), as well as genes known to be (13, 14). These findings are in line with the model that the tumor expressed specifically during prophase meiosis I (TCFL5, types originate from germ cells at different stages of maturation, CLGN, and LDHc). This is consistent with different cells of and that (most of) the characteristics found in the tumors are origin, the primordial germ cell and primary spermatocyte, intrinsic to the cell of origin (6). Finally, the seminomas/ respectively. Based on the region of amplification defined on dysgerminomas and spermatocytic seminomas show a different 9p and the associated expression plus confirmatory immuno- chromosomal constitution, as determined by DNA flow cytometry, karyotyping, and conventional comparative genomic hybridization (CGH; refs. 15, 16 and references therein). Although the seminomas Requests for reprints: Leendert H.J. Looijenga, Department of Pathology, Erasmus are consistently aneuploid (around the hypertriploid range), the Medical Center/University Medical Center Rotterdam, Josephine Nefkens Institute, spermatocytic seminomas contain tumor cells with a diploid, Room 430b, P.O. Box 1738, 3000 DR Rotterdam, the Netherlands. Phone: 31-10-40- tetraploid, and hypertetraploid DNA content (17). Gain of the short 88329; Fax: 31-10-40-88365; E-mail: [email protected]. I2006 American Association for Cancer Research. arm of chromosome 12 is the consistent finding in seminomas/ doi:10.1158/0008-5472.CAN-05-2936 dysgerminomas (see refs. 18, 19 for review), whereas additional

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Genomic Investigation of Spermatocytic Seminomas copies of chromosome 9 has been found in all spermatocytic was used as chromogene for the detection of POU5F1-OCT3/OCT4, XPA, seminomas studied to date (15, 16). DMRT1, and DAZ, resulting in a brown nuclear signal. Thus far, no systematic study has been done integrating both DNA/RNA isolation and quality control. DNA was isolated from frozen genome-wide DNA copy number profiling and gene expression tissue. For each sample, 10 pieces of 10-Am-thick histologic sections were cut analysis of spermatocytic seminomas. Therefore, we initiated a from each representative block and used for standard DNA isolation (1 mg/ mL proteinase K digestion/SDS, phenol extraction, and ethanol precipita- study on a unique set of frozen tumor samples. For the tion). Parallel 3-Am-thick sections were stained with H&E and alkaline investigation of the genomic constitution, karyotyping, spectral phosphatase enzymatic detection for histologic control in case of semi- karyotyping (SKY) as well as array CGH using a tiling-resolution 32 noma/dysgerminoma [i.e., analysis of percentage of tumor cells (>80%)]. K BAC array platform, and in situ hybridization were applied. DNA was dissolved in 10 mmol/L Tris/1 mmol/L EDTA (pH 7.5). Expression analysis was done using the genome-wide Affymetrix High-quality total RNA was extracted with RNAqueous-4PCR kit (Ambion U133 plus 2.0 array, as well as the comparative expressed sequence Europe, Huntingdon, United Kingdom) according to the manufacturer’s hybridization (CESH) technique (see ref. 20 for review). Based on instructions. RNA concentration, quality, and purity were examined using integration of data obtained through this multiplatform approach, denaturing gel analysis. None of the samples showed RNA degradation [28S/ z we show that the spermatocytic seminomas, in contrast to 18S RNA (rRNA) ratio 2] and/or contamination with DNA. cDNA synthesis and real-time quantitative PCR. seminomas/dysgerminomas, originate from primary spermato- For reverse transcription, 1 AL oligo-d(T)12-18 primers (0.6 Ag/AL; Invitrogen, Carlsbad, cytes, and that have at least initiated prophase meiosis I. In CA) and 0.5 AL oligo d(N)6 primers (0.6 Ag/AL; Amersham, Piscataway, NJ) addition, DMRT1 is identified as a likely candidate gene to explain were added to 2 Ag of total RNA, isolated as described above, in a final the selective advantage of the observed consistent gain of volume of 9 AL. Samples were subsequently incubated for 5 minutes at chromosome 9 in these tumors. 70jC, 1 minute at room temperature, and chilled on ice. Samples were spun down using centrifugation and incubated with 6 AL5Â First Strand Buffer (Invitrogen), 2 AL DTT (0.1 mol/L), 1 AL deoxynucleotide triphosphates Materials and Methods (10 mmol/L), 10 AL threhalose (1.7 mol/L; Sigma, St. Louis, MO), 0.5 AL Materials. Tissues use for the reported studies was approved by an RNaseOUT (40 units/AL; Invitrogen), and 1.5 AL Superscript II reverse institutional review board (MEC 02.981). Samples were used according to transcriptase (200 units/AL; Invitrogen). The reverse transcription reaction the ‘‘Code for Proper Secondary Use of Human Tissue in the Netherlands,’’ was carried out for 50 minutes at 45jC and terminated by incubating the developed by the Dutch Federation of Medical Scientific Societies (version samples for 10 minutes at 70jC. 2002; ref. 21). Quantitative PCR was done using the real-time PCR ABI PRISM 7700 In total, three dysgerminomas (of the ovary), four seminomas (of the sequence detector system (Applied Biosystems, Foster City, CA). The PCR testis), and five spermatocytic seminomas were included in this study. reactions were done in a final volume of 25 AL containing cDNA synthesized All were primary tumors, only treated with surgery, not with previous from 2 Ag total RNA (see above), 330 nmol/L primers, and 12.5 AL SYBR- irradiation and/or chemotherapy. The spermatocytic seminomas (except green PCR master mix (Applied Biosystems). The input of cDNA for each case 5) have been reported previously (15). Tumors were collected in the tumor sample was determined based on expression analysis of the Netherlands (Rotterdam) as well as abroad, as reported before (19). housekeeping gene hypoxanthine-guanine phosphoribosyltransferase Representative parts of the tumor were snap frozen in liquid nitrogen, and/ (HPRT), being the concentration resulting in a threshold cycle (Ct) of 23. or were fixed in 10% buffered formalin for paraffin embedding. Diagnosis After 10 minutes of denaturation and activation of the Taq-DNA was done by a pathologist experienced in GCT pathology (J.W.O.) according polymerase, PCR products were amplified in 35 cycles of 15 seconds of to the classification of the WHO (22) and supported by immunohisto- denaturation at 95jC, 30 seconds of annealing at 62jC, 10 seconds of chemistry using antibodies directed against germ cell-specific alkaline ramping to 72jC, 20 seconds of extension at 72jC, 10 seconds of ramping to phosphatase (PLAP), a-fetoprotein, human chorionic gonadotropin, stem 79jC, and 20 seconds of extension at 79jC. A dissociation curve was run at cell factor receptor (c-KIT), and the marker of pluripotency POU5F1 (OCT3/ the end of the reaction for product specificity. Quantitative values were OCT4; ref. 10). obtained from the Ct. Each target mRNA was quantified relative to HPRT Tissue microarrays (TMA) were generated including 50 primary semi- (target mRNA = 2(mean Ct HPRT À mean Ct target)). Intron spanning primers used nomas and 23 spermatocytic seminomas, according to standard proce- are DMRT1 forward, 5V-GAGAACAATGGCAGTAACCCG-3V and reverse, dures. Besides the invasive component, also the precursor lesions (i.e., 5V-ACAGAGACGGCTGGTAGAAG-3V; DMRT3 forward, 5V-CTGCGT- carcinoma in situ/intratubular germ cell neoplasia unclassified and TGGACTGCCGAG-3Vand reverse, 5V-TCCACGGACACTATCTCAG-3V; HPRT intratubular spermatocytic seminoma, respectively) were represented in forward, 5V-CGTGGGGTCCTTTTCACCAGCAAG-3Vand reverse, 5V-AATTAT- the array. Representative parts of the paraffin-embedded tissues of all GGACAGGACTGAACGTC-3V. frozen tumors investigated in this study for their expression, and genomic Gene profiling and quality control. The five spermatocytic seminomas imbalances were included in the TMA. were investigated using the CESH technique (20), as described before (24). Immunohistochemistry. Immunohistochemical staining was done as Normal testicular tissue RNA was used as reference and labeled in green, described previously (8). Three-micrometer-thick paraffin-embedded tissue whereas tumor RNA was labeled in red. Self-to-self hybridizations were used sections were incubated with the primary antibodies overnight at 4jC [PLAP to determine the cutoff ratios of 0.8 and 1.2 to define differential expression. (1:200, Cell Marque, Hot Springs, AR) and c-KIT (1:500, DakoCytomation, Multiple metaphase spreads, with a minimum of six, were investigated per Glostrup, Denmark)], or for 2 hours at room temperature [POU5F1-OCT3/ tumor. OCT4 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA)]. Antibodies All samples were analyzed using Affymetrix GeneChip Human Genome against SSX2 to SSX4, XPA, and SYCP1 were used as described before (8). In U133A plus 2.0 arrays. Each transcript on this chip is represented by a set of addition, specific antibodies against DMRT1 (kindly provided by Drs. David 11 probe pairs, called the probe set. The GeneChip contains >54,000 probe Zarkower and Vivian Bardwell; dilution 1:1000) and Deleted in Azospermia sets, representing 47,400 transcripts, including 38,500 genes. The intensity (DAZ; kindly provided by Dr. Renee Reijo Pera, Howard Hughes Medical of hybridization of labeled mRNA to these sets reflects the level of Institute, Whitehead Institute and Department of Biology, Massachusetts expression of a particular transcript. Ten micrograms of total RNA were Institute of Technology, Cambridge, MA; ref. 23; dilution 1:400) were used. used to prepare anti-sense biotinylated RNA, according to the affymetrix All slides for single staining experiments were counterstained with GeneChip eukaryotic one-cycle target preparation protocol (Affymetrix, hematoxylin. New fuchine was used as chromogene for the detection of Santa Clara, CA). In short, single-stranded cDNA was synthesized using a PLAP, c-KIT, and SSX, resulting in a red signal of cytoplasmic, membranous, T7-Oligo(dT) Promoter Primer followed by RNase H–facilitated second- and cytoplasmic/nuclear localization, respectively. 3,3V-Diaminobenzidine strand cDNA synthesis, which was purified and served as a template in www.aacrjournals.org 291 Cancer Res 2006; 66: (1). January 1, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research the subsequent in vitro transcription (IVT). The IVT reaction was carried method available in the Affymetrix Microarray Suite. For each probe set, the out in the presence of T7 RNA polymerase and a biotinylated nucleotide geometric mean of the hybridization intensities of all patient samples was analogue/ribonucleotide mix for cRNA. The biotinylated cRNA targets calculated. The level of expression of each probe set in every sample was were then cleaned up and fragmented. The quantity of the fragmented- determined relative to this geometric mean and logarithmically trans- labeled cRNA was determined using standard spectrophotometric analysis, formed (on a base log 2 scale) to ascribe equal weight to gene expression and the quality was checked on an Agilent 2100 Bioanalyzer (Agilent, levels with similar relative distances to the geometric mean. Deviation from Amstelveen, the Netherlands), using an RNA 6000 NANO assay. Fragmented the geometric mean reflects differential gene expression. The transformed biotinylated cRNA was hybridized to the GeneChip (45jC for 16 hours). expression data were subsequently imported into Omniviz software, version Staining, washing, and scanning procedures were carried out as described 3.6 (Omniviz, Inc., Mynard, MA), significance analysis of microarrays (SAM) in the GeneChip Expression Analysis Technical Manual (Affymetrix). software, version 1.21. Principle component analysis was done as part of the All GeneChips were visually inspected for irregularities. The global method Spotfire software package (Spotfire, Inc., Sommerville, MA; Spotfire decision of scaling/normalization was applied. All additional measures of quality site V8.1). [percent genes present (50.6 F 3.8), actin 3Vto 5Vratio (1.24 F 0.19), and Data analyses using Omniviz and SAM. The Omniviz package was glyceraldehyde-3-phosphate dehydrogenase 3V to 5V ratio (1.05 F 0.14)] used for performing and visualizing the results of unsupervised cluster indicated high overall sample and assay quality. analysis (an analysis that does not take into account external information Data normalization, analysis and visualization. All intensity values such as histology). Genes whose level of expression differed from the were scaled to an average value of 150 per GeneChip according to the GCOS geometric mean (reflecting up-regulation or down-regulation) in at least

Figure 1. A, principle component analysis of gene expression, indicating that the seminomas (SE1-4) cluster together with the dysgerminomas (DG1-3), whereas the spermatocytic seminomas (SS1-5) are separate. B, results of unsupervised clustering of the seminomas (SE), dysgerminomas (DG), and spermatocytic seminomas (SS) using Omniviz. The seminomas and dysgerminomas could not be separated, in contrast to the seminomas/dysgerminomas and spermatocytic seminomas. C, correlation plots of the individual tumors compared to each other, SS-3 and SS-4 are the bilateral cases. D, correlation between fold change level of expression of the differentiating genes between seminomas/dysgerminomas and spermatocytic seminomas and number of differentiating genes, based on SAM (see text). E, representative examples of immunohistochemistry for DAZ on seminoma (left) and spermatocytic seminoma (right). Note the specific positive staining in spermatocytic seminoma.

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Table 1. Overview of the top 50 discriminatory genes between seminoma/dysgerminoma and spermatocytic seminoma

Spermatocytic seminoma

Gene ID Function Affy ID x-fold

GAGE4 Spermatogenesis, CTA 207663_x_at 100 DAZ4 Spermatogenesis 216351_x_at 100 GPC4 Cell suface proteoglycan 204984_at 100 LOC81569 ? 214957_at 50 DAZ Spermatogenesis 207912_s_at 100 PEPP_2 Transcription 236920_at 50 GAGE8 Spermatogenesis, CTA 207739_s_at 50 ASB9 Signaling 205673_s_at 50 SAGE Spermatogenesis, CTA 220793_at 50 SSX1 Spermatogenesis, CTA 206626_x_at 20 SSX3 Spermatogenesis, CTA 207666_x_at 20 TEX15 Spermatogenesis 232760_at 50 PAX6 Differentiation 205646_s_at 100 SSX2 Spermatogenesis, CTA 207493_x_at 20 DAZ2 Spermatogenesis 208281_x_at 50 LOC64180 ? 220179_at 50 EYA4 Transcription, development 238877_at 20 RBMY1A1 Spermatogenesis 208307_at 20 GAGEB1 Spermatogenesis, CTA 206897_at 20 DHRS2 Carbohydrate metabolism 206463_s_at 50 RBP4 Retinol binding 219140_s_at 20 DHRS2 Carbohydrate metabolism 214079_at 100 VCX_2r unknown 207281_x_at 20 SSX4 Spermatogenesis, CTA 210394_x_at 20 TKTL1 Transketolase_like 1 216370_s_at 10 TAF7L Transcription 224380_s_at 20 CHD5 Chromatin modification 217250_s_at 20 LOC90499 ? 234016_at 20 CTCFL Spermatogenesis, CTA 1552368_at 20 HOXB6 Transcription 205366_s_at 20 HOXB8 Transcription 229667_s_at 20 DMRT3 Testis development? 231800_s_at 20 ESX1L Embryogenesis 1552445_a_at 10 FTCD Transferase/deaminase 223979_x_at 20 CLGN Spermatogenesis 205830_at 20 EYA2 Development 209692_at 20 MAGEA3 Spermatogenesis, CTA 209942_x_at 20 LOC283694 ? 1560735_s_at 20 DLK1 Neuroendocrine diff. 209560_s_at 20 OTEX Transcription 1552724_at 20 KIAA1713 ? 233536_at 20 MAGEA2 Spermatogenesis, CTA 214603_at 20 TKTL1 Transcription repression 214183_s_at 10 DKFZp686A1627 ? 1561961_at 20 FLJ23058 ? 219218_at 20 LOC139135 ? 240687_at 20 HAB1 B for mucin 239791_at 50 MSX1 Transcription 205932_s_at 20 LOC339258 ? 241828_x_at 20 FLJ23191 ? 219747_at 20 lPELI2 Signaling 219132_at 10

NOTE: CTA = Testis Cancer Antigen. Shaded areas are known as diagnostic marker. Their supposed function/pathway is indicated, as well as their fold change.

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Table 1. Overview of the top 50 discriminatory genes between seminoma/dysgerminoma and spermatocytic seminoma (Cont’d)

Seminoma/dysgerminoma

Gene ID Function Affy ID x-fold

POU5F1 Pluripotency 210905_x_at 100 Nanog Pluripotency 220184_at 100 FLJ20171 ? 219121_s_at 100 PROM1 Stem cell biology (CD133) 204304_s_at 50 LOC90625 ? 232191_at 100 GIP Insulin secretion 207899_at 50 LOC221810 ? 221911_at 50 FLJ32835 ? 1553918_at 100 ZFP42 Stem cell biology (MXF_1/mRex1) 1554776_at 50 CHI3L1 Cartilage glycoprotein 209396_s_at 100 INDO Tryptophane degradation 210029_at 50 CRACC NK- & CD8 function 222838_at 20 GPX2 Glutathion peroxidase 202831_at 50 RAB15 Vesicular trafficing 221810_at 20 CDH3 p_cadherin, adhesion 203256_at 20 SOX17 Transcription 230943_at 20 ADAMDEC1 Proteolysis/peptidolysis 206134_at 50 IGHM Immune reaction 209374_s_at 20 UBD Protein modification 205890_s_at 50 SERPINA1 Inhibit serine protease 202833_s_at 20 MX1 Against influenza infect. 202086_at 20 SFRP2 Wnt signaling 223122_s_at 50 RAI3 Differentiation 212444_at 20 FLJ25694 ? 1553614_a_at 20 MDA5 Helicase 219209_at 20 CXCL10 Chemotax. Mono_/T lymphocytes 204533_at 20 CTH Cystathione gamma_lyase 217127_at 20 CDO1 Cysteine dioxygenase I 204154_at 20 IGJ Immune system 212592_at 20 LOC222967 ? 1557417_s_at 20 GPHA2 Glycoprotein hormone alpha 2 237230_at 20 T1A_2 Membrane glycoprotein 204879_at 10 CDW52 Carbohydrate carrying and orientation 34210_at 20 FLJ22662 ? 218454_at 20 PYPAF3 ? 237461_at 20 CASP8 Apoptose 213373_s_at 20 GBP1 Glianylate binding 202270_at 20 MGC16491 ? 229518_at 10 LIN28 Cold shock domain DNA binding 219823_at 10 MGC12335 ? 229070_at 20 IL6R Interleukin signaling 226333_at 10 TRIM2 Tripartite motif 202342_s_at 20 EFEMP1 Extracellular matrix 201842_s_at 10 PLXNA2 Embryogenesis 213030_s_at 20 BK65A6.2 ? 227480_at 20 CXCL9 Chemotax. Activated T cells 203915_at 20 GBP1 Guanylate binding 231577_s_at 10 BICD1 Differentiation 1554020_at 10 IFITMI Interferon induced membrane protein 214022_s_at 10 APOC Apolipoprotein C1, chemotaxic? 204416_x_at 10 MEG3 P53 induced, maternally derived 210794_s_at 10

one patient were selected for further analysis. The clustering of molecularly All supervised analyses were done using SAM software (25). Genes recognizable specific groups of patients was investigated with each of the identified as being discriminating using Omniviz were used for the analysis. selected probe sets using the Pearson’s Correlation and Visualization tool of A supervised analysis correlates gene expression with an external variable Omniviz. The cutoff value of expression modulation was set to either 2.5- or being histology. SAM calculates a score for each gene based on the change 3.0-fold (resulting in 1,019 and 373 differentiating genes, respectively). This in gene expression relative to the SD of all measurements. The criteria for study will only focus on the data obtained from the analysis using the identifying the top 50 genes for an assigned cluster were a minimum of a 3.0-fold change. 2-, 10-, 20-, 50-, and 100-fold difference in gene expression between the

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Genomic Investigation of Spermatocytic Seminomas assigned cluster and the other samples by a factor of at least 2.0 and a As expected, both the seminomas and dysgerminomas showed the q < 2%. The q for each gene represents the probability that it is falsely called opposite pattern (see below). significantly deregulated. Expression profiling. All seminomas, dysgerminomas, and SKY analysis. Cytogenetic preparations were pretreated with RNase for spermatocytic seminomas were expression profiled using Affyme- 60 minutes at 37jC and digested with pepsin for 10 minutes. After trix U133 plus 2.0 arrays. Omniviz analysis using unsupervised washing, cells were fixed in 1% formaldehyde/50 mmol/L MgCl2 in PBS for 10 minutes, washed, and dehydrated, as reported before (19). clustering showed that the seminomas and dysgerminomas could Hybridization for SKY analysis was done according to the manufacturer’s not be distinguished based on mRNA expression (data not shown). protocol with minor adjustments (Applied Spectral Imaging, Migdal Therefore, throughout this article, these two types of tumors will Ha’Emek, Israel). In short, after 2 days of hybridization, slides were be considered together. Subsequently, the total sets of tumors washed for 10 minutes with 55% formamide/2Â SSC (pH 7.0) followed by (seminomas/dysgerminomas and spermatocytic seminomas) were 1Â SSC at 39jC and 4Â SSC/0.05% Tween 20 at room temperature. Cells compared. Principle component analysis showed that these tumors were counterstained with 4V,6-diamidino-2-phenylindole and mounted in are subdivided into defined profile entities (see Fig. 1A). This was antifade solution (Dabco-Vectashield 1:1). Using the Spectra Cube 300 confirmed using Omniviz, which separated these two histologic system and Skyview analysis software (ASI), nine metaphase cells were groups of tumors successfully based on 373 genes with an at least examined (26). 3-fold difference in expression (Fig. 1B). Correlation analyses Fluorescence in situ hybridization. For each case analyzed, 5-Am-thick tissue sections were cut and air-dried at 55jC overnight on starfrost showed that the spermatocytic seminomas were more similar to microscope slides. Two adjacent sections were used for each histologic each other than the seminomas/dysgerminomas (Fig. 1C, shown by examination. Fluorescent in situ hybridization (ISH) was done as described the various color intensities). The bilateral cases (cases 3 and 4) previously (27). The following BACs were used: RP11-117J18, RP11-621O18, were not more similar to each other than to the other tumors. This and RP11-344A7 (all three located on 9p21.3-9p23). Probes were labeled with defined set of 373 genes was used for supervised analysis (SAM), in biotin-16-dUTP and detected using streptavidin-CY3 (Jackson Immuno- which the seminomas/dysgerminomas (group 1) were compared Research Laboratories, West Grove, PA). The centromere-specific probe for with the spermatocytic seminomas (group 2). The discriminating chromosomes 9 was labeled with digoxigenin-11-dUTP (Roche, Mannheim, genes were classified based on their difference in level of expression Germany) and visualized with FITC-conjugated sheep-anti-digoxigenin (2-, 10-, 20-, 50- up to 100-fold), which revealed a log 2–association (Roche). Results were confirmed with reverse labeling. Probes were verified between the fold change and number of differentiating genes to be specific for the regions of interest on metaphase spreads isolated from in vitro cultured human lymphocytes, both male and female. (Fig. 1D). The top 50 differentiating genes [score(d) higher than Array CGH. We prepared a tiling-resolution microarray consisting of 4.00 or lower than À4.00] were investigated in detail and are listed 32,447 overlapping BAC clones selected to cover the entire human genome in Table 1. The role of the protein encoded by the gene, or the (28, 29) and available through BACPAC resources8 using methodology process in which it is involved, is indicated. essentially as described before (30). In brief, genomic target DNAs were Identification of significant biomarkers and cell of origin. isolated from 1 mL bacterial cultures using an AutogenPrep 960 (Autogen, Our expression profiling results show that some of the validated Holiston, MA), following the instructions of the manufacturer. Degenerate diagnostic markers previously identified by us and others for oligonucleotide primed (DOP) PCR was done on 50 ng DNA from all clones seminoma/dysgerminoma and spermatocytic seminomas are essentially as described before (31) with minor modifications (32). DOP-PCR indeed in the most highly ranking distinguishing genes (see products were dissolved at a concentration of 1 Ag/AL in a 30% DMSO Table 1, shaded and Table 2). These include POU5F1-OCT3/OCT4 solution and spotted onto CMT-ULTRAGAPS-coated glass slides (Corning, Acton, MA) using an Omnigrid 100 arrayer (Genomic Solutions, Ann at position 1 for seminoma/dysgerminoma, being one of the Arbor, MI). six genes showing at least a 100-fold expression difference. The Analysis of the microarray images obtained from the BAC hybridizations spermatocytic seminoma specific SSX2-SSX4 genes showed a was done using the software package GenePix Pro 5.0 (Axon Instruments, 20-fold expression difference. Although the antibody used for Inc., Foster City, CA). For each spot, the median pixel intensity minus the immunohistochemistry only detects the SSX2 to SSX4 variants, median local background for both dyes was used to obtain a genomic copy expression data clearly show that also SSX1 (at position 10 of the number ratio. Data normalization was done in the software package SAS top list of genes, P = 1.85eÀ8) and SSX5 (P = 0.004), as well as the version 8.0 (SAS Institute, Cary, NC) for each array subgrid, by applying related gene SSX2IP (P = 9.79eÀ4), are discriminators. Results of Lowess curve fitting with a smoothing factor of 0.3 to predict the log other suggested diagnostic markers are summarized in Table 2, 2–transformed test-over-reference (T/R) value based on the average of which the pattern of expression of these genes was fully in line logarithmic fluorescent intensities. All mapping information regarding clone locations, cytogenetic bands, and gene content were retrieved from with our prior expectations, thereby supporting the experimental the University of California Santa Cruz genome browser (May 2004 freeze). set-up of this study. The genes CHEK2, c-KIT, PLAP, POU5F1-OCT3/ OCT4, P53, SSX2 to SSX4, SYCP1, and XPA were indeed found to be significantly different in expression, whereas this was not the case Results for MAGEA4 and CDKN2D (33). Five spermatocytic seminomas (including one pair of bilateral As it is reasonable to assume that genes with the highest tumors from a single patient, cases 3 and 4), four testicular expression difference might be the best discriminators for seminomas, and three ovarian dysgerminomas were investigated in diagnostic purposes, we did immunohistochemistry to establish this study. All spermatocytic seminomas were diagnosed as stage I the diagnostic performance of DAZ. The gene encoding this and were found to stain positive by immunohistochemistry for the protein is in the top 5 of discriminatory genes, specifically diagnostic markers XPA, SYCP1, VASA, and SSX2 to SSX4 and expressed in spermatocytic seminomas (Table 1). As no specific negative for the markers characteristic for seminoma/dysgermi- antibodies are currently available for other interesting targets, like noma, including PLAP, c-KIT, and POU5F1-OCT3/OCT4 (7, 8, 10). GAGE4/8/B1, GPC4, PEPP2, and SAGE, these markers could not be included in this survey. Fully in line with our expression profiling–based observation that DAZ (as well as DAZ2 and 8 http://bacpac.chori.org/. DAZ4) is specifically up-regulated in spermatocytic seminoma www.aacrjournals.org 295 Cancer Res 2006; 66: (1). January 1, 2006

Table 2. Summary of diagnostic markers for seminoma/ checked the expression pattern of genes related to this specific type dysgerminoma and spermatocytic seminoma cell division in more detail (Fig. 2A and B). In contrast to SYCP1, SYCP3 showed no difference (P = 0.38; Tables 1 and 2), whereas a Gene Reported pattern P number of genes related to meiotic prophase I (TCFL5, CLGN, and LDHc) are specifically expressed in spermatocytic seminomas SE SS compared with seminomas/dysgerminomas (P = 0.0055, 2.94eÀ5, and 0.0046, respectively; Fig. 2B). TCFL5 is a transcription factor– Previous reported: type basic helix-loop-helix (36); CLGN is a chaperone, interacting CHEK2 + À 0.028 with TCFL5 (37); and LDHC is a germ cell lineage–specific variant c-KIT + À 0.014 of the lactate dehydrogenase enzyme (38). MAGEA4 À + 0.089* Genomic imbalances in spermatocytic seminomas. Four of PLAP/ALPPL2 + À 2.08E-4 the spermatocytic seminomas have previously been characterized POU5F1-OCT3/4 + À 5.58E-10 P19/CDKN2D + À 0.399* by conventional CGH analysis and one (case 1, Table 3) with P53 + À 0.0070 karyotyping, of which the results were published elsewhere (15). SSX2 À + 4.32E-7 The changes identified with conventional karyotyping are: SSX3 À + 1.22E-8 74-83<3n>:XY, add(X)(p11),+Y,+1,add(4)(p15.1),+5,+6,+add(6) SSX4 À + 5.54E-8 (q21),À7,+9,+9,+10, inv(10)(q22q24) x2,del(12)(p11.2fp12), SYCP1 À + 0.010 +14,+17,+19,+20,À22,+mar. Here, we report additional SKY analyses XPA À + 1.47E-4 on nine metaphase spreads of this previously karyotyped tumor. Novel: The results are illustrated in Fig. 3A and Table 3, of which the CTCFL À + 1.56E-6 DMRT1 À + 0.0028 description is 57-83<3n> XXY,-X[3],+Y[7],+1,À2[4],-3[3],+5[5], DMRT2 À + 0.044 +del(5q),+del(5q)[4],+6[8],À7,À7[3],À[3],+9[7],+9[3], +10,+14[5],del DMRT3 À + 4.25E-5 (14q)[2],+17[5],+19[4],+20,+20[3],À22 [cp9]. The previously repor- GAGE4 À + 5.05E-6 ted ploidy heterogeneity of spermatocytic seminomas was GAGE8 À + 1.08E-5 confirmed by the identification of tumor cells with a different GAGEB1 À + 3.0E-6 DNA content, based on metaphase spread analyses (Table 3). The MAGEA2 À + 2.69E-7 clonal relationship between this tumor cells was supported by a MAGEA3 À + 2.97E-7 common del(5q) anomaly, which was confirmed with fluorescent MAGEC3 À + 0.02 SAGE À + 9.8E-7 ISH with a whole chromosome 5 paint (data not shown). The SPANXA + À 7.98E-6 majority of changes identified using SKY were in line with the SPANXC + À 8.99E-4 results of the other approaches (see Table 3, see also below). TSP50 À + 0.019 Array CGH using a 32 K platform was done on all five spermatocytic seminomas (Fig. 3B; Table 3). The seminomas and dysgerminomas were studied using a 3.7 K array, of which the NOTE: Both previously identified and novel targets are indicated. The results will be described elsewhere, as they were in agreement with P identified based on the expression profiling data is indicated. The previously described chromosomal imbalances, including gain genes are listed in alphabetical order. of chromosome 7 and 8, as well as X, and the short arm of *Not statistically significant in this study. chromosome 12, and loss of chromosomes 4, 5 and 13 (see ref. 19 for review). Consistent with karyotyping, SKY, and conventional CGH, array CGH showed that the majority of imbalances in the compared with seminoma/dysgerminoma, immunohistochemistry spermatocytic seminomas affect entire chromosomes. Two sper- showed a specific staining for DAZ in all spermatocytic matocytic seminomas (cases 1 and 5) showed a stepwise pattern of seminomas, including their intratubular precursor, with a nuclear imbalances of multiple chromosomes. The imbalances resulting in localization (see Fig. 1E). This was found both on frozen as well the smallest numerical differences were not classified according to as paraffin-embedded tissues, including those represented in the the criteria set as significant. Some of these nonsignificant changes TMA (data not shown). affected chromosomes showing significant imbalances in the other A considerable number of CTA genes, of which the SSX genes are tumors, like gain of chromosome 1 and gain of chromosome Y in representatives, are in the top 50 list of discriminatory genes (n = case 1. Nineteen of the 21 significant changes detected by array 11, 22%), being specifically expressed in spermatocytic seminoma CGH were also found using conventional CGH (Table 3). Gain of (see Table 1). These genes are known to exhibit a maturation-stage chromosome 9 was the only consistent anomaly found, whereas specific expression pattern during spermatogenesis (Fig. 2A). gain of chromosome 20 was detected in four cases. Notably, array Therefore, they can be informative to identify the cell of origin. CGH revealed sub-chromosomal changes, being amplification of TSP50 was predominantly found in spermatocytic seminomas (P = 9p21.3-pter in tumor 4 (Fig. 3C), confirmed by fluorescent ISH 0.018), in accordance to the previously reported down-regulation of (Fig. 3D), estimated to be about 25 MB in size. In addition, tumor 2 this supposed protease in seminoma (34). CTCFL, which is also showed loss of the 19q13.32-qter region. known as BORIS (position 26, Tables 1 and 2; ref. 35), is specifically Combined genome and expression analysis and identification up-regulated in spermatocytic seminomas compared with semi- of a positional functional candidate gene on chromosome 9. nomas/dysgerminomas, whereas CTCF did not show a significant Using CESH, an overview of the expression profile on the difference (P = 0.33). The CTA gene SYCP1, known to be involved chromosomal level was obtained. The results showed that the in meiosis, is expressed (although at a rather low level) in only genomic fragment with a consistent overexpression of genes spermatocytic seminomas (ref. 8; Fig. 2A; Table 2). Therefore, we compared with normal spermatogenesis is mapped to the short

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Genomic Investigation of Spermatocytic Seminomas arm of chromosome 9, centered around band p22, but covering Affymetrix array results. In total, 186 Affy-probe sets were 9p13.3-pter. Representative examples are shown in Fig. 3E. represented in the region of interest, including known and Because of the consistent gain of chromosome 9, as well as hypothetical genes. To identify the gene(s) of interest, we compared overexpression of genes from a restricted p-region in spermatocytic the absolute expression level of the candidates in SS-4, containing seminomas, and the identification of an overlapping, subchromo- the restricted 9p amplification, to the mean of expression of the somal amplification of the 9p21.3-pter region (see above) this other four spermatocytic seminomas. Only candidates showing a genomic fragment was investigated in more detail based on the 2-fold overexpression were considered, and the candidate must

Figure 2. A, schematic representation of the expression pattern of a number of CTA and genes related to prophase meiosis I during normal spermatogenesis (middle and right). The different maturation stages of the germ cells are indicated, as well as the timing of mitosis, meiosis (I and II), and spermiogenesis (left). Genomic constitution of the germ cells at different stages of development (Ploidy). Adapted from (46, 60). B, overview of genes specifically expressed during meiotic prophase I in spermatocytic seminoma (SS-1 to SS-5) compared with seminoma/ dysgerminoma (SE-1-4/DG-1-3) as log 2 ratio (i.e., TCFL5, CLGN, and LDHC).

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Table 3. Summary of the array CGH, compared to SKY (case 1), data on the five spermatocytic seminomas

NOTE: White = not changed; light green, nonsignificant gain; dark green, significant gain; orange, nonsignificant loss; red, significant loss. Boxed are also identified by conventional CGH or karyotyping (case 5 has only be studied by aCGH); Cases 3 and 4 are bilateral from a single patient (gray boxes) One SKY results (nine metaphase spreads were analyzed). The number of metaphases showing the anomaly are indicated (+ is gain; À is loss). Codes as used in the original paper are in order of presented here 1,4, 2,3. Abbreviations: G, gain; L, loss.

show at least a 2-fold increase in level of expression in the tumor against DMRT3 is available), using an antibody directed against the with the restricted amplification compared with the nonamplified mouse protein. As expected, a consistent nuclear staining of tumor with the highest level of expression. These included 38 probe human spermatogonia and spermatocytes was observed (Fig. 4B). sets, representing 12 known and 11 hypothetical genes. Three genes Although all spermatocytic seminomas showed a positive staining, were identified by nonspecific probe sets, or the different probe the seminomas/dysgerminomas were mainly negative (Fig. 4C). sets gave inconsistent results, which were therefore excluded from This was found both on frozen tissue and formalin-fixed samples, further analysis. Finally, eight genes from the 9p21.3-pter region including those present on the TMA (data not shown). The protein showed overexpression in SS-4 compared with the others tumors: was specifically located in the nucleus. The tumor with the specific DMRT1, MPDZ, NIRF, PTPRD, RLN1, SH3GL2, SNAPC3, and MDS030. 9p amplification showed a particularly intense staining. A genetic None of these were identified as a discriminatory gene in the top 50 change, reported in 46XY sex reversal individuals (39), Pro > Leu list (Table 1). Within the set of spermatocytic seminomas, DMRT1 (codon 295, exon 4) was not found in the spermatocytic seminomas shows the highest absolute level of expression (i.e., around 2,000) as determined by PCR amplification and sequencing (data not of the candidate genes. Based on the (supposed) function of shown). the encoded protein,9 DMRT1 was indeed considered as the most interesting candidate. DMRT3, closely related to DMRT1,isa discriminatory gene between spermatocytic seminoma and semi- Discussion noma/dysgerminoma (position 31 in top 50; P = 4.25eÀ5, Tables 1 The inability of principle component, and unsupervised and 2). This in spite of the fact that this gene showed a low clustering analysis of genome-wide expression profiling data to absolute level of expression, <100, like DMRT2 (see below). The distinguish seminomas from dysgerminomas, supports their expression pattern of DMRT1 and DMRT3 has been confirmed in common origin and pathogenesis. Their similar expression profile an array technology-independent manner using quantitative PCR further supports the notion that these tumors indeed originate (Fig. 4A). Interestingly, DMRTC2, DMRTB1,andDMRT2 also from the same embryonic germ cell (primordial germ cell/ showed a significant difference between spermatocytic seminomas gonocyte), as was further supported by transcriptional interroga- and seminomas/dysgerminomas (P = 1.3eÀ4, 3.9eÀ4, and 0.044, tion of a number of stem cell markers, such as POU5F1-OCT3/OCT4 respectively). and NANOG, PROM1, ZNF42 (mRex1, also known as MZF-1), as well The presence of DMRT1 protein was investigated using as markers related to the germ line, like PLAP,c-KIT, and VASA. immunohistochemistry of tissue sections (no antibody directed Another interesting gene is NANOS3. In contrast to NANOS1 and NANOS2 (P = 0.267 and 0.18, respectively), NANOS3 is specifically up-regulated in all seminomas/dysgerminomas compared with 9 http://www.genecards.org. spermatocytic seminomas (P = 4.4eÀ4), although it was not

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Figure 3. A, representative example of SKY data of a single spermatocytic seminoma (case 1, see text and Table 3 for further information). B, results of the 32 K BAC array CGH experiments on all five spermatocytic seminomas (see Table 3 for further information). C, detailed representation of chromosome 9 pattern of case 4 showing a subchromosomal amplification of the 9p21.3-pter region. D, representative example of fluorescent ISH on the spermatocytic seminoma with the restricted 9p amplification, in which the centromeric region is visualized in green and the 9p-arm in red (BAC 344A7). Note the amplification of the red signals. E, representative examples of chromosome 9 CESH in two spermatocytic seminomas. Note the specific overexpression of genes from the short arm, band p22-p23.

amongst the top 50 genes. In mouse, this gene is expressed in different from seminomas (1–3, 7–10, 13, 15–17). The results migrating germ cells, and its absence results in complete loss provide useful biomarkers that distinguish these two histologic of germ cells (40). In this context, the apoptosis inhibiting role groups, as shown by the finding of the known diagnostic markers of POU5F1-OCT3/OCT4 in mouse primordial germ cells is also of identified previously by educated guesses or serendipitously. These interest (41). The previously reported expression of the ‘‘stem cell included POU5F1-OCT3/OCT4 (positive in seminomas/dysgermi- gene’’ HIWI (also called MIWIL) in seminoma was confirmed by nomas) and SSX2 to SSX4 (positive in spermatocytic seminomas; this study (P = 0.06; ref. 42), adding to the previously mentioned list refs. 8, 10). Of the other earlier identified genes (encoding PLAP, of stem cell–specific genes. c-KIT, XPA, SYCP1, MAGE4A2, p16INK4A, and CHEK2; ref. 33), Here, we report on the first high resolution genome-wide none are found in our top 50 list, or even in the list of 373 analysis of chromosomal anomalies, as well as transcriptional distinguishing genes; however, some of them show a significant profiling of a set of well-defined spermatocytic seminomas. difference in expression between the two histologic subgroups The expression data were compared with observations made in (Table 2). POU5F1-OCT3/OCT4 was one of the few genes showing a seminomas/dysgerminomas. Principle component and unsuper- 100-fold positive difference between seminomas/dysgerminomas vised cluster analysis showed that spermatocytic seminomas are and spermatocytic seminomas. The second best gene, NANOG, different from seminomas/dysgerminomas. Based on various also showed a high level of expression in seminoma, in agreement attributes, including morphology, immunohistochemical character- with previous studies (43–45). Immunohistochemistry on primary istics, genomic imprinting, and chromosomal constitution, it has tumors and a TMA containing 50 invasive seminomas and 23 inva- indeed been established that spermatocytic seminomas are sive spermatocytic seminomas, including their precursor lesions, www.aacrjournals.org 299 Cancer Res 2006; 66: (1). January 1, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research confirmed this finding. In conclusion, based on the availability of (the human homologues of PUF-8) in spermatocytic seminomas antibodies, POU5F1-OCT3/OCT4 is the best diagnostic marker for based on expression analysis as well as immunohistochemistry seminoma/dysgerminoma, in line with previous observations, and (data not shown). Interestingly, DAZ and DAZL have been reported SSX2 to SSX4, XPA, and DAZ have high diagnostic value for to interact with PUM1 and PUM2 (48). This might be of relevance spermatocytic seminoma. because we observed specific up-regulation of a number of DAZ In contrast to the observed pronounced expression of stem cell– genes in spermatocytic seminoma (DAZ, P = 3.29eÀ6; DAZ2, specific related genes in seminoma/dysgerminoma, spermatocytic P = 6.07eÀ6; DAZ4, P = 7.34eÀ6). The up-regulation of DAZ was seminomas are characterized by expression of CTAs. These genes subsequently confirmed by immunohistochemistry and found to be are of particular interest, because they can be used to identify the an informative biomarker (see above). Although this provides an cell of origin and because the expression of CTAs is highly specific interesting novel diagnostic marker, the functional consequences of and tightly regulated during spermatogenesis (see ref. 46 for this observation remains to be elucidated. Besides the C. elegans review). Based on the consistent pattern of expression of these germ cell tumors, also the spontaneously occurring canine genes, it prompted our conclusion that spermatocytic seminomas seminomas deserve further investigation as an animal model for indeed show characteristics of germ cells at the differentiational spermatocytic seminoma (17, 49). stage of spermatocytes. The expression pattern of SYCP1, CTCFL, Another striking difference between seminoma/dysgerminoma TCFL5, CLGN, and LDHc suggests that the tumor cells at least enter and spermatocytic seminoma is the specific expression of genes meiotic prophase I. These findings strongly suggest that human encoding proteins known to be chemotaxic for macrophages and spermatocytic seminomas mimic germ cell tumors generated in (activated) T lymphocytes, including CRACC, CXCL10, and CXCL9 Caenorhabditis elegans by knocking out of the PUF8 gene (47). (refs. 50–52; Table 1). Indeed, the seminomas/dysgerminomas are These tumor cells undergo meiosis but do not finish meiotic characterized by a consistent presence of lymphocytic infiltrates, division but, instead, return to the mitotic cell cycle. However, we which are absent in spermatocytic seminomas. The specific did not observe absence of expression of PUM1 and PUM2 genes expression of APOC in seminomas/dysgerminomas might also be

Figure 4. A, quantitative PCR results of analysis of DMRT1 (left) and DMRT3 (right) in spermatocytic seminomas (SS), seminomas (SE), and dysgerminomas (DG). B, representative example of immunohistochemistry of DMRT1 in normal spermatogenesis, in which the spermatogonia and spermatocytes are stained positively. C, representative examples of immunohistochemistry of DMRT1 in seminoma (upper) and spermatocytic seminoma (lower), at low (Â20) and high (Â40) amplification (left and right, respectively).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Genomic Investigation of Spermatocytic Seminomas explanatory for this difference, which has been reported to act of chromosome 9 in the majority of these tumors (6). The ability as an attractant for these cells of the immune system (53). The of the antibody directed against the mouse Dmrt1 protein to presence of IGHM and IGJ is most likely due to the presence of recognize the human protein, allows a careful investigation of infiltrating lymphocytes in the seminomas/dysgerminomas. DMRT1 presence during normal male and female development, Besides the unique data on expression profiling, this study and this is currently under investigation. In addition, the exact also reports on the first comprehensive genomic screen for role of DMRT1 in the development of spermatocytic seminomas imbalances in spermatocytic seminomas, using a combination of remains to be elucidated. complementary molecular cytogenetic approaches. All techniques In summary, this study is the first comprehensive analysis of applied (conventional and spectral karyotyping as well as array spermatocytic seminomas that includes genome wide analysis of CGH) led to the same conclusion: spermatocytic seminomas genomic changes as well as expression profiling. The integrated are characterized by recurrent chromosomal imbalances that results clearly illustrate the distinct pathogenesis of spermatocytic mainly affect complete chromosomes. Gain of chromosome 9 was seminomas and profound differences compared with seminomas/ the only consistent imbalance. The specific amplification of a dysgerminomas. Spermatocytic seminomas express genes related subchromosomal region on the short arm of chromosome 9 to specific stages of post-pubertal male germ cell maturation, (p21.3-pter), as well as the CESH results showing overexpression which is in line with the testis-specific occurrence of this cancer. of genes from this region relative to normal testis, led us to Our data identify the spermatocyte as the cell of origin for this type hypothesize that a gene or genes of interest resides within this of germ cell tumor. Additional, supportive evidence is presented specific chromosomal fragment. Therefore, we systematically that these tumor cells undergo partial meiosis, possibly followed by compared expression data with chromosomal data and found a reentry into the mitotic cycle. Furthermore, spermatocytic that a number of 9p genes were indeed up-regulated in the seminomas show a specific pattern of chromosomal imbalances, spermatocytic seminomas with the restricted 9p amplification in which gain of chromosome 9 is consistent, likely related to compared with those with only gain of the whole chromosome 9. DMRT1, a gene expressed during embryogenesis and male In particular, the DMRT genes are of interest. Various DMRT germ cell maturation. Lastly, this study not only confirms known genes have been identified, of which DMRT1, DMRT2, and DMRT3 diagnostic markers but also discovers new markers that are map to the region of interest. DMRT stands for Doublesex and available for immunohistochemical analysis and further biological mab-related transcription factor. These genes are related to sex studies. determination, and at least DMRT1 and DMRT2 are candidates to In conclusion, spermatocytic seminomas are a separate entity of explain 46XY sex reversal in individuals with monoallelic loss of germ cell tumors, originating from primary spermatocytes, to be one of the 9p24.3 regions during embryogenesis (54–56). In recognized as such in the upcoming WHO classification system. addition, DMRTC2 (mapping to 19p13.3), DMRTB1 (mapping to This study shows that a genome-wide investigation on well- 1p32), and DMRT2 (mapping to 9p24.3) all show a significant characterized tumors is informative, even when done on a limited transcriptional up-regulation in spermatocytic seminoma (P = number of cases. 1.3eÀ4, 3.0eÀ4, and 0.044). This might be of interest because of the evolutionary link between DMRT1 and DMRTC2 and DMRTB1 (57). DMRT1 is present during embryogenesis, in Sertoli and germ Acknowledgments cells (58, 59). Thus far, the presence of DMRT1 during human Received 8/17/2005; accepted 10/26/2005. development and adult life has not been elucidated yet. All Grant support: Dutch Cancer Society, the Daniel den Hoed Cancer Center, Cancer Research UK, Lance Armstrong Foundation, and German Cancer Aid Max-Eder grant three DMRT genes (DMRT1, DMRT2,andDMRT3)showed (D.T. Schneider). overexpression in spermatocytic seminomas, although the last The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance two are at low level. Only DMRT1 was also overexpressed in the with 18 U.S.C. Section 1734 solely to indicate this fact. tumor with the subchromosomal amplification. Indeed, the We thank Prof. Dr. A. Grootegoed (Department of Reproduction and Development, protein was consistently detected by immunohistochemistry in Erasmus Medical Center Rotterdam) for the stimulating discussions regarding meiosis and Drs. David Zarkower and Vivian Bardwell (Department of Genetics, Cell Biology, spermatocytic seminomas and more heterogeneously in semi- and Development, and Cancer Center, University of Minnesota, Minneapolis, MN) for noma/dysgerminoma, possibly related to the underrepresentation making the antibody directed against DMRT1 available for this study.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Genomic and Expression Profiling of Human Spermatocytic Seminomas: Primary Spermatocyte as Tumorigenic Precursor and DMRT1 as Candidate Chromosome 9 Gene

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