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 kar- yotyping, 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 Cancer Res 2006; 66: (1). January 1, 2006 290 www.aacrjournals.org 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