—  SYMPOSIUM #34  —

Testicular Neoplasia
Moderators: Dr. Gregor Mikuz and Dr. Victor E. Reuter

Section 3 - Cytogenetics of Germ Cell and Sex Cord-stromal Tumors of the Testis

Gregor Mikuz and Irmgard Verdorfer
Institute of Pathology
Medical University
Innsbruck , Austria


Since the advent of cytogenetic analysis, knowledge about fundamental aspects of cancer biology has increased allowing the process of cancer development and progression to be better understood. Many tumors show specific gene mutations, amplifications, deletions and translocations (Ewing sarcoma, synovial sarcoma, translocation carcinoma of the kidney etc.) as well as a specific gain or loss of entire chromosomes. These chromosomal aberrations can be used in the morphological diagnostic in cases with ambiguous morphology or in completely undifferentiated tumors.

Classical cytogenetics of solid tumors has been extremely difficult in the past because only living cells in the metaphase could be analysed, which means that tumor cells have to be cultivated. Modern molecular techniques allow cytogenetic analyses even in interphase cells and in paraffin embedded tissue. For the analysis of chromosomes the most used methods are the fluorescence in situ hybridization (FISH) and the comparative genomic hybridization (CGH). FISH is particularly useful for gene mapping and for identifying chromosomal abnormalities, whereas, CGH allows the genome wide detection of numerical chromosomal changes without the need for metaphase spread of tumor cells.

Cytogenetic analyses of testicular germ cell tumors (TGCTs) of the testis are of scientific interest, because they allow a deeper insight into their histogenesis. For diagnostic purposes the cytogenetic of TGCTs is, however, useless because, with few exceptions, the chromosomal aberrations are not specific for the single tumors. Thus the morphological diagnosis of TGCTs is still based on their morphological features.

In 1982 Atkin and Baker [1] described a chromosomal change, that they supposed to be specific for testicular tumors – the isochromosome i(12p), which shows a gain of the short arm (Fig.1).


Fig.1 Isochromosome i(12p) left CGH preparation with gain of short arm (short bar) and right the classical metaphase karyogram.

i(12p) is present in about 80% of the TGCT [2] – more often in non seminomatous germ cell tumors (NSGCT) than in seminomas. Extragonadal as well as ovarian GCTs also show this chromosomal anomaly. TGCT without i(12p) have an amplification of genetic material of chromosome 12 [3] The gain of genetic material on chromosome 12 seems to be crucial for the transition from the undifferentiated intratubular germ cell neoplasia to invasive GCTs [3, 4, 5].

Classic seminoma are aneuploid tumors. Previous reports on diploid cases detected by flow cytometry have most likely been caused by extensive lymphocytic infiltration [6]. Cytogenetic studies almost constantly revealed numerical chromosomal aberrations in seminoma. CGH analysis show chromosomal imbalances including gain of parts of 7,8,12,14 and X, and losses of parts of 3,4,5,10,11,16,18,22,and Y [7].


Fig.2. CGH of a classic (blue bars ) – and spermatocytic seminoma (pink bars). The bars on the right mean gain, those on the left losses of chromosomes.

Of all TGCT spermatocytic seminoma show the most specific chromosomal imbalances – the gain of chromosome 9 [8, 9], which is related to the gene DMRT1. Based on the region of amplification defined on 9p and the associated expression plus confirmatory immunohistochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for involvement in the development of spermatocytic seminomas [9]. NSGCT show very heterogeneous patterns of chromosomal imbalance. In combined tumors (embryonal carcinoma, teratoma and yolk sac tumor) all histological components show gains and losses in a very variable amount. Most frequent gain of parts of 1q, 17, 19p, 20q and 22, and losses of parts of 4, 5, 9p, 13 and 18q have been observed [7]. In our material seminoma combined with embryonal carcinomas show a marked gain of X chromosome.

Since sex cord-stromal tumors of the testis account for 3-4% of all testicular neoplasms the rarity [10, 11] of genetic analysis of such tumors is not really surprising. Leydig cell tumors (LCT) of the testis represent only 1-3% of testicular neoplasms. These tumors occur over a wide range of age, from childhood to senior adulthood. They are most common in the third to sixth decade, approximately one fourth of the reported cases occurred


Fig 3.

FISH of Leydig cell tumors: gain of X chromosome before puberty. The histomorphology of these tumors is well known but no reports of cytogenetic data are available in literature. In our material the majority of the 25 analyzed LCT [12] showed chromosomal imbalances (21/25, 84%). The total number of DNA imbalances per tumor varied from case to case. A high genetic heterogeneity and involvement of chromosomes were found. Gain on chromosome X (Fig.3) was detected at a high frequency (56%), followed by gain of chromosome 19 or 19p in 28% and losses on material of chromosome 1 (24%) and 8 or 8p in 16%. The frequent finding of gain of chromosome X by CGH in the present study was confirmed by FISH, with mainly two copies of chromosome X, showing XXY/XX/XXYY or XXY/XXYY mosaicism. The fact that in 56% of our cases of LCT showed gain on chromosome X is of interest, because overexpression of chromosome X was also a frequent observation in seminomas, nonseminomas and spermatocytic seminomas (21-23). The biological and clinical significance of numerical increase in X chromosomes in testicular germ cell tumors was suggested by enhanced expression of the 2 X-linked oncogenes ARAF1 and EKL1 [13].

Sertoli cell tumors (SCT) are even more rare than LCT. Their morphology is also extremely heterogeneous. In most cases, however, a tubular differentiation permits a correct classification. Even in less differentiated cases with solid growth patterns some tubules can be detected. In cases with entirely solid pattern the diagnosis is made when "the appearance is inconsistent with any other plausible diagnosis" [14] , which is also supported by appropriate immunostains.

Cytogenetic data about human SCT of the testis are very rare. Aly et al. [15] reported loss of chromosome Y as the sole aberration in a 5-year-old boy with malignant SCT.


Fig 4. CGH of Sertoli cell tumors – right bars gain, left bars losses

Few genetic data are reported in Sertoli cell tumors of the ovary [16, 17, 18]. Only a single CGH analysis of an ovarian SCT is reported in the literature [18] with amplification of 1q21.3.31, 6q and deletion of 7q32-35. Formation of an i(1q) was reported [20] as the sole karyotypic abnormality in a peritoneal metastasis of an ovarian Sertoli cell tumour.

Gain on copy number of chromosome X (Fig.4), was detected as the most frequent aberration in our study (58%) and was confirmed by FISH [21]. In most of the analysed tumour tissues we found tripartite mosaicisms (XY/XXY/XXYY). The loss of the entire or part of chromosome 2 is the second most frequent (30% of cases) aberration in our material.

Two cases of malignant Sertoli-Leydig cell tumors of the testis and one ovarian SLCT with benign behavior showed copy number changes of chromosome 1, 8, 9p, 10, 11, 12, 16, 19, 22 and X [21].

References:
  1. Atkin NB, Baker MC, Specific chromosome change, i(12p), in testicular tumours? Lancet. 1982 ;2:1349.

  2. van Echten J, Oosterhuis JW, Looijenga LH, van de Pol M, Wiersema J, te Meerman GJ, Schaffordt Koops H, Sleijfer DT, de Jong B, No recurrent structural abnormalities apart from i(12p) in primary germ cell tumors of the adult testis.Genes Chromosomes Cancer. 1995 ; 14:133-44.

  3. Skotheim RI, Lothe RA, The testicular germ cell tumour genome. APMIS 2003; 111: 136-51.

  4. Looijenga LHJ, Oosterhuis JW, Pathogenesis of testicular germ cell tumours. Rev Reprod 1999; 4:90-100.

  5. Reuter VE, Origins and molecular biology of testicular germ cell tumors. Modern Pathology 2002; 18:S51-S60.

  6. Hittmair A, Rogatsch H, Feichtinger H, Hobisch A, Mikuz G, Testicular seminomas are aneuploid tumors.Lab Invest. 1995 72:70-4.

  7. Looijenga LH, Rosenberg C, van Gurp RJ, Geelen E, van Echten-Arends J, de Jong B, Mostert M, Wolter Oosterhuis J, Comparative genomic hybridization of microdissected samples from different stages in the development of a seminoma and a non-seminoma.J Pathol. 2000 ;191:187-92.

  8. Verdorfer I, Rogatsch H, Tzankov A, Steiner H, Mikuz G, Molecular cytogenetic analysis of human spermatocytic seminomas.J Pathol. 2004 ;204):277-81.

  9. Looijenga LH, Hersmus R, Gillis AJ, Pfundt R, Stoop HJ, van Gurp RJ, et al., Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. Cancer 2006; 66:290-302.

  10. Hirakawa T, Ascoli M: A constitutively active somatic mutation of the human lutropin receptor found in Leydig cell tumors activates the same families of G proteins as germ line mutations associated with Leydig cell hyperplasia. Endocrinology 2003;.144: 3872-3878.

  11. Coppes MJ, Ye Y, Rackley R, Zhao XL, Liefers GJ, Casey G, Williams BR, Analysis of WT1 in granulosa cell and other sex cord-stromal tumors. Cancer Res 1993; 53: 2712-14.

  12. Verdorfer I, Horst D, Höllrigl A, Susani M, Hartmann A, Rogatsch H, Mikuz G, Leydig cell tumors of the testis. A molecular cytogenetic study based on series of 25 patients.- submitted.

  13. Kawakami T, Okamoto K, Sugihara H, Hattori T, Reeve AE, Ogawa O, Okada Y: The roles of supernumerical X chromosomes and XIST expression in testicular germ cell tumors. J Urol 2003; 169: 1546-1552.

  14. Ulbright TM, Amin MB, Young RH : Tumors of the testis, adnexa, spermatic cord, and scrotum . AFIP Atlas of Tumor Pathology. 3rd Series Fascicle 25 Washington D.C. 199.

  15. Aly MS, Dal Cin P, Moerman P, De Wever I, Devriendt K, Brock P, Casteels-Van Daele M, Van den Berghe H, Loss of the Y-chromosome in a malignant Sertoli tumor. Cancer Genet 1993; Cytogenet 65:104-106.

  16. Kato N, Fukase M, Ono I, Matsumoto K, Okazaki E, Motoyama T, Sertoli-stromal cell tumor of the ovary: immunohistochemical, ultrastructural, and genetic studies. Hum Pathol 2001; 32: 796-802.

  17. Kato N, Fukase M, Motoyama T (2004) Expression of a transcription factor, SOX9, in Sertoli-stromal cell tumors of the ovary. Int J Gynecol Pathol 23:180-181.

  18. Kato N, Romero M, Catasus L, Prat J (2004) The STK11/LKB1 Peutz-Jegher gene is not involved in the pathogenesis of sporadic sex cord-stromal tumors, although loss of heterozygosity at 19p13.3 indicates other gene alteration in these tumors. Hum Pathol 35:1101-1104.

  19. Pejovic T, Heim S, Alm P, Iosif S, Himmelmann A, Skjaerris J, Mitelman F (1993) Isochromosome 1q as the sole karyotypic abnormality in a Sertoli cell tumor of the ovary. Cancer Genet Cytogenet 65:79-80.

  20. Verdorfer I, Höllrigl A, Strasser U, Susani M, Hartmann A, Rogatsch H, Mikuz, Molecular cytogenetic characterisation of sex cord tumors: CGH analysis in Sertoli cell tumors of the testis. - submitted.

  21. Verdorfer I, Horst D, Höllrigl A, Rogatsch H, Mikuz G, Sertoli-Leydig cell tumors of the ovary and testis:A molecular cytogenetic study - Virchows Archiv - in press