Germ Cell Tumors Cytogenetic Analysis of 124 Prospectively Ascertained Male

We report the cytogenetic analysis of 124 adult male germ cell tumors ascertained consecutively at the Memorial Sloan-Kettering Cancer Center between 1988 and 1990. Biopsies from testicular and extragonadal pri mary and metastatic lesions studied included all histolÃ3gica!subtypes of germ cell tumors and cases of malignant transformation. Nonrandom numerical and structural chromosomal abnormalities including i(12p), the previously described characteristic marker of these tumors, were determined, and their frequency was compared between histolÃ3gica! subtypes, between gonadal and extragonadal lesions, and between pri mary and transformed lesions. The frequency and copy number of i(12p) were found to be higher in nonseminomas compared with seminomas. Nonrandom sites of chromosome rearrangements associated with specific histologies comprised lp32-36 and 7ql 1.2 in teratomas and Ip22 in yolk sac tumors. Some tumors that underwent malignant differentiation exhib ited chromosome changes previously described to be nonrandomly asso ciated with de novo tumors with the same histolÃ3gica!characteristics. Cytological evidence of gene amplification in the form of homogeneously staining regions and/or double minutes was detected in 24% of extrago nadal lesions, mainly metastatic tumors, suggesting amplification of a gene(s) associated with metastatic progression of these tumors. While a number of previous small cytogenetic series or individual case reports of germ cell tumors identified several of the features of these tumors reported here, this series comprises analysis of the largest group of tumors ascertained consecutively at a single institution, defines the inci dence of nonrandom abnormalities in tumor subsets, and addresses their biological significance. INTRODUCTION Adult male GCTs3 are a heterogenous group of neoplasms. Their biological properties and responsiveness to chemotherapy make them a model system for the study of differentiation, malignant transformation, and drug sensitivity. GCTs may originate in the testis or at extragonadal sites (mediastinum, retroperitoneum, pineal). Histologically, they comprise two main entities: seminomas, composed of neoplastic germ cells which mimic gametogenesis and act as immature spermatogenic cells; and nonseminomas, composed of embryonic neoplastic germ cells which mimic histogenesis of the early embryo (1). Among nonseminomas, embryonal carcinoma is a pluripotent tumor which may progress along extraembryonic or trophoblastic lineages to form yolk sac tumor or choriocarcinoma or along the embryonic lineage to form teratoma. Both seminomas and nonseminomas have been suggested to originate from a common precursor cell, carcinoma in situ of the testis, which is the neoplastic counterpart of a gonocyte (2, 3). Individual tumors may present several histologies: when they contain only nonseminomatous components, they are termed mixed GCTs; and when they present seminomatous and nonseminomatous elements, they are termed combined GCTs (4). Occasionally, Received 9/24/91; accepted 2/4/92. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by NIH Research Grant CA-05826. 2To whom requests for reprints should be addressed, at Box 391, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. 3The abbreviations used are: GCT, germ cell tumor; ERMS, embryonal rhabdomyosarcoma; HSR, homogeneously staining region. teratomatous components of nonseminomatous GCTs undergo malignant differentiation into tumors typically encountered in other organs and tissues such as sarcoma, adenocarcinoma, neuroblastoma, or myeloid leukemia (5-7). Cytogenetic analyses of adult male GCTs have identified i(12p) as a common marker to all histologies and sites, occur ring in over 80% of described cases (8-15). However, recurrent abnormalities other than i(12p) remain to be defined in GCT. We report here the cytogenetic analysis of 124 consecutively ascertained specimens of GCTs derived from 106 patients and representing all described histological subtypes. The tumors studied included testicular and extragonadal presentation of primary as well as metastatic lesions biopsied prior to and after treatment. This series comprises the largest cytogenetic series of GCTs analyzed so far and enabled us to examine the asso ciation between nonrandom chromosome abnormalities and histological and biological features of these tumors. MATERIALS AND METHODS Tumor Ascertainment and Cell Culture. The tumors studied represent a consecutive ascertainment of all GCTs seen at the Memorial SloanKettering Cancer Center between January 1988 and December 1990. A total of 124 specimens from 106 patients were processed for culture and cytogenetic analysis. Upon receipt in the laboratory, each biopsy specimen was finely minced with scalpels into 2to 3-mm pieces and digested in growth medium containing collagenase II (GIBCO) at a concentration of 200 units/ml for 4 to 16 h. After disaggregation, cells were centrifugea, transferred to fresh medium, and cultured in a 5% CO2 atmosphere at 37°C. The growth medium comprised RPMI 1640 (500 ml), 15% fetal calf serum, 1% 200 m\i L-glutamine, 5 Mg/ml of insulin, 5 ng/ml of transferrin, 5 Mg/ml of sodium satinate, and 50 ng/ ml of penicillin/streptomycin. Tumors were staged according to the criteria of Hendry et al. (16). Diagnostic histopathology was performed according to conventional methods. Information on the clinical status and laboratory data on the patients including serum levels of a-fetoprotein, human chorionic gonadotropin, and láclatedehydrogenase were obtained at the time of tumor resection. Cytogenetic Analysis. Cells for cytogenetic analysis were harvested following short-term culture (1 to 10 days after seeding of the cells) by conventional methods. Metaphases were accumulated by Colcemid treatment (final concentration, 0.01 to 0.02 Mg/ml) that ranged from 6 to 14 h and stained to reveal Qor G-banding patterns. Karyotypes were described according to the International System for Human Cy togenetic Nomenclature (17). Clonality was defined by the detection of two cells with the same structural abnormality or nonrandom gain of the same chromosome or three cells with loss of the same chromosome. RESULTS Of the 124 specimens processed for cytogenetic analysis, abnormal clones were found in 65 biopsies from 58 patients; 29 showed normal karyotypes and 30 did not yield dividing cells. Of the 65 specimens with clonal abnormalities, 40 were derived from metastatic lesions, and 25 were from primary tumors; 34 specimens were from patients who underwent chemotherapy, and 31 were from previously untreated patients; 15 specimens were primary extragonadal in origin (in each of these cases, an occult testicular lesion was ruled out by sonog2285 American Association for Cancer Research Copyright © 1992 on February 23, 2013 cancerres.aacrjournals.org Downloaded from MALE GERM CELL TUMOR CYTOGENETIC ANALYSIS raphy), and the remaining were of testicular origin. Table 1 records the cytogenetic success rate in each of the histological subsets and shows that abnormal clones were less frequently detected in seminomas, yolk sac tumors, and mixed tumors compared with teratomas, embryonal carcinomas, combined tumors, and tumors exhibiting malignant transformation. The cytogenetic data on 27 of these specimens with clonal chromosome abnormalities were published by us previously (7, 14, 18). Table 2 summarizes the clinical, histological, and karyotypic data on the remaining 38 specimens with clonal chromosome abnormalities. The karyotypes in general were characterized by gains of chromosomes and the presence of marker chromosomes. The modal chromosome numbers ranged from 46 to 124; one tumor was pseudodiploid, 23 tumors were hyperdiploid, 31 tumors were near-triploid, 5 tumors were near-tetraploid, and 2 tumors were near-pentaploid. In an additional 2 tumors, 2 related abnormal clones in the diploid and tetraploid ranges were detected. Finally, an additional tumor presented two abnormal clones in the triploid and pentaploid ranges. The mean of the modal numbers of the short and long arms of chromosomes (including those in structural abnormalities when a complete arm could be identified) was calculated and shown in Fig. 1. Most autosomal arms were in the near-triploid range except 12p which was overrepresented. In nonseminomas the X chromosome was represented by more copies (mean, 1.66) than the Y chromosome (mean, 1.24), whereas in semi nomas the Y chromosome (mean, 1.71) was represented by more copies than the X chromosome (mean, 1.29). The i(12p) marker, characteristic of germ cell tumors, was observed in 56 (86%) tumors; its copy number varied from 1 to 4. Seminomas had an average of 1.2 i(12p) copies per cell which was seen in 71% of the specimens. Teratomas and embryonal carcinomas had an average of 2 i(12p)s per cell which was seen in 89% of the former and 83% of the latter. Yolk sac tumors had an average of 1.4 i(12p) copies per cell which was seen in 100% of the specimens. Deletions or translocations affecting the 12ql 125 region were observed in 11 specimens from 10 patients, 9 of these also having one or more copies of i(12p). The relevant breakpoints comprised one at 12qll, 3 at 12ql3, 5 at 12ql5, and one each at 12q21, 12q22, 12q24, and 12q25. A total of 9 specimens did not exhibit an i(12p). Of these, 6 had abnormal ities of chromosome 12 which comprised a del(12q) in one (No. 176A), translocations affecting 12p in 2 (Nos. 231A and 251 A), a der(12) chromosome with a translocation affecting both the p and q arms in one (No. 240A), while 2 showed monosomy 12 Table 1 Cytogenetic success rate among the histological subsets of GCT patients studied Histological subsetTeratomaSeminomaEmbryonal carcinomaYolk sac tumorChoriocarcinomaMixed tumorCombined tumorMalignant transformationLeukemiaNeuroepitheliomaEmbryonal rhabdomyosarcomaOther sarcomaSquamous carcinomaUndifTerentiated carcinomaNo. of patients 125252574185114122No. of Biopsies 125252594195214122CytogeneticsFailure Normal Abnormal 9552700000005970061001000187134264213122 Total 120 124 30 29 65 (Nos. 207A and 249A). The remaining 3 specimens (Nos. 178A, 178B, and 238A) from two other patients had no re arrangements affecting chromosome 12; however, 2 specimens exhibited aberrantly banded marker chromosomes of unknown origin (Nos. 178A and 238A). Fig. 2 illustrates chromosomal sites involved in clonal re arrangements 3 or more times each, and Figs. 3 and 4 are representative of full karyotypes from an embryonal carcinoma (242A) and a teratoma (231 A). Fig. 5 is an idiogram of the human karyotype showing all the breakpoints encountered in this series according to histology and comparing primary with metastatic lesions. Some breakpoints were nonrandomly asso ciated (P < 0.05) with certain histologies, e.g., lp32-36 (7 of 18 in teratomas compared with one of 32 in the rest of the tumors with single histology); 7qll.2 (3 of 18 in teratomas compared with 0 of 32 in the rest of the tumors with single histology); and Ip22 (2 of 4 in yolk sac tumors compared with 2 of 48 in the rest of tumors with single histology). We were able to isolate regions of pure yolk sac and pure teratoma and perform cytogenetic analysis from one patient (No. 235; Table 2). The only difference in the karyotype between the two his tologies was the presence of a del(l)(p22) in the yolk sac which was absent in the teratoma. Some breakpoints were found only in metastatic lesions, e.g., 10pl3 (4 of 40), 7qll.2 (3 of 40), and 12pll-ql3 (7 of 40). Interestingly, in Patient 159, a 10pl3 rearrangement was seen only in the metastatic lesion (No. 159B) but not in the primary lesion (No. 159A). Some breakpoints were significantly more frequent in posttherapy specimens (P < 0.05), e.g., 6q21 (6 of 34 posttherapy, one of 31 pretherapy), while other breakpoints were seen only in posttherapy specimens, e.g., Ip36 (6 of 34), 7ql3 (3 of 34), and 12qll-13 (5 of 34). Another finding of interest was cytological evidence of gene amplification in the form of HSRs and/or double minute chro mosomes in 6 of the tumors (Nos. 23A, 51D, 74A, 154A, 221 A, 240A). An additional, 6 tumors presented aberrantly banded marker chromosomes with a repeating banding pattern of un known origin which may also represent amplification (Nos. 76C, 178A, 220A, 238A, 242A, 248A) (Fig. 1). Of these, 8 were resected from the retroperitoneum, 3 from lymph nodes, and one from the mediastinum. In this series, we had the opportunity to study chromosome changes associated with malignant transformation of GCTs. A total of 9 tumors presented with transformed histologies. In 2 i(12p)-positive teratomas, we were able to study both the pri mary and malignant components. In the first case (No. 149), a patient with primary mediastinal teratoma/yolk sac tumor de veloped an acute nonlymphocytic leukemia 18 mo following the initial GCT; the leukemia retained the i(12p) of the primary t mor and, in addition, acquired a del(5)(q 13) (6). In the second case (No. 203), a mediastinal GCT comprised discreet segments of teratoma and ERMS. Clonal relatedness was demonstrated by the presence of i(12p) in both, and the ERMS in addition acquired a der(2) t(2;?)(q37;?) (7). In an additional case (No. 243), only the transformed state was available for cytogenetic analysis, a neuroepithelioma which exhibited both an i(12p) and a der(l 1) t(l I;?)(q24;?). In addition to the above, 5 mixed tumors (Nos. 159A, 2ISA, 244A, 246A, and 262A) exhibited malignant differentiation. In one of these (No. 246A), the transformation was to ERMS which showed a der(2) t(2;l)(p21;qll) and multiple copies of chromosome 2. In an other of these (No. 215A), the transformation was to a squa2286 American Association for Cancer Research Copyright © 1992 on February 23, 2013 cancerres.aacrjournals.org Downloaded from MALE GERM CELL TUMOR CVTOGENETIC ANALYSIS Table 2 Clinical, histological, and karyotypic data on germ cell tumors with donai chromosome abnormalities Tumor Prior Tumor no. Stage therapy Histology Site" state155B IV CT¿ T, EC Lung M 175A III N EC Testis PI78B III N EC, S Testis P 179A II CT EC Ascites M184B