Chromosomal Abnormalities of a Mediastinal Embryonal Cell Carcinoma in a Patient with 47,XXY Klinefelter Syndrome: Evidence for the Premeiotic Origin of a Germ Cell Tumor

Barry D. Mann, Robert S. Sparkes, David H. Kern, and Donald L. Morton

ABSTRACT: Trypsin-Giemsa banding studies were performed on 30 tumor cells from an embryonal cell carcinoma originating in the mediastinum of an 18-year-old male with the Klinefelter syndrome (47,XXY). All tumor cells revealed an X X Y chromosomal pattern with the addition of extra chromosomes. Electrophoretic patterns of the patient's red blood cells and tumor cells were identical.

These data suggest that this germ cell tumor originated from a primordial germ cell in which oncogenesis had occurred prior to meiotic division.

INTRODUCTION

The Klinefelter syndrome, the most common cause of male hypogonadism, has an estimated incidence of 0.06% [1]. Recently, this syndrome was reported to be asso- ciated with gonadal and extragonadal germ cell tumors [2-5], this association being attributed to an increased malignant potential of the dysgenetic gonad.

Early studies of the nuclear sex of teratomas and other germ cell neoplasms em- phasized discrepancies between the nuclear sex of the tumor and host. These dis- parities were explained by several theories of germ cell tumor origin, inc luding the hypothesis that these tumors may originate from haploid gametes. More recent ex- perimental evidence, however, has led authorities to believe that embryonal neo- plasms arise from diploid origins. We report a case of embryonal cell carcinoma of the mediast inum in an 18-year-old male with the XXY Klinefelter syndrome in which, for the first time, a banded chromosomal analysis of both the tumor and the host is included. The consistent XXY karyotype in this germ cell tumor is further evidence that these tumors arise from a primordial germ cell prior to meiotic division.

From the Divisions of Surgical Oncology and Medical Genetics, UCLA School of Medicine, Los Angeles, and Surgical Service, Veterans Administration Medical Center, Sepulveda, California.

Address requests for reprints to Dr. Barry D. Mann, Division of Surgical Oncology, UCLA School of Medicine, Los Angeles, CA 90024.

Received March 15, 1982; accepted June 23, 1982.

© Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, NY 10017

1 9 1 Cancer Genetics and Cytogenetics 8, 191-196 (1983) 0165-4608/83/030191-06503.00

192 B.D. Mann et al.

CASE REPORT

An 18-year-old male had a 3-month history of intermittent anterior chest pain ra- diating to the right shoulder and a 3-week history of a tender protrusion over the superior aspect of the sternum.

In appearance, the patient was tall and slender. He had a eunuchoid habitus and his testicles were small. A 3 x 5-cm firm, smooth, subcutaneous mass protruded anteriorly at the level of the manubriosternal angle. The mass was fixed to the un- derlying bone. Chest X ray revealed a large mediastinal mass occupying much of the left hemithorax and eroding anteriorly through the manubr ium and sternum. The lateral film showed a soft tissue mass anterior to the sternum. An incisional biopsy of the sternal mass identified an embryonal cell carcinoma (Fig. 1). Markedly elevated ~-fetoprotein of greater than 320 mg/ml (normal -> 20 mg/ml) and serum [3-human chorionic gonadotropin ([3-HCG) of 30 mIU/ml (normal -> 5 mIU/ml) were consistent with this histologic type of tumor.

The patient was treated preoperatively with c/s-platinum, vinblastine, and bleo- mycin with no decrease in the tumor mass or in ~-fetoprotein or [3-HCG. At opera- tion, a 600-g, 26 x 8.5 x 11.0-cm anterior mediastinal mass was resected en bloc with the manubr ium, superior half of the sternum, and the medial halves of the clavicles. Portions of the pericardium and left upper lobe, which were contiguous with the tumor, were also resected. Following surgery the ~-fetoprotein and [3-HCG serum levels were wi thin normal range. The patient remained clinically free of dis- ease for 8 months but died 10 months following surgery as the result of local recur- rence and metastatic disease.

METHODS

Following resection of the embryonal cell carcinoma, 6 g of tissue was minced and digested with 0.14% collagenase II (Sigma Chemical Company) for cell suspension. The tumor cells were cultured in CEM medium (Microbiological Associates) sup- plemented with 20% fetal calf serum. Peripheral blood cultures were established with the whole blood microtechnique of Arakaki and Sparkes [6], with RPMI 1640 (Microbiological Associates) medium containing 25% fetal calf serum and antibiot- ics. Skin fibroblasts were grown in Ham's F-12 medium containing 15% fetal calf serum and 1% penic i l l in-s t rep tomycin antibiotics.

Tryps in-Giemsa banding of chromosomes was accomplished using the method of Seabright [7].

Electrophoresis of polymorphic enzyme markers on red blood cells and cultured tumor cells used standard methods [8].

RESULTS

Cytogenetic analysis of peripheral blood cultures in the presence of phytohemag- glut inin (PHA) showed 47 chromosomes with an XXY male sex chromosome pat- tern. This abnormal karyotype, typical of the Klinefelter syndrome, was confirmed by t ryps in-Giemsa banding. The same 47,XXY pattern was seen in a culture of fibroblasts derived from biopsied forearm skin used as a control (Table 1). All lym- phocytes and fibroblasts were 47,XXY. No diploid cells were seen.

Trypsin-Giemsa banding studies of 30 tumor cells from culture revealed 27 with 50 chromosomes and 1 cell each with 48, 49, and 51 chromosomes. All cells with 50 chromosomes displayed the following abnormalities: an extra chromosome #7, an extra chromosome #21, an extra marker chromosome that appeared to consist of an isochromosome of the long arms of chromosome 21, and the extra X chromo-

193

C

F i g u r e 1 (a-c) Endodermal, mesodermal, and ectodermal elements within the patient 's tu- mor. (d) High-power view of poorly differentiated cells compatible with embryonal cell car- cinoma.

194 B.D. Mann et al.

Table 1 Cultures confirming karyotype typical of the Klinefelter syndrome

Number of cells Source Chromosome pattern

15 Lymphocytes 47,XXY 15 Fibroblasts 47,XXY 27 Embryonal cell 50,XXY, + 7, + 21, + marker

1 carcinoma 49,XXY, + 7, + 21, + marker 1 48,XXY, + 7, + 21, + marker 1 51,XXY, + 7, + 21, + marker

some typical of the Klinefelter syndrome. The cell wi th 51 chromosomes had a quest ionable addi t ional chromosome, whi le the cells wi th less than 50 showed the same basic pat tern wi th random chromosomal loss. A typical banding pat tern is seen in Figure 2 wi th the karyotype designat ion of 50,XXY, + 7, + 21, + marker. Late- labeling studies using bromodeoxyur id ine incorporat ion showed one late-labeling X chromosome in the tumor cell.

Electrophoresis showed a homozygous pat tern for adenylate kinase, phospho- gluconate dehydrogenase, adenos ine deaminase, phosphoglucomutase-1 , and ester- ase D. The pat tern for glyoxalase-1 showed a heterozygous 2-1 type. The pat terns were ident ical in the pat ient ' s red blood cells and tumor cells from t issue culture.

DISCUSSION

The increased associat ion between the Klinefelter syndrome and extragonadal germ cell tumors has been recent ly recognized [2-4]. This associat ion appears to be more than coincidenta l and suggests that the dysgenetic germ cell t issue in the Klinefelter syndrome has an increased mal ignant potent ial [3].

It is general ly accepted that teratomatous mal ignancies arise from pr imi t ive germ cells; however, their occurrence as pr imary medias t ina l tumors has not been en- t irely expla ined. Several theories have been proposed. Earliest specula t ions of their genesis postula ted an " inc luded twin" (fetus in fetu), and later theories favored a dis turbance in the deve lopment of the branchial clefts [9]. More recent investigators propose that extragonadal teratomas originate from undifferent ia ted ceils of the pr imit ive streak that have escaped the influence of embryonic organizers [10]. Oth- ers see the appearance of the germ cell in the med ias t inum as an incomple te migra- t ion of the pr imordia l germ cell along the urogenital r idge from its origin in the endoderm of the yolk sac to the gonad [9, 11, 12]. The extension of the urogenital ridge from the sixth cervical vertebra to the third lumbar vertebra could expla in the occurrence of germ cell tumors in the medias t inum.

Since the method for nuclear sexing was original ly descr ibed by Barr and Ber- tram [13], tumors of somatic t issues have usual ly been found to be of the same nuclear sex as the pat ients in whom they arose [14]. Invest igat ions of various go- nadal tumors, however, have shown female sex chromat in pat terns in some terato- mas from male pat ients [14-17]. For example, in a large series of test icular tumors repor ted by Theiss et al. [15], none of 50 seminomas, 25 interst i t ial cell tumors, or 23 gonadal stromal tumors showed a female chromat in pattern; however, in 29/96 test icular teratomas, 12/50 embryonal cell carcinomas, and 4/25 medias t ina l terato- mas from male patients, female nuclear sex chromat in was identified. The existence of female nuclear sex chromat in in test icular teratomas was original ly interpreted as suppor t for the theory that teratomas of the testis arise from the fusion or auto-

Chromosomal Abnormal i t ies of an Embryonal Cell Carcinoma 195

2 3 4 5

6 7 11 t2 X 8 9 10

13 14 15 16 17 18

J

19 20 21 22 Y

Figure 2 Trypsin-Giemsa chromosome banding of tumor cell in culture. Note the extra chromosomes, #7, X, and #21. The arrow points to a marker chromosome which appears to consist of two long arms of chromosome #21.

fert i l ization of two haplo id cells [10] or that a single hap lo id cell has undergone a chromosomal redupl ica t ion by mitosis wi thout nuclear divis ion (parthenogenetic division) [14]. Whereas recent invest igat ions have shown that benign ovarian tera- tomata are indeed par thenogenic tumors that arise from a single germ cell after the first meiot ic d ivis ion [18] evidence has accumula ted that ovarian, testicular, and extragonadal teratomata have different behaviors and origins [19]. Experiments in a mouse model have shown that embryonal cell carcinoma can be p roduced by trans- plant ing single (diploid) cells of 7-day embryos to immunologica l ly pr ivi leged sites [20]. The embryonal cell carc inoma in our pat ient appears to have or iginated from such a premeiot ic precursor. The consis tent XXY pat tern strongly suggests that this tumor arose from a pr imord ia l germ cell that d id not undergo meiosis pr ior to on- cogenesis. It is un l ike ly that a consis tent XXY pat tern would have resul ted from either the recombinat ion or redupl ica t ion of abnormal hap lo id gametes. The f inding of ident ical heterozygous or homozygous enzyme patterns in both the pat ient ' s tu- mor cells and red blood cells strengthens the conclus ion that the tumor and the normal somatic t issues or iginated from ident ical premeiot ic genetic materal.

The appearance of female nuclear sex chromat in in teratomata from male pa- t ients in the earl ier s tudies poses a quest ion as to whether any of these pat ients had the extra X chromosome of the Klinefelter syndrome. Unfortunately, none of these studies inc luded a full chromosomal analysis, and it is possible that these were false posi t ives related to the aneup lo idy known to occur in mal ignant teratomata. Their value is l imited as wel l by the general problems of accurate sex chromat in determinat ion.

196 B . D . M a n n et al.

Because th i s is t he first r e p o r t in w h i c h a b a n d e d c h r o m o s o m e a n a l y s i s of a m e d i a s t i n a l e m b r y o n a l cel l c a r c i n o m a has b e e n i n c l u d e d , we c a n n o t be su re of t he s ign i f i cance of the p a r t i c u l a r c h r o m o s o m e a d d i t i o n s . T h e a n e u p l o i d c h a n g e s are in k e e p i n g w i t h t he n e o p l a s t i c state, b u t w h e t h e r the c h a n g e s are spec i f ic for t h i s tu- m o r t y p e c a n n o t be d e t e r m i n e d f rom a s ing le case.

This work was supported by Grant CA-09Ol0 from the National Cancer Institute, DHEW, and by the Medical Research Service of the Veterans Administration.

REFERENCES

1. Gerald PS (1976)'. Current concepts in genetics--Sex chromosome disorders. New Engl J Med 294, 706-708.

2. Turner AR, Macdonald RN (1981): Mediastinal germ cell cancers in Klinefelter's syndrome (letter) Ann Intern Med 94, 279.

3. Sogge MR, McDonald SD, Cofotd PB (1979): The malignant potential of the dysgenetic germ cell in Klinefelter's syndrome. Am J Med 66, 515-518.

4. McNeil MB, Leong A, Sage RE (1981}: Primary mediastinal embryonal carcinoma in asso- ciation with Klinefelter's syndrome. Cancer 47, 343-345.

5. Schellhas HF {1974): The malignant potential of the dysgenetic gonad. Obstet Gynecol 44, 298-309.

6. Arakaki DT, Sparkes RS {1963}: Microtechnique for culturing leukocytes from whole blood. Cytogenetics 2, 57-60.

7. Seabright M (1971}: A rapid banding technique for human chromosomes. Lancet 2, 971- 972.

8. Harris H, Hopkinson DA (1976}: Handbook of Enzyme Electrophoresis in Human Genetics. North Holland Publishing Company, Amsterdam.

9. Schlumberger HG (1946): Teratoma of the anterior mediast inum in the group of military age. Arch Pathol 41, 398.

10. Hunter WF, Lennox B (1954}: The Sex of Teratoma. Lancet 2 ,633-634.

11. Recondo J, Libshitz HI (1978}: Mediastinal extragonadal germ cell tumors. Urology 11, 369-375.

12. Johnson BE, Laneri JP, Mountain RF, Luna M (1973}: Extragonadal germ cell tumors. Sur- gery 73, 85-90.

13. Barr ML, Bertram JEG (1949}: A morphological distinction between neurones of the male and female, and the behavior of the nucleolar satellites during accelerated nucleoprotein synthesis. Nature 163, 676-677.

14. Tavares AS (1955): On the sex of cancer and teratoma ceils. Lancet 2 ,948-949.

15. Theiss EA, Ashley D, Mostofi KF (1960}: Nuclear sex of testicular tumors and some related ovarian and extragonadal neoplasms. Cancer 13,323-327.

16. Myers LM (1959}: Sex chromatin in teratomas. J Pathol Bacteriol 78, 43-55.

17. Atkin NB (1973}: High chromosome numbers of seminomata and malignant teratomata of the testis: A review of data on 103 tumors. Br J Cancer 28, 275-279.

18. Linder D, McCaw BK, Hecht F (1975): Parthenogenetic origin of benign ovarian teratomas. N e w Engl J Med 292, 63-66.

19. Erickson RP , Condos B {1976}: Alternative explanations of the differing behavior of ovar- ian and testicular teratomas. Lancet 1 ,407-409.

20. Damjanov I, Solter D, Belicza M, Skreb N (1971}: Teratomas obtained through extrauterine growth of seven-day mouse embryos. J Natl Cancer Inst 48,471-475.