GENES, CHROMOSOMES & CANCER 2:21&216 (1990)

Malignant Rhabdoid Tumor: A Highly Malignant Childhood Tumor With Minimal Karyotypic Changes Edwin C. Douglass, Marc Valentine, Susan T. Rowe, David M. Parham, JudithA. Wilirnas, JoannM. Sanders, and Peter J. Houghton Departments of HematologylOncology (E.C.D., M.V., S.T.R., J.A.W.), Pathology and Laboratory Medicine (D.M.P.), and Pharmacology (P.J.H.), St. Jude Children’s Research Hospital and Division of Hematology/Oncology. Department of Pediatrics (E.C.D..J.A.W.) and Department of Pathology, University of Tennessee College of Medicine, Memphis: Department of HematologylOncology, Cook-Ft. Worth Children’s Medical Center, Fort Worth, Texas (J.M.S.)

Malignant rhabdoid tumors (MRT) are rare; thus very few cytogenetic studies of this type of tumor have been performed. We report the results of cytogenetic studies of I0 MRTs from various anatomic primary sites. Six cases had normal diploid karyotypes with no detectable rearrangements or aneuploidy except for occasional tetraploid cells. In 4 of these cases the tumor phenotype was verified by electron microscopic studies. In a seventh case only normal cells were identified in short-term culture, but a del( I3)(q 14) appeared after 4 months in culture. A soft tissue MRT contained a translocation, t(8; IS)(q I2;p I I), and a liver MRT containeda de1(3)(q21) or t(3;?)(q21;?). The single case of a primary brain MRT had monosomy 22 with deletion of part of the remaining chromosome 22. Our findings indicate that visible chromosomal rearrangements occur in fewer than half of MRTs. When combined with other reported series, our study indicates that monosomy 22 is a non-random chromosomal abnormality in primary MRT of the brain.

INTRODUCTION

Originally described as an unfavorable variant of Wilms’ tumor (Beckwith and Palmer, 1978; Palmer and Sutow, 1983), malignant rhabdoid tumors (MRT) have more recently been recognized as a separate tumor type that can also occur in other organs such as liver (Parham et al., 1988), brain (Biggs et al., 1987), and soft tissues (Tsuneyoshi e t al., 1985). These tumors are aggressive, are often widely metastatic at diagnosis, respond poorly to therapy, and are uniformly fatal, except in children with localized disease (Weeks et al., 1989). T h e cell of origin is unknown, but ultrastructural studies and the presence of cytokeratins and epithelial membrane antigen suggest that M R T may be a very primitive epithelial neoplasm (Parham e t al., 1988; Vogel et al., 1984). We have performed cytogenetic studies on several of these very rare tumors and have found, surprisingly, that they are characterized by few or no detectable chromosomal rearrangements. This is in contrast to other childhood malignancies such as Wilms’ tumor and neuroblastoma, in which aggressive malignant behavior and resistance to therapy are correlated with increased frequency of chromosomal rearrangements. 0 1990 WILEY-LISS, INC.

MATERIALS AND METHODS Patient Population

Patient age, sex, and primacy tumor site are all given in Table 1. Six patients were less than 1 year of age at the time of diagnosis. All tumors were obtained prior to therapy. All patients have died of their disease except for patient 9, who is stili alive with disease. Cytogenetic Studies

We attempted cytogenetic analysis of 12 MRTs, and obtained karyotypes in 10 cases. Tumor material for chromosomal analysis was finely minced and placed in cell culture with RPMI 1640 supplemented with 10% fetal calf serum. Primacy cultures were harvested directly and at 1, 2, 4, and 7 days, and cell lines were harvested in log growth phase to obtain adequate numbers of metaphases for analysis. Colcemid (0.05 pg/ml) was added to the flasks 30 to 60 min before adding trypsin and KCI (0.075 M). Cells were fixed several times in methanol: Received March 21, 1990; accepted June 22, 1990. Address reprint requests to Dr. Edwin C. Douglass, Department of Hematology/Oncology, St. Jude Children’s Research Hospital, P.O. Box 318, Memphis, T N 38101.

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TABLE I . Karvotmes of Malienant Rhabdoid Tumors Case

Agelsex

I 2 3 4 5

5 molf 3 molf I9 molm 3 yrlf 2 molm

6 7

4 molm 9 molf

8 9 10

I 5 yrlm 9 molm 2 yrlf

a

Primary site

Specimen

Time in culture

Karyotype (cells)

Kidney Kidney Kidney Kidney Kidney Kidney Liver Liver Liver Liver Liver Liver Shoulder Neck Brain

Tumor Tumor TumoP TumoP Tumor Tumor Tumor Ascites Ascites Tumor Tumor Xenograft Tumor Tumor Tumor

2 month 25 days 4 days 3 days 6 days 4 months I 3 days Direct I 3 days 32 days 10 months I 3 months Direct 4 days Direct

46,XX (9)/92,XXXX (I) 46,XX (I0 ) 46,XY (4) 46,XX (5) 46,XY (20) 46,XY,del( I3)(q 14) (20) 46,XY,de1(3)(q2I) or t(3;?)(q21;?) (6) 46,XX (5) 46,XX (10) 46,XX (5) 46,XX (5)/92,XXXX (2) 46,XX (6)/92,XXXX (4) 46,XY,t(8; I5)(q I2;pl I) (4) 46,XY (I5) 46,XY,+?20,-22,de1(22)(q I I) (5)

Not verified by EM.

Figure 1. Electron micrograph of a cell from case 7 (46,XX), which displays large nucleoli and large cytoplasmic bundles filled with intermediate filaments.

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acetic acid (3:l by volume), and the slides were prepared by air drying before staining with Giemsa and trypsin-Giemsa. Every G-banded metaphase spread (4 to 20 metaphases per case) was counted and karyotyped. A missing chromosome in three or more metaphases or an extra chromosome or structural abnormality in two or more metaphases defined a clonal abnormality. Cells from one tumor (case 7) were studied both from initial explants, after 10 months of cell culture, and from a xenograft that originated from the cell culture. Electron microscopic studies were performed on cultured tumor cells from each case to verify their origin with the exception of cases 3 and 4 whose cells could not be maintained in culture for an adequate time to allow study. Histologic Criteria

T h e diagnosis of malignant rhabdoid tumor was

Figure 2.

established by the presence of sheets of cells with oval, eccentric nuclei, prominent nucleoli, and eosinophilic cytoplasm that often contained hyaline inclusions (Weeks et al., 1989). T h e histologic diagnosis was supported in most cases by the immunohistochemical findings of vimentin positivity or coexpression of vimentin and cytokeratin in the cytoplasm and by electron microscopic findings as described below. Electron Microscopy

For electron microscopy, cells were harvested and centrifuged at 180g for 5 minutes. T h e resultant pellet was overlaid with 2.5% glutaraldehyde for 2 hours, rinsed in 0.1 M sodium phosphate buffer, and minced into fine pieces. T h e fragments were placed in osmium tetroxide solution for 1 hour and then rinsed again in buffer. They were then dehydrated through a graded series of alcohol

Karyotype of case 5 after 4 months in culture, demonstrating del( I3)(q14).

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rinses before being embedded in Spurr resin. Thick sections were cut and stained with toluidine blue, and optimal sections were placed onto copper grids, stained with uranyl acetate and lead citrate, and examined using a Phillips 301 transmission electron microscope. RESULTS Cytogenetic Studies

T h e karyotypes are presented in Table 1. Tumor cells from six of the ten cases (1, 2, 3 , 4 , 7, and 9) contained only 46 chromosomes with no detectable rearrangements or aneuploidy except for occasional tetraploid cells. In four of these cases (1, 2, 7, and 9) we verified the tumor phenotype of the cells by electron microscopy (Fig. 1). In a seventh case (No. 5), only normal cells were identified in short-term culture; however, after 4 months of culture a de1(13)(q14) appeared (Fig. 2 ) , presumably from expansion of a clone not identified in the primary culture. A soft tissue MRT, case 8, contained a single rearrangement, t(8; lS)(qlZ;pll) (Fig. 3), and a liver MRT, case 6, contained a

Figure 3.

de1(3)(q21) or t(3;?)(q21;?) (Fig. 4). T h e single case of a primary brain MRT, case 10, had monosomy 22 with a deletion at 22ql1, and an extra chromosome identified as a possible 20 (Fig. 5). Electron Microscopy

Electron microscopic examination disclosed a population of undifferentiated tumor cells in all four cases studied. These cases also revealed numerous cells with eccentric nuclei containing prominent nucleoli. T h e cytoplasm generally contained sparse organelles, primarily mitochondria and lysosomes, and cytoplasmic bundles of intermediate filaments measuring 8 to 10 nm. T h e number of cells containing these filaments varied from few to many, depending on the case. There were no features of neurons, muscle, or epithelium, i.e., dense core granules, microtubules, sarcomeres, or tonofilaments, in any of the cells. DISCUSSION

No chromosomal abnormalities were detected in 6 of the 10 cases we studied. Three of the other 4

Karyotype of case 8 demonstrating t(6 15)(q IZ;p I I).

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Figure 4.

Karyotype of case 6 demonstrating de1(3)(q2I) or t(%?)(q2l;?).Missing chromosome 22 from random loss in this cell.

cases had only a single rearrangement with a single deletion in 2 cases and a single translocation in the third. T h e single case of M R T originating in brain demonstrated monosomy 22 and probable trisomy 20. Similarly, few abnormalities have been noted in published cytogenetic case studies. Dao and coworkers reported the karyotypes of 12 Wilms’ tumors and a single MRT of the kidney (Dao et al., 1987) that had a normal diploid (46,XY) karyotype. Three reports of cytogenetic studies of MRT have appeared in abstract form. Karnes has studied tumor material from a 21 month old male with a retroperitoneal MRT and found a single translocation, t(11;22)(p15.5;ql1.23) (Karnes et al., 1989). Hayashi and coworkers described a case of M R T of the kidney and found del(ll)(pl3) as the only abnormality (Hayashi et al., 1985). Most intriguingly, Biegel and coworkers have found a specific abnormality, monosomy 2 2 , in three brain tumors characterized as MRT or atypical teratoid tumors (Bie-

gel et al., 1989a,b). The single M R T of the brain in our series, case 10, is consistent with monosomy 22 as a nonrandom abnormality in primary brain tumors with rhabdoid morphology. Normal karyotypes in cells from solid tumor cultures are often dismissed as resulting from overgrowth of normal cells. In the present series, however, electron microscopic study of the monomorphic cell cultures done simultaneously with cytogenetic studies have demonstrated the exclusive presence of tumor cells. T h e lack of aneuploidy in MRT has also been reported in a study by Schmidt and coworkers where flow cytometric analysis disclosed only diploid stem lines in 11 cases of M R T (Schmidt et al., 1989). Other pediatric malignancies have typically been associated with increased numbers of chromosomal rearrangements, i.e., translocations, corresponding with increased degrees of malignancy. Examples include Wilms’ tumor (Douglas et al., 1986), neuroblas-

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Figure 5. Karyotype of case 10 demonstrating monosomy 22 with de1(22)(q I I) and +?20 in this primary brain MRT. The missing I4 is due t o random loss.

toma (Hayashi et al., 1989), and acute lymphoblastic leukemia (Look et al., 1985). Although M R T certainly has marked genetic aberrations as expressed by its clinical aggressiveness and resistance to therapy, in contrast to other pediatric tumors, these changes are not reflected by visible chromosomal abnormalities. ACKNOWLEDGMENTS

We wish to thank Sharon Nooner for tissue culture of the cell lines, Linda Daniels for editorial assistance, and Alice Slusher and Victoria Denison for preparation of the electron microscopic materials. This work was supported in part by grants CA 23099 and CA 21765 from the National Cancer Institute, National Institutes of Health, and by the American Lebanese Syrian Associated Charities. REFERENCES Beckwith JB, Palmer N F (1978) Histopathology and prognosis of

Wilms’ tumor. Results from the First National Wilms’ Tumor Study. Cancer 41:1937-1948. Biegel JA, Rorke LB, Emanuel BS (1989a) Monosomy 22 in rhabdoid or atypical teratoid tumors of the brain. N Engl J Med 32 1:906. Biegel JA, Rorke LB, Packer RJ, Emanuel BS (1989b) Monosomy 22 in atypical teratoid or rhabdoid tumors of the brain. Am J Hum Genet [Suppl] 45(4):A15 (Abstract). Biggs PJ, Garen PD, Powers JM, Garvin AJ (1987) Malignant rhabdoid tumor of the central nervous system. Hum Pathol 18:332337. Dao DT, Schroeder WT, Chao L-Y, Kikuchi H, Strong LC, Riccardi VM, Pathak S, Nichols WW, Lewis WH, Saunders GF (1987) Genetic mechanisms of tumor-specific loss of l l p DNA sequences in Wilms tumor. Am J Hum Genet 41:202-217. Douglass EC, Look AT, Webber B, Parham D, Wilimas JA, Green AA, Roherson PK (1986) Hyperdiploidy and chromosomal rearrangements define the anaplastic variant of Wilms’ tumor. J Clin Oncol 4:975-981. Hayashi Y, Hanada R, Yamamoto I