GENES, CHROMOSOMES & CANCER 11:146152 (1994)

Rhabdoid Tumor of the Kidney With Primitive Neuroectodermal Tumor of the Central Nervous System: Associated Tumors With Different Histologic, Cytogenetic, and Molecular Findings

Daniel W. Fort, Vijay S. Tonk, Gail E. Tomlinson, Charles F. Timmons, and Nancy R. Schneider

Simmons Cancer Center (G.E.T.) and Departments of Pediatrics (D.W.F., G.E.T.) and Pathology (V.S.T., C.F.T., N.R.S.), University of Texas Southwestern Medical Center, Dallas, Texas

Rhabdoid tumor of the kidney (RTK) is associated with tumors of the central nevous system (CNS) in approximately 15% of cases. We describe the clinical features, histologic and cytogenetic findings, and molecular analysis of renal and CNS tumors from the same patient. The histology of the renal tumor was consistent with rhabdoid tumor. The CNS tumor was a primitive neuroectodermal tumor (PNET). The karyotype of the RTK was normal male. The PNET of the brain demonstrated monosomy 22 as the only cytogenetic abnormality, similar t o reported cases of malignant rhabdoid tumor of the brain, but dissimilar t o nonrandom cytogenetic findings in other CNS PNETs. Molecular cytogenetic and DNA marker studies confirmed loss of chromosome 22 in this patient’s brain tumor. DNA allelotyping showed retention of both parental chromosome 22 alleles in the RTK and loss of the maternal allele in the PNET. Evaluation of additional RTKs and brain tumors occurring in the same patient may provide insight into the origins and relationships of these enigmatic tumors. Genes Chrornosorn Cancer I 1:146-152 (1994). 0 1994 Wiley-Liss, Inc.

INTRODUCTION

Rhabdoid tumor of the kidney (RTK) is a rare, deadly malignant tumor of infancy and early child- hood originally described as a rhabdomyosarcoma- toid variant of Wilms’ tumor (Beckwith and Palmer, 1978). More recently RTK has been de- fined as a distinct entity (Haas e t al., 1981). Tu- mors with rhabdoid appearance have been reported in extrarenal sites, including soft tissues of various sites (Ekfors e t al., 1985; Frierson et al., 1985; Douglass et al., 1990), heart (Small et al., 1985), and the central nervous system (CNS) (Biggs et al., 1987). There is a well-established association be- tween RTK and primary nonrhabdoid embryonal tumors arising in the CNS (Bonnin et al., 1984; Weeks et al., 1989). CNS tumors associated with RTK are usually histologically distinct from the renal tumors. They may resemble medulloblasto- mas, primitive neuroectodermal tumors (PNET), ependymomas, gliomas, or pinealomas (Bonnin et al., 1984).

Cytogenetic abnormalities of RTK and of extra- renal malignant rhabdoid tumors are uncommon; most have a normal karyotype (Douglass et al., 1990). Monosomy 22 has been described in iso- lated rhabdoid tumors of the CNS and in one case of CNS rhabdoid tumor associated with an RTK that was not karyotyped (Biegel et al., 1990; Dou- glass et al., 1990). Shashi et al. have described a

translocation involving chromosome 22 in an RTK (Shashi et al., 1994).

We report a case of RTK with synchronous P N E T of the CNS in a 6-month-old infant. T h e tumors differed by histologic, cytogenetic, and mo- lecular analysis. This is, to our knowledge, the first case in which such analysis has been performed on an RTK and a CNS tumor from the same patient.

MATERIALS AND METHODS

Case Report

This child was healthy until he developed he- maturia at 6 months of age. He was referred to this institution after a large mass in the right kidney was detected by ultrasonography. T h e left kidney was normal. There was no evidence of pulmonary, ab- dominal, or bone disease. Computerized tomogra- phy (CT) of the head was normal; however, mag- netic resonance imaging (MRI) of the brain showed a nonenhancing cystic lesion in the pineal area. T h e tumor was not surgically accessible. T h e kid- ney tumor was treated by right radical nephrec- tomy and local irradiation of the right renal bed followed by systemic chemotherapy consisting of

Received November 17, 1993; accepted May 14, 1994. Address reprint requests to Daniel W. Fort, M.D., U.T. South-

western Medical Center, Department of Pediatrics, 5323 Harry Hines Boulevard, Dallas, T X 75235-9063, U.S.A.

0 1994 Wiley-Liss, Inc.

RHABDOID KIDNEY TUMOR AND CNS PNET 147

ifosfamide, etoposide, and carboplatin. Prior to the fourth course of chemotherapy, MRI of the brain demonstrated enlargement of the pineal mass. T h e patient underwent craniotomy with gross total re- section of the pineal tumor.

Postoperatively he was treated according to an institutional phase I protocol using methotrexate and zidovudine. After completion of 4 weeks of therapy, C T of the head revealed recurrent tumor in the pineal area. Subsequent MRIs of the brain showed continued growth of the tumor. T h e pa- tient died at the age of 22 months.

lmmunohistochemistry

Tissue for immunohistochemistry was routinely fixed in 10% buffered formalin prior to paraffin embedding. Sections of 5 pm thickness on poly- L-lysine-coated slides were exposed in 2-hour in- cubations at room temperature to antibodies to the following antigens: desmin, epithelial membrane antigen (EMA), leukocyte common antigen (LCA), alpha and gamma isotypes of actin (HHF35), HMB-45 antigen, glial fibrillary acidic protein (GFAP), S-100 protein, and neuron-spe- cific enolase (NSE) from BioGenex, synaptophysin from Zymed, p30/32”’c2 (HBA-71) from Signet, vimentin from Dako, and a wide molecular weight range cocktail of cytokeratins (40-68 kD) from Biomeda.

Cytogenetic and Molecular Cytogenetic Evaluation

The methods of tissue culture and chromosome preparation have been described earlier (Tonk et al., 1992). GTG-banding was performed using con- ventional techniques. Interpretation of the karyo- types followed the International System for Hu- man Cytogenetic Nomenclature (ISCN, 1991 1.

A biotin-labeled a-satellite DNA probe specific for chromosomes 14 and 22 (Oncor, Gaithersburg, h4D) was hybridized to standard interphase and metaphase cell preparations from cultured brain tu- mor and was subsequently visualized by fluores- cence microscopy using reagents and a protocol provided by Oncor. One hundred interphase cells were scored. No material from the renal tumor was available for in situ hybridization.

Molecular Analysis

T o confirm cytogenetic findings and to assess for possible submicroscopic loss of genetic material in the renal tumor, allelotyping was done using highly informative dinucleotide repeat markers mapping to chromosome band 22ql1, a region altered in CNS rhabdoid tumors (Biegel et al., 1992).

DNA was extracted using conventional methods from tumor tissue frozen at the time of surgery and from peripheral blood lymphocytes. Primer pairs for polymerase chain reaction (PCR) were those described by Marineau and Rouleau (1991) and Marineau et al. (1991). Genomic DNA (0.5 pg) was amplified by PCR using Taq polymerase (Promega) according to the instructions of the man- ufacturer. PCR products were analyzed on a 5% polyacrylamide denaturing gel.

RESULTS

Histologic Evaluation

T h e upper pole of the right kidney contained a 5.0 X 4.2 X 3.5 cm tumor. Histologic sampling included renal hilum, margins of hilar vessels and ureters, residual normal kidney, and 12 blocks of the tumor itself, each examined at a minimum of two levels. T h e cells of the kidney tumor displayed some of the features of the classic rhabdoid phe- notype, namely, abundant cytoplasm, large oval nuclei, and prominent nucleoli (Fig. 1). However, there were several atypical features, most notably the scarcity of classic cytoplasmic inclusions. T h e predominant architectural pattern was one of sheets of large cells; this with a paucity of inclu- sions comprises the “lymphomatoid” pattern of malignant rhabdoid tumor described among cases of the National Wilms’ Tumor Study (NU’TS) by Weeks et al. (1989). Additional patterns described by Weeks et al. were seen focally in the present case, including especially “spindled” areas and “histiocytoid” areas. T h e histiocytoid foci lacked reactive giant cells but had large numbers of reac- tive eosinophils.

T h e kidney tumor was weakly but diffusely pos- itive for vimentin. Negative results were achieved with desmin, actin, LCA, HMB-45, GFAP, S-100, NSE, synaptophysin, and cytokeratin. Rare, iso- lated cells appeared positive for EMA.

By electron microscopy, cytoplasmic filaments were unevenly distributed. Focally they formed parallel bundles. Some cells exhibited round ag- gregates of filaments (Fig. 2 ) within the eccentric cytoplasm, similar to the published ultrastructure of rhabdoid tumor, though smaller and less dense than those usually pictured, perhaps accounting for the difficulty of recognizing them by light micros- copy. Cell junctions were numerous, although small. Other organelles included moderately abun- dant mitochondria and rough endoplasmic reticu- lum, occasionally dilated, with cytoplasmic glyco- gen and occasional lipid droplets.

FORT ET AL.

Figure I. Light microscopy of the formalin-fixed kidney tumor demonstrates sheets of large, somewhat dixohesive cells with oval vesicular nuclei, prominent nucleoli, and ample cytoplasm. Cytoplasmic inclusions are not a significant feature of the light microscopy in this case. Occasional small, mature lymphocytes are interspersed (H&E. x 600).

T h e diagnosis of malignant rhabdoid tumor of kidney, rendered without knowledge of any ac- companying intracranial pathology, was based on the following criteria, drawn from the analysis of Weeks et al. (1989). T h e nuclear features were those of classic rhabdoid tumor. T h e architectural patterns were among those observed by Weeks et al. Although cytoplasmic inclusions were not well delineated by light microscopy, round aggregates of filaments were observed by ultrastructure. Im- munohistochemistry is consistent in that vimentin is positive; and although cytokeratin is classically also seen, it is frequently reported as negative (Seo et al., 1988; Weeks et al., 1989). There is no strong evidence by immunohistochemistry or electron mi- croscopy to indicate another diagnosis.

T h e brain tumor biopsy showed a proliferation of small cells quite distinct from the kidney tumor (Fig. 3 ) . T h e nuclei were smaller and more irreg- ular, with inconspicuous nucleoli and dispersed rather than vesicular chromatin. T h e cytoplasm was sparse and indistinct; the cells appeared to lie

in a loose extracellular matrix with a suggestion of intervening cell processes. The re was strong im- munohistochemical positivity for vimentin, and fo- cal positivity for NSE and S-100 protein. Cytoker- atin, HBA-71, synaptophysin, GFAP, and desmin were negative.

Electron microscopy of the brain tumor (Fig. 4) showed cells with angulated nuclei, dispersed chromatin, and usually two small nucleoli attached to the nuclear membrane. T h e cytoplasm lacked the condensations of filaments seen in the kidney tumor. Rough endoplasmic reticulum was abun- dant but not generally dilated. Mitochondria were variable in number. Occasional membrane-bound, dense granules, consistent with neurosecretory granules, were seen. Cells abutted with straight, well-defined borders with occasional small cell junctions. Other cell surfaces gave rise to long cell processes containing abundant microtubules.

T h e brain mass was considered a primitive neu- roectodermal tumor because of its composition of small, poorly differentiated cells, ultrastructural

RHABDOID KIDNEY TUMOR AND CNS PNET 149

Figure 2. Electron microscopy of the kidney tumor. Adjacent to the oval nucleus with its prominent nucleolus (N), the cytoplasm contains a disorganized but discrete bundle of cytoplasmic filaments (*). Such in- c lus iw were frequently visible by EM in some areas of the tumor; other

areas lacked such inclusions ( x 13,600). insert frame shows at higher magnification the simple cell junctions (arrows) that were common throughout the tumor ( X 36,000).

features consistent with neurosecretory granules and neuritic processes with microtubules, and pos- itivity for neuron-specific enolase, without evi- dence of other differentiation.

T h e two tumors were quite distinct in nuclear morphology, cell shape, cytoplasmic constituents visible by ultrastructure, and patterns of immuno- histochemical reactivity. There was no indication of a metastatic relationship.

Cytogenetic and Molecular Evaluation

Cytogenetic studies revealed a 45,XY, -22 karyotype in 14 metaphase cells from the brain tu- mor. Only cells with a normal male karyotype were found in the patient’s kidney tumor. In situ hy- bridization revealed only three hybridization sig- nals per cultured brain tumor cell in 99% of the cells, consistent with two copies of chromosome 14 and only one copy of chromosome 22 (Fig. 5 ) . Thus, in situ hybridization results are consistent with conventional cytogenetic results.

Allelotyping with both DNA markers at the

22qll locus revealed retention of both paternally and maternally derived alleles in the kidney tumor, confirming the normal cytogenetic findings, and loss of a 22qll allele in the CNS tumor. T h e lost allele was of maternal origin, as demonstrated in Figure 6.

DISCUSSION

T h e association of embryonal tumors originating in the kidney and brain was first described by Bon- nin et al. (1984). Weeks (1989) reported a large series of RTKs in which 13.5% were associated with CNS tumors. CNS tumors associated with RTKs tend to occur in the midline and have no specific temporal relation to the renal tumors. They may have the histologic appearance of PNET, ependymoma, or glioma, or occasionally may contain rhabdoid elements.

T h e histogenesis of rhabdoid tumors remains enigmatic. Recent investigations have revealed a nonrandom cytogenetic abnormality, monosomy 22, in CNS rhabdoid tumors and in CNS tumors of

Figure 3. Light microscopy of the primitive neuroectodermal tumor of the brain demonstrates cells with somewhat irregular hyperchromatic nuclei, inconspicuous nucleoli, and scant cytoplasm. The cells cohere in a loose, fibrillary matrix (H&E, x 600).

Figure 4. Electron microscopy of the brain tumor shows a cluster of tumor cells adjacent to an area occupied by neuritic processes (P), corresponding to the fibrillary background of the tumor. Occasional

membrane-bound, electron-dense cytoplasmic structures consistent with neurosecretory granules are noted (arrows) ( x 13,600). The pro- cesses contain abundant microtubules. enlarged in the inset ( x 27.000).

RHABDOID KIDNEY TUMOR AND CNS PNET

Figure 5. Molecular cytogenetic study by fluorescence in situ hy- bridization of the brain tumor revealed only three hybridization signals per cell, consistent with two copies of Chromosome # 14 and only one copy of chromosome #22 in 99% of the cells.

infants with mixed histology, including rhabdoid cells, called atypical teratoid tumor of infancy by Lefkowitz et al. (1987). Monosomy 22 may be helpful in differentiating these tumors from other embryonal tumors of the CNS (Biegel et al., 1990). PNETs in the CNS frequently have an isochromo- some 17q, abnormalities of chromosome 1 1 (Griffin et al., 1988), or no cytogenetic abnormality, but the); rarely have chromosome 22 abnormalities.

Monosomy 22 in the CNS tumor from our pa- tient suggests an origin more closely related to the pure rhabdoid tumor and atypical teratoid tumors of the CNS, and possibly to peripheral PNET, than to CNS PNETs. An etiology or histogenesis dis- tinct from other CNS PNETs is also suggested by its clinical association with RTK and its aggressive clinical behavior.

Figure 6. Allelotyping gel using dinucleotide repeat polymorphism at the D22S264 locus. The two alleles observed in patient’s lymphocyte D N A (L) are retained in the renal tumor (RT); however, only one allele is observed in the brain tumor (BT). Analysis of parental lymphocyte D N A (mother, M; father, F) demonstrates that the lost allele is of maternal origin.

Loss or disruption of chromosomal material on chromosome 22 in tumors of neural origin is not unique to CNS rhabdoid tumors, or, as in our case, brain tumors associated with R K T (Ransom et a]., 1992; Rouleau et al., 1993). Thus, one or more important regulatory genes that serve as a tumor suppressor in the CNS mav exist on chromosome 22. Biegel et al. (1992) have demonstrated prelim- inary refinement of the critical region involved in rhabdoid tumors to chromosome band 22qll. Ad- ditional studies at the molecular level will further define the position of a relevant gene(s) at this locus.

Study of tumors in which there is consistent loss of heterozygosity at a specific locus, as in ret- inoblastoma, has led to the discovery of predis- position genes. The association of multiple pri- mary renal and CNS tumors in very young chil- dren, as in this case, suggests a genetic predispo- sition to malignancy that may be related to a locus on chromosome 22. T h e repeated observation of monosomy 22 may serve as a marker for highly malignant CNS tumors with a distinct relationship to RTK.

This is the first report of cytogenetic analysis of tumors from the same patient with RTK and CNS PNET. It is also the first report of parental origin of chromosome 22 loss in a CNS PNET. Whether the maternal origin of the chromosomal loss has significance in rhabdoid related tumors, as i t does in other pediatric malignancies, such as Wilms’ tu- mor (Schroeder et al., 1987) or retinoblastoma (Toguchida et al., 1989), will depend on future analysis of additional rhabdoid tumors, including parental DNA. One study of chromosome 22 loss in sporadic neuromas suggests a preference for loss of maternally derived alleles (Fontaine et al., 1991).

I52 FORT ET AL.

ACKNOWLEDGMENTS

The authors wish to acknowledge Dr. J. Bruce Beckwith for histologic review of the renal tumor, Dr. Dennis Burns for histologic review of the CNS tumor, Dr. Derek Bruce for providing tumor tissue for analysis, Elizabeth Chappell for expert techni- cal assistance, and Pat Ellisor for secretarial assis- tance.

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