Histopathology 1992. 20, 157-165
Malignant rhabdoid tumour of the liver. A case report M.P .FOSCHINI, P .VAN EYKEN, P .R.BROCK*, M.CASTEELS-VAN DAELE*, R.DE VOS, P.DAL CINt, H.VAN DEN BERGHEt & V.J.DESMET Laboratory for Histo- and Cytochemistry, Pathology Department 11, UZ St Rafael, *Paediatrics Department, UZ Gasthuisberg and tCentre for Human Genetics, Catholic University of Leuven, Leuven, Belgium Date of submission 14 May 1991 Accepted for publication I 9 August 1991
FOSCHINI M . P . , V A N EYKEN P., BROCK P . R . , CASTEELS-VAN DAELE M., DE VOS R . , DAL CIN P . , V A N DEN BERGHE H . & DESMET V . J .
(1992) Histopathology 20, 157-165
Malignant rhabdoid tumour of the liver. A case report A case of primary malignant rhabdoid tumour of the liver occurring in a 5-month-old girl is reported. Histologically the neoplasm presented the ‘typical’ features as described in malignant rhabdoid tumour arising in the kidney. In addition, some areas of the tumour showed a ‘pseudoglandular’growth pattern. Immunohistochemistry revealed the neoplastic cells to be intensively positive for vimentin and for cytokeratin 19. Variable immunoreactivity for cytokeratin polypeptides 7, 8 and 19 was also detected. On electronmicroscopy the neoplastic cells contained bundles of intermediate filaments, tonofilaments and intercellular junctions. These findings are suggestive of a primitive epithelial differentiation.
Keywords: malignant rhabdoid tumour, liver, cytokeratin, vimentin
Introduction
Case history
Malignant rhabdoid tumour is a rare tumour with an extremely aggressive behaviour pattern. Histologically. it is characterized by large cells with eosinophilic inclusions and nuclei with prominent nucleoli. Malignant rhabdoid tumour mainly affects the kidney, but it has also been described in several other organs, including the liver’-4. Both renal and extrarenal tumours share the same histology. Most authors report immunoreactivity of the tumour cells for vimentin and often for cytokeratins. The light and electronmicroscopic features of renal and extrarenal malignant rhabdoid tumour have both been extensively documented. Immunohistochemical data on primary liver tumours are scarce and a detailed analysis of cytokeratin expression does not appear to have been performed. We report on the electronmicroscopic and immunohistochemical features of a case of malignant rhabdoid tumour arising in the liver.
A 5-month-old baby girl presented with a 3-week history of pallor, intermittent fever, poor appetite and vomiting. On clinical examination a normally developed baby was seen with a large, hard, knobbly mass filling the epigastrium and the right hypochondrium, extending to the umbilicus. On ultra-sound examination the mass was solid and situated in the left lobe of the liver. Abdominal CT-scan showed a hypodense lesion measuring 7.1 cm by 6.9 cm, which on selective angiography was poorly vascularized. Brain CT-scan was normal. Peripheral blood examination revealed anaemia with a haemoglobin level of 5.0 g/dl; the reticulocyte count was 4.3/1, the platelet count was raised to 840 x 1O9/l and the lactate dehydrogenase (LDH) level was raised at 1 350 U/1. The a-fetoprotein level was, however, normal for age. Clinically, the mass was increasing rapidly in size. Laparotomy revealed a heterogeneous, multinodular left liver lobe, which was totally resected. Postoperative recovery was straightforward. The serum LDH decreased progressively to reach normal levels 2 weeks post-laparotomy. Chemotherapy was then started with a combination of cisplatin ( 3 mg/kg per dose) given 3weekly as a 24 h i.v. infusion, and vincristine (1.5 mg/
Address for correspondence: Dr P.Van Eyken, Laboratorium VOOI Histo-en-Cytochemie,Universitair Ziekenhuis Sint-Rafael, Minderbroedersstraat 12. 8-3000 Leuven. Belgium.
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m2 per dose) given initially weekly and then 3-weekly5. After three courses of cisplatin and eight doses of vincristine, she relapsed on treatment with a fast growing hard mass in the right lobe of the liver, haemorrhagic ascites and an increased serum LDH (564 U/l). In spite of further chemotherapy, including carboplatin and etoposide, and intensive symptomatic support, she died of progressive disease 5 months from diagnosis.
Materials and methods The hemihepatectomy specimen was received fresh. Part of the tumour was snap-frozen in liquid nitrogen cooled isopentane and stored at -7OOC until use. Small fragments were glutaraldehyde-fixed, post-fixed in
Antibody
Sectionst
6 B10 RCK 105
F F
CK 4$ CK 7
CK 8 CK 18 RKSE 60 1c 7 CK RCK 107 A 5 3 JBA2 CK KL1 CAM 5.2 EMA* Albumin* a-fetoprotein* P-HCG*
F
Chromogranin Chromogranin Desmin Desmin LCA Leu 19 Myoglobin* NCAM Vimentin Vimentin KP 1
F P F
CK 8 CK 18 CK 10 CK 13 CK 14 CK 19 55-57 kDa cytokeratin CK 8, 18, 19 Milk fat globulin Albumin a-fetoprotein P-human chorionic gonadotrophin Chromogranin Chromogranin Desmin Desmin CD 45 Neural cell adhesion molecule Myoglobin CD 56 Vimentin Vimentin CD 68
F
F F F
F P
P P
FJP P P
P F
F FJP F
F P P
Specificity
* Polyclonal: all others monoclonal. t F =frozen: P =paraffin. $ Cytokeratin number according to the catalogue of Molly.
osmium tetroxide and epon-embedded for electronmicroscopy. Cytogenetic analysis was performed on a fresh tumour sample according to previously described procedures6. Parts of the remaining tissue were fixed in B5 or in formalin and processed to paraffin. For routine histological examination sections were stained with haematoxylin & eosin, picro Sirius, silver impregnation for reticulin fibres, PAS after diastase digestion, Perls’ Prussian blue, and Hall’s stain for bilirubin. Serially cut cryostat sections were immunostained using a three step indirect immunoperoxidase technique as described by Mason and co-workers’. On serially cut paraffin sections an unlabelled enzyme antibody (PAP) method was performeds. All the primary antibodies used, their sources and specificities are listed in Table 1. Controls, which were invariably negative, consisted of omission of the primary antibody.
Source
Dilution
1:10 1:100
Eurodiagnostics F.C.S. Ramaekers, Nijmegen Amersham Amersham Eurodiagnostics Eurodiagnostics F.C.S. Ramaekers Karsten Immunotech Becton-Dickinson Dako Nordic Dako
1:10 1:10 1:s 1:lO Undiluted Undiluted 1:50 1:10 1:lOO 1:4000 1 :500
Dako Clonatec, Pasture Sanbio Boehringer Eurodiagnostic Dako Becton-Dickinson Dako Marseille Lab Systems Amersham Dako
1: 1000 1:10 1:5 1:10 1:lO 1:10 1:160 1:250 1: 500 1:2O 1:2O 1 3
Table 1. Antibodies used in this study
Malignant rhabdoid tumour of the liver
Results MACROSCOPIC A N D LIGHT MICROSCOPIC FEATURES
The left hemihepatectomy specimen, which weighed 336 g, measured 1 3 x 10 x 8 cm. The external surface showed a large, lobulated mass. The liver capsule was macroscopically uninvolved. On the cut surface, the tumour comprised about 90% of the liver parenchyma. The tumour had irregular margins and was mainly composed of whitish, soft tissue. Large areas of haemorrhage and necrosis gave a pseudocystic appearance to the neoplastic mass (Figure 1). The bulk of the tumour had the typical appearance of malignant rhabdoid tumour (Figure 2a), being composed of large, polygonal cells, with abundant and eosinophilic cytoplasm. Numerous cells contained cytoplasmic inclusions, which stained positively with PAS after diastase digestion. The neoplastic cells had a large and vesicular nucleus, with a prominent nucleolus. They were organized in sheets and large nests, surrounded by fibrous strands: focal areas of sclerosis were observed. A small part presented a ‘pseudoglandular’ configuration and was composed of smaller cells, with deeply eosinophilic cytoplasm, and a large nucleus with imperceptible nucleolus (Figure 2b). Mitoses were numerous (8 per 10 high-power fields). Neoplastic intravascular thrombi were observed. Necrotic foci were
interspersed among the neoplastic mass. The liver capsule was not infiltrated. Within the tumour, welldifferentiated hepatocytes were seen either as isolated cells or arranged in small clusters. They were interpreted as normal liver parenchymal cells entrapped in the tumour. Extramedullary haemopoiesis was not seen. The macroscopically uninvolved liver parenchyma contained microscopic foci of neoplastic cells. The resection margin was free of tumour. IMMUNOHISTOCHEMICAL PINDXNGS
The immunohistochemical staining results are summarized in Table 2. Frozen material contained only the tumour cells with the typical rhabdoid appearance. The paraffin-embedded tissue used for immunohistochemistry contained both the rhabdoid and the ‘pseudoglandular’ parts. The anti-vimentin antibodies decorated all the neoplastic cells with rhabdoid features, on frozen and paraffin sections. In the ‘pseudoglandular’ component about 50% of the neoplastic cells were vimentin positive. Positivity was localized in the cytoplasmic inclusions. Diffuse cytoplasmic staining was also seen (Figure 3a). Almost all the large neoplastic cells stained positively for cytokeratin (CK9) 1 9 (Figure 3b) and CK 18 was present in about 80%of these. About SO% of the tumour
Figure 1. On cut section the liver parenchyma is almost completely replaced by a tumour composed of whitish tissue, with large, necrotic
areas.
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h4.P. Foschini et al.
Figure 2. a The bulk of the tumour shows the typical rhabdoid appearance, being composed of large cells with eosinophilic cytoplasm and nuclei with prominent nucleoli. A number of tumour cells have eosinophilic cytoplasmic inclusions. b Only a small part of the tumour showed a 'pseudoglandular' pattern and was composed of smaller cells, with large nucleus and inconspicuous nucleolus. H & E. x 400.
cells were positive for CK 8. About 20% of the neoplastic cells were immunoreactive for CK 7. Positivity, when present, was observed in the cytoplasmic inclusions, or as a diffuse cytoplasmic staining. Bile ducts and normal hepatocytes adjacent to or entrapped within the tumour were stained by monoclonal antibodies directed against CK 8 and CK 18; in addition bile ducts showed positivity for CK 7 and CK 19. On paraffin sections, the entire
Virnentin CK 19 CK 18 CK 8 CK 7 Milk fat globulin (EMA) Neural cell adhesion molecule KP 1 CD 45 S-100 proteins Other markers listed in Table 1
cytoplasm, the cytoplasmic inclusions of about 70% of the large neoplastic cells and about 30% of the small neoplastic cells were stained using the monoclonal antibodies CAM 5.2 and KL1. Normal hepatic parenchyma present in the section was also stained by both of these antibodies. In the neoplastic population with rhabdoid appearance the anti-EMA antibody showed the same distribution, as was observed with the anti-
+ +
+ (80%) + (50%) + (20%)
+ + (focal positivity) + (variable positivity) + (occasional cell) + (occasional cell, mainly in cytoplasmic inclusion) -
Table 2. Immunohistochemical staining pattern of rhabdoid tumour cells
Malignant rhabdoid tumour of the liver
161
Figure 3. a The neoplastic cells are intensely immunoreactive for vimentin. b Most neoplastic cells are also positive for CK 19. Immunoperoxidase counterstained with Mayer’s haemalum. a x 640. b x 400.
cytokeratin antibodies. In the ‘pseudoglandular’component of the tumour, about 10%of the neoplastic cells were positive for EMA. In the normal liver parenchyma, anti-EMA antibody was localized on the luminal surface of the bile duct cells. Clusters of neoplastic cells were positive with the Leu 19 antibody. Using the monoclonal antibody KP1, immunostaining of macrophages present in the necrotic areas was observed. However, numerous neoplastic cells were also positive. In addition occasional neoplastic cells were positive for leukocyte common antigen (LCA). S-100 protein was positive in the cytoplasmic inclusions of occasional large neoplastic cells. All other markers tested on frozen and paraffin sections were negative.
Elect ronmicroscopy The tumour cells were large and oval in shape with a peripherally located oval to irregularly indented nucleus with prominent nucleolus. The voluminous cytoplasm contained many organelles: cisternae of rough endoplasmic reticulum-often slightly dilated; round to oval mitochondria: small heterogeneous lysosomes; several Golgi areas: and numerous ribosomes (Figure 4). Most
striking were masses of intermediate filaments (Figure 4) among which bundles of tonofilaments were apparent (Figure 5a). Intercellular junctions often of considerable length were found (Figure 5b).
Cytogenetic analysis Three out of 2 0 G-banded metaphases obtained from a 6-day-old culture displayed a translocation between chromosomes 1 and 8, at bands p36 and q24 respectively, as the only chromosomal change. A normal female karyotype was found in the remaining cells.
Discussion Malignant rhabdoid tumour is a rare and aggressive neoplasm, first described by Beckwith & Palmer” as an aggressive variant of Wilms’ tumour. Subsequent observations”.l2 suggested that it was a distinct neoplasm, different in nature and behaviour from Wilms’ tumour. Numerous cases have been described in various organs, but only four cases have been reported arising in the liver’-4. All cases of primary hepatic malignant rhabdoid tumour reported up to now have shown a ‘classical’
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M.P.Foschini et al.
Figure 4. Electronmicrographof a tumour cell. Intermediate filaments are conspicuous (arrow). x 9200.
rhabdoid appearance. The tumour reported here was also mainly composed of large polygonal cells, with vesicular nuclei and prominent nucleoli, and the typical eosinophilic PAS positive cytoplasmic inclusions. Interestingly, however, a small part of the present tumour presented a ‘pseudoglandular’ component and was composed of smaller cells. Recently, Weeks et al.13 have drawn attention to the various histological appearances of renal malignant rhabdoid tumours. The histological
patterns observed in the present case are consistent with their observations”. Immunohistochemical studies performed on renal and extrarenal malignant rhabdoid tumours constantly report positivity for vimentin in almost all cases and for cytokeratins in numerous cases13-15.Single cases have been reported to contain other markers13-16.Nevertheless, in none of the reported cases has the expression of different cytokeratins been studied in detail. In the
Malignant rhabdoid turnour of the liver
163
Figure 5. Electronmicrograph of part of a tumour cell. a Bundles of tonofilaments are evident (arrow). b Adjacent tumour cells forming intercellular junctions (arrow). a x 14 500. b x 18 400.
present case immunohistochemistry proved the neoplastic cells to be intensely positive for vimentin and CK 19. CK 8 and CK 18 were expressed by numerous neoplastic cells, whereas CK 7 was only detected in a small number
of neoplastic cells. No staining for CK 4, CK 10 or CK 1 3 was seen. On paraffin sections, vimentin, CAM 5.2, KL1 and EMA antibodies stained the neoplastic cells of the rhabdoid and ‘pseudoglandular’ components. Positivity
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M.P.Foschini et al.
in the 'pseudoglandular' component was confined to a smaller number of cells. The expression of the same immunohistochemical profile in the two components of the present tumour was considered as proof that they are different morphological aspects of the same neoplasm. Immunostaining for /?-HCG and a-fetoprotein, and for markers for muscular and neural differentiation was negative. The only exception was focal S-100 protein positivity. A similar cytokeratin profile was observed in the embryonal component of hepat~blastoma'~. The coexpression of cytokeratins and vimentin has been suggested to be indicative of cells in a very early stage of differentiation''. In addition, Stosiek et have demonstrated that CK 1 9 is localized in all the hepatic progenitor cells at the very early phases of human liver organogenesis. Subsequently, parenchymal cells lose CK 19. but remain positive for CK 8 and CK 18, whereas cells differentiating towards bile ducts express in addition CK 7'". Positivity for EMA has been previously reported in renal and extrarenal malignant rhabdoid t u m o ~ r s ' ~ .including '~, one case arising in the liver2. The presence of EMA was considered a n argument in favour of epithelial differentiation2.'3. The positive staining for LCA, KP1 and S-100 protein was unexpected. Positivity was mainly, if not exclusively, localized in the cytoplasmic inclusions and may represent nonspecific staining. An alternative explanation would be the aberrant expression of various genes by the tumour. The immunoreactivity of cytokeratins, EMA and vimentin, together with the electronmicroscopic features, argue against a true histiocytic or neural differentiation. Electronmicroscopy showed the neoplastic cells to contain bundles of variously arranged intermediate filaments, tonofilaments and intercellular junctions, suggestive of epithelial differentiation. These findings are consistent with previous observationsl 1.12.1 5.1 6 Weeks and colleague^'^, on the basis of a careful review of the literature and a study of a large series of renal cases, proposed to consider renal malignant rhabdoid tumour as a separate entity, and malignant rhabdoid tumour in extrarenal sites as a simple phenotype. Renal variants have been shown to contain a 5 4 kDa cytokeratin, that could correspond to CK 8". and a 5557 kDa cyt~keratin'~.No data are reported on the expression of other cytokeratins in renal cases. Cytokeratins were studied during kidney organogenesis. In the developing kidney CK 8 and CK 1 8 appear at the early phases and CK 1 9 and vimentin are transiently expressed by the collecting ducts22.CK 7 has not been studied during kidney development. There are only a few reports on cytogenetic analysis of extrarenal rhabdoid tumours (reviewed by Douglass et
al. in 199023),and so far only primary brain tumours with a rhabdoid morphology seem to be characterized by the loss of chromosome 22. The outcome of published cases of extrarenal malignant rhabdoid tumour, including ours, is uniformly fatal, despite aggressive treatment. Short-term responses in cases of relapse appear to be anecdotal. Survival has been noted in malignant rhabdoid tumour of the kidney following complete excision in localized disease with negative nodes13. In conclusion, on the basis of the electronmicroscopic and immunohistological data, the present case of malignant rhabdoid tumour arising in the liver seems to have a very primitive epithelial differentiation, with an 'attempt' at differentiation towards bile ductal cells. These findings could support the hypothesis of Weeks and colleagues' 3,24 that extrarenal malignant rhabdoid tumour is a 'phenotype' shared by poorly differentiated neoplasms, arising in different organs.
Acknowledgements Peter Van Eyken is a research assistant of the Belgian National Fund for Scientific Research. This study was supported in part by the Interuniversity Network for Fundamental Research sponsored by the Belgian government (1987-1991). The authors gratefully acknowledge the technical assistance of Suzanne Taelemans and Bernadette Smets, and the photographical assistance of Michel Rooseleers. They are also greatly indebted to Professor F.C.S.Ramaekers, Maastrict, The Netherlands and Dr U.Karsten, Berlin Buch, Germany who generously supplied the monoclonal antibodies RCK 105, RCK 107 and A53/BA2 respectively. Our thanks also go to Professor H.Devlieger and to Professor F.de Zegher for their share in the intensive care of this child and to Professor I.De Wever for his surgical skills.
References 1 . Gonzales-Crussi F. Goldschmidt RA, Hsueh W. Trujillo YP. Infantile sarcoma with intracytoplasmic filamentous inclusions. Cancer 1982: 49: 2365-2375. 2 . Parham DM. Peiper SC, Robicheaux G. Ribeiro RC. Douglass EC. Malignant rhabdoid tumour of the liver. Evidence of epithelial differentiation. Arch. Pathol. Lab. Med. 1988: 112; 61-64. 3. Chang CH. Ramirez N. Sakr WA. Primitive neuroectodermal tumor of the brain associated with malignant rhabdoid tumor of the liver: a histologic, immunohistochemical and electron microscopic study. Pediatr. Pathol. 1989: 9; 307-319. 4. Hunt SJ, Anderson W. Malignant rhabdoid tumor of the liver. A distinct clinicopathologic entity. Am. 1. Clin. Pathol. 1990: 94: 645-648. 5 . Brock PR. Yeomans E. Beltman S.Pritchard J, Cisplatin therapy in infants: short and long-term morbidity. Br. 1. Cancer (in press).
Malignant rhabdoid tumour of the liver
6. Limon J , Dal Cin P. Sandberg AA. Application of long-term collagenase disaggregation for the cytogenetic analysis of human solid tumors. Cancer Genet. Cytogenet. 1986: 23: 305-31 3. 7. Mason DY, Naiem M. Abdulaxiz 2. Nash JKG, Gatter KC, Stein H. Immunohistological applications of monoclonal antibodies. In McMichael AJ, Fabre JW eds. Monoclonal Antibodies in Clinical Medicine. London: Academic Press, 1982: 585-635. 8. Sternberger LA. The unlabelled antibody enzyme method. In Sternberger LA ed. lmmunohistochemistry. Englewood Cliffs, NJ: Prentice Hall, 1974: 129-171. 9 . Moll R. Franke WW. Schiller D. Geiger B. Krepler R. The catalog of human cytokeratins: pattern of expression in normal epithelia, tumors and cultured cells. Cell 1982; 31: 11-24. 1 0 . Beckwith JB. Palmer NF. Histopathology and prognosis of Wilms' tumor. Results from the Erst National Wilms' tumor study. Canrer 1978: 41; 1937-1948. 1 1 . Haas JE. Palmer NF. Weinberg AG. Beckwith JB. Ultrastructure of malignant rhabdoid tumor of the kidney. A distinctive renal tumor ofchildren. Hum. Pathol. 1981: 12; 646-657. 12. Fung CH. Gonzales-Crussi F. Yonan TN, Martinez N. 'Rhabdoid' Wilms' tumor. An ultrastructural study. Arch. Pathol. Lab. Med 1981: 105; 521-523. 13. Weeks DA, Beckwith JB, Mireau GW. Luckey OW. Rhabdoid tumor of the kidney. A report of 1 1 1 cases from the National Wilms' Tumor Study Pathology Center. A m . 1. Surg. Puthol. 1989: 13: 439-458. 14. Sotelo-Avila C , Gonzales-Crussi F, deMello D et a!. Renal and extrarenal rhabdoid tumors in children: a clinicopathologic study of 1 4 patients. Semin. Diagn. Pathol. 1986: 3: 151-163. 15. Schmidt D, Leuschner 1. Harms D. Sprenger E, Schlfer HJ. Malignant rhabdoid tumor. A morphological and flow cytometric study. Pathol. Res. Pract. 1989; 184: 202-210.
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16. Mireau GW. Weeks DA. Beckwith JB. Anaplastic Wilms' tumor and other clinically aggressive childhood renal neoplasms: ultrastructural and immunocytochemical findings. Ultrastruct. Pathol. 1989: 13: 225-248. 17. Van Eyken P. Sciot R, Callea F, Ramaekers F. Schaart G, Desmet VJ. A cytokeratin-immunohistochemical study of hepatoblastoma. Hum. Pathol. 1990; 21; 302-308. 18. Abenoza P, Manivel JC, Wick MR, Hagen K. Dehner LP. Hepatoblastoma: a n immunocytochemical and ultrastructural study. Hum. Pathol. 187: 18; 1025-1035. 19. Stosiek P, Kasper M. Karsten U. Expression of cytokeratin 19 during human liver organogenesis. Liver 1990: 1 0 59-63. 20. Van Eyken P, Sciot R. Callea F. Van Der Steen K. Moerman P, Desmet VJ. The development ofthe intrahepatic bile ducts in man: a keratin-immunohistochemical study. Hepatology 1988: 6: 1586-1595. 21. Vogel AM, Gown AM, Caughlan J, Haas JE. Beckwith JB. Rhabdoid tumors of the kidney contain mesenchymal specific and epithelial specific intermediate filament proteins. Lab. Invest. 1984: 5 0 232-238. 22. Oosterwijk E, Van Muijen GNP, Oosterwijk-Wakka JC, Warnaar SO. Expression of intermediate-sized filaments in developing and adult human kidney and in renal cell carcinoma. 1. Histochem. Cytochem. 1990: 38; 385-392. 23. Douglass EC. Valentine M. Rowe ST et al. Malignant rhabdoid tumor: a highly malignant childhood tumor with minimal karyotypic changes. Genes Chromosomes Cancer 1990; 2; 210216. 24. Weeks DA. Beckwith JB, Mireau GW. Rhabdoid tumor. An entity or a phenotype? Arch. Pathol. Lab. Med. 1989: 113; 113-114.
Malignant rhabdoid tumour of the liver. A case report M.P .FOSCHINI, P .VAN EYKEN, P .R.BROCK*, M.CASTEELS-VAN DAELE*, R.DE VOS, P.DAL CINt, H.VAN DEN BERGHEt & V.J.DESMET Laboratory for Histo- and Cytochemistry, Pathology Department 11, UZ St Rafael, *Paediatrics Department, UZ Gasthuisberg and tCentre for Human Genetics, Catholic University of Leuven, Leuven, Belgium Date of submission 14 May 1991 Accepted for publication I 9 August 1991
FOSCHINI M . P . , V A N EYKEN P., BROCK P . R . , CASTEELS-VAN DAELE M., DE VOS R . , DAL CIN P . , V A N DEN BERGHE H . & DESMET V . J .
(1992) Histopathology 20, 157-165
Malignant rhabdoid tumour of the liver. A case report A case of primary malignant rhabdoid tumour of the liver occurring in a 5-month-old girl is reported. Histologically the neoplasm presented the ‘typical’ features as described in malignant rhabdoid tumour arising in the kidney. In addition, some areas of the tumour showed a ‘pseudoglandular’growth pattern. Immunohistochemistry revealed the neoplastic cells to be intensively positive for vimentin and for cytokeratin 19. Variable immunoreactivity for cytokeratin polypeptides 7, 8 and 19 was also detected. On electronmicroscopy the neoplastic cells contained bundles of intermediate filaments, tonofilaments and intercellular junctions. These findings are suggestive of a primitive epithelial differentiation.
Keywords: malignant rhabdoid tumour, liver, cytokeratin, vimentin
Introduction
Case history
Malignant rhabdoid tumour is a rare tumour with an extremely aggressive behaviour pattern. Histologically. it is characterized by large cells with eosinophilic inclusions and nuclei with prominent nucleoli. Malignant rhabdoid tumour mainly affects the kidney, but it has also been described in several other organs, including the liver’-4. Both renal and extrarenal tumours share the same histology. Most authors report immunoreactivity of the tumour cells for vimentin and often for cytokeratins. The light and electronmicroscopic features of renal and extrarenal malignant rhabdoid tumour have both been extensively documented. Immunohistochemical data on primary liver tumours are scarce and a detailed analysis of cytokeratin expression does not appear to have been performed. We report on the electronmicroscopic and immunohistochemical features of a case of malignant rhabdoid tumour arising in the liver.
A 5-month-old baby girl presented with a 3-week history of pallor, intermittent fever, poor appetite and vomiting. On clinical examination a normally developed baby was seen with a large, hard, knobbly mass filling the epigastrium and the right hypochondrium, extending to the umbilicus. On ultra-sound examination the mass was solid and situated in the left lobe of the liver. Abdominal CT-scan showed a hypodense lesion measuring 7.1 cm by 6.9 cm, which on selective angiography was poorly vascularized. Brain CT-scan was normal. Peripheral blood examination revealed anaemia with a haemoglobin level of 5.0 g/dl; the reticulocyte count was 4.3/1, the platelet count was raised to 840 x 1O9/l and the lactate dehydrogenase (LDH) level was raised at 1 350 U/1. The a-fetoprotein level was, however, normal for age. Clinically, the mass was increasing rapidly in size. Laparotomy revealed a heterogeneous, multinodular left liver lobe, which was totally resected. Postoperative recovery was straightforward. The serum LDH decreased progressively to reach normal levels 2 weeks post-laparotomy. Chemotherapy was then started with a combination of cisplatin ( 3 mg/kg per dose) given 3weekly as a 24 h i.v. infusion, and vincristine (1.5 mg/
Address for correspondence: Dr P.Van Eyken, Laboratorium VOOI Histo-en-Cytochemie,Universitair Ziekenhuis Sint-Rafael, Minderbroedersstraat 12. 8-3000 Leuven. Belgium.
157
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M.P.Foschini et al.
m2 per dose) given initially weekly and then 3-weekly5. After three courses of cisplatin and eight doses of vincristine, she relapsed on treatment with a fast growing hard mass in the right lobe of the liver, haemorrhagic ascites and an increased serum LDH (564 U/l). In spite of further chemotherapy, including carboplatin and etoposide, and intensive symptomatic support, she died of progressive disease 5 months from diagnosis.
Materials and methods The hemihepatectomy specimen was received fresh. Part of the tumour was snap-frozen in liquid nitrogen cooled isopentane and stored at -7OOC until use. Small fragments were glutaraldehyde-fixed, post-fixed in
Antibody
Sectionst
6 B10 RCK 105
F F
CK 4$ CK 7
CK 8 CK 18 RKSE 60 1c 7 CK RCK 107 A 5 3 JBA2 CK KL1 CAM 5.2 EMA* Albumin* a-fetoprotein* P-HCG*
F
Chromogranin Chromogranin Desmin Desmin LCA Leu 19 Myoglobin* NCAM Vimentin Vimentin KP 1
F P F
CK 8 CK 18 CK 10 CK 13 CK 14 CK 19 55-57 kDa cytokeratin CK 8, 18, 19 Milk fat globulin Albumin a-fetoprotein P-human chorionic gonadotrophin Chromogranin Chromogranin Desmin Desmin CD 45 Neural cell adhesion molecule Myoglobin CD 56 Vimentin Vimentin CD 68
F
F F F
F P
P P
FJP P P
P F
F FJP F
F P P
Specificity
* Polyclonal: all others monoclonal. t F =frozen: P =paraffin. $ Cytokeratin number according to the catalogue of Molly.
osmium tetroxide and epon-embedded for electronmicroscopy. Cytogenetic analysis was performed on a fresh tumour sample according to previously described procedures6. Parts of the remaining tissue were fixed in B5 or in formalin and processed to paraffin. For routine histological examination sections were stained with haematoxylin & eosin, picro Sirius, silver impregnation for reticulin fibres, PAS after diastase digestion, Perls’ Prussian blue, and Hall’s stain for bilirubin. Serially cut cryostat sections were immunostained using a three step indirect immunoperoxidase technique as described by Mason and co-workers’. On serially cut paraffin sections an unlabelled enzyme antibody (PAP) method was performeds. All the primary antibodies used, their sources and specificities are listed in Table 1. Controls, which were invariably negative, consisted of omission of the primary antibody.
Source
Dilution
1:10 1:100
Eurodiagnostics F.C.S. Ramaekers, Nijmegen Amersham Amersham Eurodiagnostics Eurodiagnostics F.C.S. Ramaekers Karsten Immunotech Becton-Dickinson Dako Nordic Dako
1:10 1:10 1:s 1:lO Undiluted Undiluted 1:50 1:10 1:lOO 1:4000 1 :500
Dako Clonatec, Pasture Sanbio Boehringer Eurodiagnostic Dako Becton-Dickinson Dako Marseille Lab Systems Amersham Dako
1: 1000 1:10 1:5 1:10 1:lO 1:10 1:160 1:250 1: 500 1:2O 1:2O 1 3
Table 1. Antibodies used in this study
Malignant rhabdoid tumour of the liver
Results MACROSCOPIC A N D LIGHT MICROSCOPIC FEATURES
The left hemihepatectomy specimen, which weighed 336 g, measured 1 3 x 10 x 8 cm. The external surface showed a large, lobulated mass. The liver capsule was macroscopically uninvolved. On the cut surface, the tumour comprised about 90% of the liver parenchyma. The tumour had irregular margins and was mainly composed of whitish, soft tissue. Large areas of haemorrhage and necrosis gave a pseudocystic appearance to the neoplastic mass (Figure 1). The bulk of the tumour had the typical appearance of malignant rhabdoid tumour (Figure 2a), being composed of large, polygonal cells, with abundant and eosinophilic cytoplasm. Numerous cells contained cytoplasmic inclusions, which stained positively with PAS after diastase digestion. The neoplastic cells had a large and vesicular nucleus, with a prominent nucleolus. They were organized in sheets and large nests, surrounded by fibrous strands: focal areas of sclerosis were observed. A small part presented a ‘pseudoglandular’ configuration and was composed of smaller cells, with deeply eosinophilic cytoplasm, and a large nucleus with imperceptible nucleolus (Figure 2b). Mitoses were numerous (8 per 10 high-power fields). Neoplastic intravascular thrombi were observed. Necrotic foci were
interspersed among the neoplastic mass. The liver capsule was not infiltrated. Within the tumour, welldifferentiated hepatocytes were seen either as isolated cells or arranged in small clusters. They were interpreted as normal liver parenchymal cells entrapped in the tumour. Extramedullary haemopoiesis was not seen. The macroscopically uninvolved liver parenchyma contained microscopic foci of neoplastic cells. The resection margin was free of tumour. IMMUNOHISTOCHEMICAL PINDXNGS
The immunohistochemical staining results are summarized in Table 2. Frozen material contained only the tumour cells with the typical rhabdoid appearance. The paraffin-embedded tissue used for immunohistochemistry contained both the rhabdoid and the ‘pseudoglandular’ parts. The anti-vimentin antibodies decorated all the neoplastic cells with rhabdoid features, on frozen and paraffin sections. In the ‘pseudoglandular’ component about 50% of the neoplastic cells were vimentin positive. Positivity was localized in the cytoplasmic inclusions. Diffuse cytoplasmic staining was also seen (Figure 3a). Almost all the large neoplastic cells stained positively for cytokeratin (CK9) 1 9 (Figure 3b) and CK 18 was present in about 80%of these. About SO% of the tumour
Figure 1. On cut section the liver parenchyma is almost completely replaced by a tumour composed of whitish tissue, with large, necrotic
areas.
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Figure 2. a The bulk of the tumour shows the typical rhabdoid appearance, being composed of large cells with eosinophilic cytoplasm and nuclei with prominent nucleoli. A number of tumour cells have eosinophilic cytoplasmic inclusions. b Only a small part of the tumour showed a 'pseudoglandular' pattern and was composed of smaller cells, with large nucleus and inconspicuous nucleolus. H & E. x 400.
cells were positive for CK 8. About 20% of the neoplastic cells were immunoreactive for CK 7. Positivity, when present, was observed in the cytoplasmic inclusions, or as a diffuse cytoplasmic staining. Bile ducts and normal hepatocytes adjacent to or entrapped within the tumour were stained by monoclonal antibodies directed against CK 8 and CK 18; in addition bile ducts showed positivity for CK 7 and CK 19. On paraffin sections, the entire
Virnentin CK 19 CK 18 CK 8 CK 7 Milk fat globulin (EMA) Neural cell adhesion molecule KP 1 CD 45 S-100 proteins Other markers listed in Table 1
cytoplasm, the cytoplasmic inclusions of about 70% of the large neoplastic cells and about 30% of the small neoplastic cells were stained using the monoclonal antibodies CAM 5.2 and KL1. Normal hepatic parenchyma present in the section was also stained by both of these antibodies. In the neoplastic population with rhabdoid appearance the anti-EMA antibody showed the same distribution, as was observed with the anti-
+ +
+ (80%) + (50%) + (20%)
+ + (focal positivity) + (variable positivity) + (occasional cell) + (occasional cell, mainly in cytoplasmic inclusion) -
Table 2. Immunohistochemical staining pattern of rhabdoid tumour cells
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Figure 3. a The neoplastic cells are intensely immunoreactive for vimentin. b Most neoplastic cells are also positive for CK 19. Immunoperoxidase counterstained with Mayer’s haemalum. a x 640. b x 400.
cytokeratin antibodies. In the ‘pseudoglandular’component of the tumour, about 10%of the neoplastic cells were positive for EMA. In the normal liver parenchyma, anti-EMA antibody was localized on the luminal surface of the bile duct cells. Clusters of neoplastic cells were positive with the Leu 19 antibody. Using the monoclonal antibody KP1, immunostaining of macrophages present in the necrotic areas was observed. However, numerous neoplastic cells were also positive. In addition occasional neoplastic cells were positive for leukocyte common antigen (LCA). S-100 protein was positive in the cytoplasmic inclusions of occasional large neoplastic cells. All other markers tested on frozen and paraffin sections were negative.
Elect ronmicroscopy The tumour cells were large and oval in shape with a peripherally located oval to irregularly indented nucleus with prominent nucleolus. The voluminous cytoplasm contained many organelles: cisternae of rough endoplasmic reticulum-often slightly dilated; round to oval mitochondria: small heterogeneous lysosomes; several Golgi areas: and numerous ribosomes (Figure 4). Most
striking were masses of intermediate filaments (Figure 4) among which bundles of tonofilaments were apparent (Figure 5a). Intercellular junctions often of considerable length were found (Figure 5b).
Cytogenetic analysis Three out of 2 0 G-banded metaphases obtained from a 6-day-old culture displayed a translocation between chromosomes 1 and 8, at bands p36 and q24 respectively, as the only chromosomal change. A normal female karyotype was found in the remaining cells.
Discussion Malignant rhabdoid tumour is a rare and aggressive neoplasm, first described by Beckwith & Palmer” as an aggressive variant of Wilms’ tumour. Subsequent observations”.l2 suggested that it was a distinct neoplasm, different in nature and behaviour from Wilms’ tumour. Numerous cases have been described in various organs, but only four cases have been reported arising in the liver’-4. All cases of primary hepatic malignant rhabdoid tumour reported up to now have shown a ‘classical’
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Figure 4. Electronmicrographof a tumour cell. Intermediate filaments are conspicuous (arrow). x 9200.
rhabdoid appearance. The tumour reported here was also mainly composed of large polygonal cells, with vesicular nuclei and prominent nucleoli, and the typical eosinophilic PAS positive cytoplasmic inclusions. Interestingly, however, a small part of the present tumour presented a ‘pseudoglandular’ component and was composed of smaller cells. Recently, Weeks et al.13 have drawn attention to the various histological appearances of renal malignant rhabdoid tumours. The histological
patterns observed in the present case are consistent with their observations”. Immunohistochemical studies performed on renal and extrarenal malignant rhabdoid tumours constantly report positivity for vimentin in almost all cases and for cytokeratins in numerous cases13-15.Single cases have been reported to contain other markers13-16.Nevertheless, in none of the reported cases has the expression of different cytokeratins been studied in detail. In the
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Figure 5. Electronmicrograph of part of a tumour cell. a Bundles of tonofilaments are evident (arrow). b Adjacent tumour cells forming intercellular junctions (arrow). a x 14 500. b x 18 400.
present case immunohistochemistry proved the neoplastic cells to be intensely positive for vimentin and CK 19. CK 8 and CK 18 were expressed by numerous neoplastic cells, whereas CK 7 was only detected in a small number
of neoplastic cells. No staining for CK 4, CK 10 or CK 1 3 was seen. On paraffin sections, vimentin, CAM 5.2, KL1 and EMA antibodies stained the neoplastic cells of the rhabdoid and ‘pseudoglandular’ components. Positivity
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in the 'pseudoglandular' component was confined to a smaller number of cells. The expression of the same immunohistochemical profile in the two components of the present tumour was considered as proof that they are different morphological aspects of the same neoplasm. Immunostaining for /?-HCG and a-fetoprotein, and for markers for muscular and neural differentiation was negative. The only exception was focal S-100 protein positivity. A similar cytokeratin profile was observed in the embryonal component of hepat~blastoma'~. The coexpression of cytokeratins and vimentin has been suggested to be indicative of cells in a very early stage of differentiation''. In addition, Stosiek et have demonstrated that CK 1 9 is localized in all the hepatic progenitor cells at the very early phases of human liver organogenesis. Subsequently, parenchymal cells lose CK 19. but remain positive for CK 8 and CK 18, whereas cells differentiating towards bile ducts express in addition CK 7'". Positivity for EMA has been previously reported in renal and extrarenal malignant rhabdoid t u m o ~ r s ' ~ .including '~, one case arising in the liver2. The presence of EMA was considered a n argument in favour of epithelial differentiation2.'3. The positive staining for LCA, KP1 and S-100 protein was unexpected. Positivity was mainly, if not exclusively, localized in the cytoplasmic inclusions and may represent nonspecific staining. An alternative explanation would be the aberrant expression of various genes by the tumour. The immunoreactivity of cytokeratins, EMA and vimentin, together with the electronmicroscopic features, argue against a true histiocytic or neural differentiation. Electronmicroscopy showed the neoplastic cells to contain bundles of variously arranged intermediate filaments, tonofilaments and intercellular junctions, suggestive of epithelial differentiation. These findings are consistent with previous observationsl 1.12.1 5.1 6 Weeks and colleague^'^, on the basis of a careful review of the literature and a study of a large series of renal cases, proposed to consider renal malignant rhabdoid tumour as a separate entity, and malignant rhabdoid tumour in extrarenal sites as a simple phenotype. Renal variants have been shown to contain a 5 4 kDa cytokeratin, that could correspond to CK 8". and a 5557 kDa cyt~keratin'~.No data are reported on the expression of other cytokeratins in renal cases. Cytokeratins were studied during kidney organogenesis. In the developing kidney CK 8 and CK 1 8 appear at the early phases and CK 1 9 and vimentin are transiently expressed by the collecting ducts22.CK 7 has not been studied during kidney development. There are only a few reports on cytogenetic analysis of extrarenal rhabdoid tumours (reviewed by Douglass et
al. in 199023),and so far only primary brain tumours with a rhabdoid morphology seem to be characterized by the loss of chromosome 22. The outcome of published cases of extrarenal malignant rhabdoid tumour, including ours, is uniformly fatal, despite aggressive treatment. Short-term responses in cases of relapse appear to be anecdotal. Survival has been noted in malignant rhabdoid tumour of the kidney following complete excision in localized disease with negative nodes13. In conclusion, on the basis of the electronmicroscopic and immunohistological data, the present case of malignant rhabdoid tumour arising in the liver seems to have a very primitive epithelial differentiation, with an 'attempt' at differentiation towards bile ductal cells. These findings could support the hypothesis of Weeks and colleagues' 3,24 that extrarenal malignant rhabdoid tumour is a 'phenotype' shared by poorly differentiated neoplasms, arising in different organs.
Acknowledgements Peter Van Eyken is a research assistant of the Belgian National Fund for Scientific Research. This study was supported in part by the Interuniversity Network for Fundamental Research sponsored by the Belgian government (1987-1991). The authors gratefully acknowledge the technical assistance of Suzanne Taelemans and Bernadette Smets, and the photographical assistance of Michel Rooseleers. They are also greatly indebted to Professor F.C.S.Ramaekers, Maastrict, The Netherlands and Dr U.Karsten, Berlin Buch, Germany who generously supplied the monoclonal antibodies RCK 105, RCK 107 and A53/BA2 respectively. Our thanks also go to Professor H.Devlieger and to Professor F.de Zegher for their share in the intensive care of this child and to Professor I.De Wever for his surgical skills.
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16. Mireau GW. Weeks DA. Beckwith JB. Anaplastic Wilms' tumor and other clinically aggressive childhood renal neoplasms: ultrastructural and immunocytochemical findings. Ultrastruct. Pathol. 1989: 13: 225-248. 17. Van Eyken P. Sciot R, Callea F, Ramaekers F. Schaart G, Desmet VJ. A cytokeratin-immunohistochemical study of hepatoblastoma. Hum. Pathol. 1990; 21; 302-308. 18. Abenoza P, Manivel JC, Wick MR, Hagen K. Dehner LP. Hepatoblastoma: a n immunocytochemical and ultrastructural study. Hum. Pathol. 187: 18; 1025-1035. 19. Stosiek P, Kasper M. Karsten U. Expression of cytokeratin 19 during human liver organogenesis. Liver 1990: 1 0 59-63. 20. Van Eyken P, Sciot R. Callea F. Van Der Steen K. Moerman P, Desmet VJ. The development ofthe intrahepatic bile ducts in man: a keratin-immunohistochemical study. Hepatology 1988: 6: 1586-1595. 21. Vogel AM, Gown AM, Caughlan J, Haas JE. Beckwith JB. Rhabdoid tumors of the kidney contain mesenchymal specific and epithelial specific intermediate filament proteins. Lab. Invest. 1984: 5 0 232-238. 22. Oosterwijk E, Van Muijen GNP, Oosterwijk-Wakka JC, Warnaar SO. Expression of intermediate-sized filaments in developing and adult human kidney and in renal cell carcinoma. 1. Histochem. Cytochem. 1990: 38; 385-392. 23. Douglass EC. Valentine M. Rowe ST et al. Malignant rhabdoid tumor: a highly malignant childhood tumor with minimal karyotypic changes. Genes Chromosomes Cancer 1990; 2; 210216. 24. Weeks DA. Beckwith JB, Mireau GW. Rhabdoid tumor. An entity or a phenotype? Arch. Pathol. Lab. Med. 1989: 113; 113-114.
