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Establishment of a rhabdoid tumor cell line with a specific chromosomal abnormality, 46,XY,t(11;22)(p15.5;q11.23)
Establishment of a Rhabdoid Tumor Cell Line with a Specific Chromosomal Abnormality, 46,XY,t(11;22)(p15.5;q11.23) Pamela S. Karnes, Tuan N. Tran, Mei Ying Cui, Emil Bogenmann, Hiroyuki Shimada, and Kuang Lin Ying
ABSTRACT: The malignant rhabdoid tumor is a rare, poor/y understood tumor which occurs primarily in children. The kidney is a frequent primary site of origin, but the tumor has arisen in other mesodermally derived tissues as well. Controversy exists regarding the embryonic origin of the rhabdoid tumor and recent histopathologic studies suggest that it may be of neuroepithelial origin. Our immunohistochemical and electron micrographic studies support this theory. No consistent chromosome abnormalities have been reported in this tumor and no cell lines are available for study. We have established and characterized the first rhabdaid tumor cell line. It possesses a specific chromosomal abnormality, 46,XY, t(11;22)(p15.5;ql 1.23). The translocation may provide an important clue to the pathogenesis of the tumor as well as an opportunity for further study of the involved chromosome regions.
INTRODUCTION The malignant rhabdoid tumor is a soft-tissue sarcoma which occurs primarily during infancy and childhood [1]. Originally described in the kidney by Beckwith et al. [2], it is a highly malignant tumor with a poor prognosis. Most cases have originated in the kidney, but other sites have included the liver, chest wall, neck, pelvis, thymus, heart, upper arm, brain, and paravertebral soft tissues [3-7]. Controversy exists over the embryonic origin of the rhabdoid tumor. The cellular ultrastructure was initially thought to be of rhabdomyoblastic differentiation [2, 8, 9], but recent histopathologic studies suggest a neuroepithelial origin [2]. Several rhabdoid tumors have been analyzed cytogenetically and a consistent pattern has not emerged, although chromosome 22 abnormalities have been noted by several workers. To our knowledge, the present report is the first to describe the establishment and characterization of a rhabdoid tumor cell line. The cell line has histopathologic characteristics suggestive of neuroepithelial origin. It possesses a specific chromosomal abnormality, 46,XY, t(ll;22)(p15.5;qll.23). The breakpoints may provide an important clue to the pathogenesis of this tumor, as well as an opportunity for further study of these chromosome regions. From the Divisions of Medical Genetics (P. S. K., T. N. T., M. Y. C., K. L. Y.), Hematology-Oncology (E. B.), and Pathology (H. S.}, The Childrens Hospital, Los Angeles, California.
Address reprint requests to: Kuang Lin Ying, Ph.D., Childrens Hospital Los Angeles, Division of Medical Genetics, 4650 Sunset Boulevard, Los Angeles, CA 90027. Received November 2, 1990; accepted March 1, 1991.
31 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 56:31-38 (1991] 0165-4608/91/$03.50
32
P.S. Karnes et al.
CASE REPORT
This 21-month-old Caucasian male (J. S.) presented with a 3-week history of gait disturbance and irritability. An MRI of the abdomen showed a retroperitoneal mass and J. S. was transferred to Childrens Hospital, Los Angeles for further evaluation and management. Physical examination revealed a nondysmorphic, alert toddler with generalized decreased muscle mass, and absent deep tendon reflexes and sensation in the lower extremities. Abdominal CT and MR! showed a retroperitoneal tumor which extended from the lower thoracic to upper lumbar levels (T9 to L1-2). The tumor invaded the spinal canal from T l l to L1, causing marked cord compression. Pathologic diagnosis of abdominal tumor biopsy revealed malignant rhabdoid tumor. Chemotherapy and radiation to the tumor bed were initiated. A large pleural effusion developed and histologic examination of the cells revealed malignant rhabdoid tumor. Despite aggressive medical management, meningeal metastases developed and the patient died within 2 months of initial presentation. MATERIALS AND METHODS
Tumor cells from the patient's retroperitoneal mass were received prior to institution of chemotherapy or radiation therapy. Fresh tumor was washed in McCoys medium (GIBCO, Grand Island, NY), minced, and digested in collagenase. Two-thirds of the cells were resuspended in McCoys medium and 1/3 in a I : I mixture of a-MEM (Irvine Scientific, Irvine, CA) plus Chang's medium (Hana Biologics, Alameda, CA). The cells were plated in T25 Falcon plastic flasks (Falcon Plastics, Oxnard, CA) and incubated at 37°C in 5% CO2. When at least 5 mitotic cells per low-power field were seen, colchicine (Colcemid, GIBCO, Grand Island, NY) was added at a final concentration of 0.02 ~g/ml and the cells were incubated for an additional 2 hours before harvesting. The cultures were harvested by trypsin-EDTA disaggregation. The cells were treated with hypotonic solution (equal parts 0.075 M KC1 and 0.97% sodium citrate) for 20 minutes at 37°C. Fixation was accomplished with two passes of a 3 : 1 methanol/acetic acid mixture. The chromosome preparations were aged in a 50°C incubator for 12-18 hours followed by 15 minutes of incubation at 75°C. Chromosomes were stained using GTG banding [10]. Tumor cells from the patient's malignant pleural effusion were received after institution of chemotherapy and radiation therapy. They were processed, cultured, and harvested in the above-described manner. Some tumor cells were passaged in culture (3-5 × ) to increase their number and subcutaneous injection into nude mice was performed. A tumor was harvested from one nude mouse and washed using Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY), supplemented with 10% human serum, insulin (10/~g/ml), transferrin (50 ~g/ml), penicillin (100 U/ml), and streptomycin (100 ~g/ml) after the method of Bogenmann et al. [11]. The tumor was dissociated by mincing and the cells were innoculated onto collagen-elastin matrices [12]. Some nude mause-derived tumor cells were also plated onto T25 Falcon flasks using Chang medium, aMEM, or a combination of both. Mitotic tumor cells were harvested using the previously described method. Samples from each specimen were viably frozen in 10% DMSO/90% complete medium in 1-mL vials and stored in liquid nitrogen for future studies. Population doubling time was determined by plating 200,000 cells from passage 22 into each of 16 wells (35 mm, Falcon Plastics, Oxnard, CA). Cells from one well were trypsinized and counted every 12 hours. The process was repeated in quadruplicate. Time at which doubling occurred was calculated. Cloning efficiency was determined by suspending single cells in agar at a concentration of 30,000 cells/ cc (15,000 cells per tissue culture well). Colonies consisting of 5 or more cells were counted at 14 days [13].
t(11;22) in a Rhabdoid Tumor Cell Line
33
Figure I Lightmicroscopic appearance of primary retroperitoneal tumor. Note characteristic intracytoplasmic hyaline-like inclusions (arrows); H&E staining; original × 200.
RESULTS Microscopic sections from the tumor revealed sheets of medium-to-large tumor cells which contained granular cytoplasm (Fig. 1). Areas of the tumor exhibited a reticular pattern with cords of tumor separated by an edematous myxomatous intercellular matrix. Some tumor cells contained eosinophilic hyaline intracytoplasmic inclusions. Using hematoxylin and eosin stain, the cytoplasm had a fibrillar appearance. The nuclei varied from round to irregular, with prominent nucleoli. Special staining studies were positive for vimentin, S-100, EMA, neuron-specific enolase, and negative for desmin, leukocyte common antigen, cytokeratin, and alpha-fetoprotein. Electron microscopic examination revealed bundles or whorls of intermediate cytoplasmic filaments, dilated RER, cytoplasmic glycogen, and cilia (Fig. 2). No thick or thin filaments, microtubules, or neurosecretory granules were seen. Chromosome studies on cultured cells from the primary tumor specimen revealed that the modal number was 46 chromosomes. However, out of 30 cells examined, 23 cells were normal 46,XY, whereas seven cells had a translocation involving chromosomes 11 and 22. Analysis of the pleural effusion cells confirmed the translocation in all 50 cells examined. Although a blood specimen was not obtained (due to the death of the patient), the presence of a majority of cells with a normal 46,XY karyotype in the first cultured specimen suggested that it was the patient's constitutional karyotype. The translocation abnormality was specific to the tumor. Tumor cells obtained after passage through the nude mouse revealed the same translocation in 97% of cells
34
1~. s. Karnes et al.
Figure 2 Electronmicroscopic appearance of cultured cell. Note the presence of intracytoplasmic whorled filamentous structures and eccentrically located nucleus (Nu).
examined (n ~ 100), with 3% of cells showing the mouse karyotype (Fig. 3). Additional studies using high-resolution banding further defined the breakpoints in the translocation and the tumor karyotype is 46,XY,t(11;22)(p15.5;q11.23) (Fig. 4). The translocation appeared to be reciprocal although a deletion at the molecular level could not be ruled out. Table 1 illustrates the properties of the cell line. The cell line originated from metastatic pleural effusion cells which were cultured for 2 weeks and injected into nude mice. The resulting tumor was harvested and cell cultures were established. Cells have been carried for more than 30 passages over 2 years with persistence of the stem line chromosome abnormality, 46,XY,t(11;22)(p15.5;q11.23). Electron microscopic and immunohistochemical studies from the cell line and the original tumor specimen are identical. The cell line exhibits a 45-hour doubling time under optimal culture conditions (37°C, 5-8% CO2, 200,000 cells/mL cultured). Cloning efficiency is 4.4% when 30,000 cells/mL are placed in soft agar. The cells adhere to culture flasks and exhibit independent growth. In agar medium they form colonies.
DISCUSSION
The malignant rhabdoid tumor has been well described histologically. The tumor contains oval to polygonal cells with eccentric nuclei, multiple prominent nucleoli, and acidophilic filament-laden cytoplasm [14]. The filaments are intermediate in size
35
~p! X
O
~m m~S
0
4 m~il
llm m a O
t t J
~
t J
0
I~
II
N
I~
36
P.S. Karnes et al.
a)
~ ~; ] ~;. .~ ,, ,~ 2 ~, ) 2~ ~ ?~ ~ ~ ; 2), )12 2] ~
~."
~:~
der(11)
der(22)
11
,~ ~ ,~ ~
22
Figure 4 a. High-resolution banding of derivative chromosomes 11 and 22 and their normal homologues from two different cells, b. Arrows indicate breakpoints identified in the translocation, t(ll;22)(p15.5;ql 1.23). and thus not thin actin or thick myosin filaments, as would be expected in tumors of rhabdomyoblastic origin [2]. Results of immunohistochemical stains have been inconsistent. Zanetti et al. concluded that the tumor was rhabdomyoblastic in origin because staining with antimyoglobin antisera was demonstrated [8]. However, Tsuneyoshi et al. noted no staining with antimyoglobin or antineurofilament [14]. He noted staining with anticytokeratin and antivimentin, both of which are expressed by intermediate filaments. Rutledge et al. verified Tsuneyoshi's work by demonstrating an absence of immunoperoxidase staining for myoglobin [15]. The failure to detect myoglobin by histochemical staining techniques or primitive sarcomeres by electron microscopy suggest that this tumor is not rhabdomyoblastic. Table 1
Properties of cell line
Cell line designation Tumor type Origin of cell line Electron microscopy Immunohistochemistry
Population doubling time (passage 22) Claning efficiency Tumorigenic in nude mice Karyotype
Tm87-16 Malignant rhabdoid tumor (retroperitoneal) Metastatic pleural effusion Intermediate filaments Positive: vimentin, S-100, EMA, and neuron-specific enolase Negative: desmin, leukocyte common antigen, cytokeratin, and alpha-fetoprotein 45 hours 4.4% Yes 46,XY,t(11;22)(p15.5;q11.23)
t(11;22) in a Rhabdoid Tumor Cell Line
37
In support of a neuroepithelial origin of this tumor, Blatt et al. reported staining with neuron-specific enolase [3]. We note staining of our tumor and cell line with antibodies to this protein. The cytoplasmic inclusions in rhabdoid tumors resemble those seen in amine-precursor-uptake-and-decarboxylation tumors (APUD tumors). Many of these tumors are thought to be of neuroepithelial origin [2, 16]. On electron microscopic examination, the cytoplasmic inclusions are characteristic intermediate filaments. The thick filaments and thin filaments characteristic of primitive sarcomeres or myoblastic differentiation are not seen. Our initial tumor sample and cell line demonstrate whorls of intermediate filaments and staining with neuron-specific enolase, supportive of a neuroepithelial origin for this tumor. Cytogenetic data on this diverse tumor are beginning to accumulate and consistent findings have yet to emerge. We propose that this may be due to the broad spectrum of tumors sharing the rhabdoid diagnosis. Recently, Biegel et al. noted m o n o s o m y 22 in two malignant rhabdoid tumors of the brain [17]. Douglass et al. [18] reported loss of one chromosome 22 and a partial long arm deletion of the homologous chromosome 22 in a rhabdoid tumor of the brain. The breakpoint, 22q11, was similar to that seen in our retroperitoneal rhabdoid tumor, 22q11.23. Cytogenetic abnormalities were present in three of the other nine rhabdoid tumors reported by Douglass et al. The abnormalities were varied and did not involve chromosome 22 [18]. We have demonstrated a consistent chromosome abnormality in our rhabdoid tumor cell line involving chromosomes 11 and 22, that is, 46,XY,t(11;22)(p15.5;q11.23). Thus, four rhabdoid tumors that have been analyzed cytogenetically exhibit abnormalities of chromosome 22. Deletion of material on chromosome 22 was seen in three rhabdoid tumors and is possible in our tumor if one postulates that exchange of DNA in our tumor translocation was not precisely reciprocal. An oncogene on the homologous 22 could have been unmasked. It is unlikely that the breakpoint cluster region (bcr) on chromosome 22 is involved because the breakpoint in our tumor is distal to bcr. Alternatively, the breakpoint on chromosome 11 in our tumor is at the site of the HRAS1 protoooncogene. The role of HRAS1 in this tumor remains to be elucidated, but it may be translocated to chromosome 22. A change in regulatory control might then take place, leading to tumorigenesis. Much remains to be understood about this chromosome translocation and its relationship to the development of the rhabdoid tumor. This cell line provides a model for study of the rhabdoid tumor and, by exhibiting precise breakpoints, it provides a unique clue to the pathogenesis of the tumor. The authors gratefully acknowledge the assistance of Dr. Hart Isaacs, Helen Ho, and Daisy Boquiren.
REFERENCES
1. Beckwith JB 11983): Wilm's tumor and other renal tumors of childhood: A selective review from the National Wilm's Tumor Study Pathology Center. Hum Pathol 14:481-492. 2. Haas JE, Palmer NF, Weinberg AG, Beckwith JB (1981): Ultrastructure of malignant rhabdoid tumor of the kidney. Hum Pathol 12:646-857. 3. Blatt J, Russo P, Taylor S (19681: Extrarenal rhabdoid sarcoma. Med Pediatr Oncol 14:221-226. 4. Small EJ, Gordon GJ, Dahms BB (1985): Malignant rhabdoid tumor of the heart in an infant. Cancer 55:2850-2853. 5. Biggs PJ, Garen PD, Powers JM, Garvin AJ (19871: Malignant rhabdoid tumor of the central nervous system. Hum Pathol 18:332-337. 8. Frierson HF, Mills SE, Innes DJ 119851: Malignant rhabdoid tumor of the pelvis. Cancer 55:1963-1967.
38
P . S . Karnes et al.
7. Lynch HT, Shurin SB, Dahms BB, Izant RJ, Lynch ], Danes BS (1983): Paravertebral malignant rhabdoid tumor in infancy. Cancer 52:290-296. 8. Zanetti G, Giangaspero F (1982): Rhabdomyoblastic nature of cytoplasmic inclusions in malignant rhabdoid tumor. Hum Pathol 13:410. 9. Beckwith JB, Palmer NF (1978): Histopathology and prognosis of Wilms' tumor. Cancer 41:1937-1948. 10. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971-972. 11. Bogenmann E, Moghadam H, DeClerck YA, Mock A (1987): c-Myc amplification and expression in newly established human osteosarcoma cell lines. Cancer Res 47:3808-3814. 12. Jones PA, Scott-Burden T, Gevers W/1979/: Glycoprotein, elastin and collagen secretion by rat smooth muscle cells. Proc Natl Acad Sci USA 76:353-357. 13. McAllister RM, Isaacs H, Rongey R, Peer M, Au W, Soukup SW, Gardner MB (19771: Establishment of a human medulloblastoma cell line. Int J Cancer 20:206-212. 14. Tsuneyoshi M, Daimaru Y, Hashimoto H, Enioii M/1985): Malignant soft tissue neoplasms with the histologic features of renal rhabdoid tumors: An ultrastructural and immunohistochemical study. Hum Pathol 16:1235-1242. 15. Rutledge J, Beckwith JB, Beniamin D, Haas JE (1983): Absence of immunoperoxidase staining for myoglobin in the malignant rhabdoid of the kidney. Pediatr Pathol 1:93-98. 18. Carstens PH, Broghamer WL (1978): Duodenal carcinoid with cytoplasmic whorls of microfilaments. J Pathol 124:235-238. 17. Biegel JA, Rorke LB, Emanuel BS (19891: Monosomy 22 in rhabdoid or atypical teratoid tumors of the brain. N Engl J Med 321:906. 18. Douglass EC, Valentine M, Rowe ST, Parham DM, Wilimas JA, Sanders JA, Houghton PJ (1990): Malignant rhabdoid tumor (MRT): A highly malignant childhood tumor with minimal karyotypic changes. Genes Chromo Cancer 2:210-216.
ABSTRACT: The malignant rhabdoid tumor is a rare, poor/y understood tumor which occurs primarily in children. The kidney is a frequent primary site of origin, but the tumor has arisen in other mesodermally derived tissues as well. Controversy exists regarding the embryonic origin of the rhabdoid tumor and recent histopathologic studies suggest that it may be of neuroepithelial origin. Our immunohistochemical and electron micrographic studies support this theory. No consistent chromosome abnormalities have been reported in this tumor and no cell lines are available for study. We have established and characterized the first rhabdaid tumor cell line. It possesses a specific chromosomal abnormality, 46,XY, t(11;22)(p15.5;ql 1.23). The translocation may provide an important clue to the pathogenesis of the tumor as well as an opportunity for further study of the involved chromosome regions.
INTRODUCTION The malignant rhabdoid tumor is a soft-tissue sarcoma which occurs primarily during infancy and childhood [1]. Originally described in the kidney by Beckwith et al. [2], it is a highly malignant tumor with a poor prognosis. Most cases have originated in the kidney, but other sites have included the liver, chest wall, neck, pelvis, thymus, heart, upper arm, brain, and paravertebral soft tissues [3-7]. Controversy exists over the embryonic origin of the rhabdoid tumor. The cellular ultrastructure was initially thought to be of rhabdomyoblastic differentiation [2, 8, 9], but recent histopathologic studies suggest a neuroepithelial origin [2]. Several rhabdoid tumors have been analyzed cytogenetically and a consistent pattern has not emerged, although chromosome 22 abnormalities have been noted by several workers. To our knowledge, the present report is the first to describe the establishment and characterization of a rhabdoid tumor cell line. The cell line has histopathologic characteristics suggestive of neuroepithelial origin. It possesses a specific chromosomal abnormality, 46,XY, t(ll;22)(p15.5;qll.23). The breakpoints may provide an important clue to the pathogenesis of this tumor, as well as an opportunity for further study of these chromosome regions. From the Divisions of Medical Genetics (P. S. K., T. N. T., M. Y. C., K. L. Y.), Hematology-Oncology (E. B.), and Pathology (H. S.}, The Childrens Hospital, Los Angeles, California.
Address reprint requests to: Kuang Lin Ying, Ph.D., Childrens Hospital Los Angeles, Division of Medical Genetics, 4650 Sunset Boulevard, Los Angeles, CA 90027. Received November 2, 1990; accepted March 1, 1991.
31 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 56:31-38 (1991] 0165-4608/91/$03.50
32
P.S. Karnes et al.
CASE REPORT
This 21-month-old Caucasian male (J. S.) presented with a 3-week history of gait disturbance and irritability. An MRI of the abdomen showed a retroperitoneal mass and J. S. was transferred to Childrens Hospital, Los Angeles for further evaluation and management. Physical examination revealed a nondysmorphic, alert toddler with generalized decreased muscle mass, and absent deep tendon reflexes and sensation in the lower extremities. Abdominal CT and MR! showed a retroperitoneal tumor which extended from the lower thoracic to upper lumbar levels (T9 to L1-2). The tumor invaded the spinal canal from T l l to L1, causing marked cord compression. Pathologic diagnosis of abdominal tumor biopsy revealed malignant rhabdoid tumor. Chemotherapy and radiation to the tumor bed were initiated. A large pleural effusion developed and histologic examination of the cells revealed malignant rhabdoid tumor. Despite aggressive medical management, meningeal metastases developed and the patient died within 2 months of initial presentation. MATERIALS AND METHODS
Tumor cells from the patient's retroperitoneal mass were received prior to institution of chemotherapy or radiation therapy. Fresh tumor was washed in McCoys medium (GIBCO, Grand Island, NY), minced, and digested in collagenase. Two-thirds of the cells were resuspended in McCoys medium and 1/3 in a I : I mixture of a-MEM (Irvine Scientific, Irvine, CA) plus Chang's medium (Hana Biologics, Alameda, CA). The cells were plated in T25 Falcon plastic flasks (Falcon Plastics, Oxnard, CA) and incubated at 37°C in 5% CO2. When at least 5 mitotic cells per low-power field were seen, colchicine (Colcemid, GIBCO, Grand Island, NY) was added at a final concentration of 0.02 ~g/ml and the cells were incubated for an additional 2 hours before harvesting. The cultures were harvested by trypsin-EDTA disaggregation. The cells were treated with hypotonic solution (equal parts 0.075 M KC1 and 0.97% sodium citrate) for 20 minutes at 37°C. Fixation was accomplished with two passes of a 3 : 1 methanol/acetic acid mixture. The chromosome preparations were aged in a 50°C incubator for 12-18 hours followed by 15 minutes of incubation at 75°C. Chromosomes were stained using GTG banding [10]. Tumor cells from the patient's malignant pleural effusion were received after institution of chemotherapy and radiation therapy. They were processed, cultured, and harvested in the above-described manner. Some tumor cells were passaged in culture (3-5 × ) to increase their number and subcutaneous injection into nude mice was performed. A tumor was harvested from one nude mouse and washed using Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY), supplemented with 10% human serum, insulin (10/~g/ml), transferrin (50 ~g/ml), penicillin (100 U/ml), and streptomycin (100 ~g/ml) after the method of Bogenmann et al. [11]. The tumor was dissociated by mincing and the cells were innoculated onto collagen-elastin matrices [12]. Some nude mause-derived tumor cells were also plated onto T25 Falcon flasks using Chang medium, aMEM, or a combination of both. Mitotic tumor cells were harvested using the previously described method. Samples from each specimen were viably frozen in 10% DMSO/90% complete medium in 1-mL vials and stored in liquid nitrogen for future studies. Population doubling time was determined by plating 200,000 cells from passage 22 into each of 16 wells (35 mm, Falcon Plastics, Oxnard, CA). Cells from one well were trypsinized and counted every 12 hours. The process was repeated in quadruplicate. Time at which doubling occurred was calculated. Cloning efficiency was determined by suspending single cells in agar at a concentration of 30,000 cells/ cc (15,000 cells per tissue culture well). Colonies consisting of 5 or more cells were counted at 14 days [13].
t(11;22) in a Rhabdoid Tumor Cell Line
33
Figure I Lightmicroscopic appearance of primary retroperitoneal tumor. Note characteristic intracytoplasmic hyaline-like inclusions (arrows); H&E staining; original × 200.
RESULTS Microscopic sections from the tumor revealed sheets of medium-to-large tumor cells which contained granular cytoplasm (Fig. 1). Areas of the tumor exhibited a reticular pattern with cords of tumor separated by an edematous myxomatous intercellular matrix. Some tumor cells contained eosinophilic hyaline intracytoplasmic inclusions. Using hematoxylin and eosin stain, the cytoplasm had a fibrillar appearance. The nuclei varied from round to irregular, with prominent nucleoli. Special staining studies were positive for vimentin, S-100, EMA, neuron-specific enolase, and negative for desmin, leukocyte common antigen, cytokeratin, and alpha-fetoprotein. Electron microscopic examination revealed bundles or whorls of intermediate cytoplasmic filaments, dilated RER, cytoplasmic glycogen, and cilia (Fig. 2). No thick or thin filaments, microtubules, or neurosecretory granules were seen. Chromosome studies on cultured cells from the primary tumor specimen revealed that the modal number was 46 chromosomes. However, out of 30 cells examined, 23 cells were normal 46,XY, whereas seven cells had a translocation involving chromosomes 11 and 22. Analysis of the pleural effusion cells confirmed the translocation in all 50 cells examined. Although a blood specimen was not obtained (due to the death of the patient), the presence of a majority of cells with a normal 46,XY karyotype in the first cultured specimen suggested that it was the patient's constitutional karyotype. The translocation abnormality was specific to the tumor. Tumor cells obtained after passage through the nude mouse revealed the same translocation in 97% of cells
34
1~. s. Karnes et al.
Figure 2 Electronmicroscopic appearance of cultured cell. Note the presence of intracytoplasmic whorled filamentous structures and eccentrically located nucleus (Nu).
examined (n ~ 100), with 3% of cells showing the mouse karyotype (Fig. 3). Additional studies using high-resolution banding further defined the breakpoints in the translocation and the tumor karyotype is 46,XY,t(11;22)(p15.5;q11.23) (Fig. 4). The translocation appeared to be reciprocal although a deletion at the molecular level could not be ruled out. Table 1 illustrates the properties of the cell line. The cell line originated from metastatic pleural effusion cells which were cultured for 2 weeks and injected into nude mice. The resulting tumor was harvested and cell cultures were established. Cells have been carried for more than 30 passages over 2 years with persistence of the stem line chromosome abnormality, 46,XY,t(11;22)(p15.5;q11.23). Electron microscopic and immunohistochemical studies from the cell line and the original tumor specimen are identical. The cell line exhibits a 45-hour doubling time under optimal culture conditions (37°C, 5-8% CO2, 200,000 cells/mL cultured). Cloning efficiency is 4.4% when 30,000 cells/mL are placed in soft agar. The cells adhere to culture flasks and exhibit independent growth. In agar medium they form colonies.
DISCUSSION
The malignant rhabdoid tumor has been well described histologically. The tumor contains oval to polygonal cells with eccentric nuclei, multiple prominent nucleoli, and acidophilic filament-laden cytoplasm [14]. The filaments are intermediate in size
35
~p! X
O
~m m~S
0
4 m~il
llm m a O
t t J
~
t J
0
I~
II
N
I~
36
P.S. Karnes et al.
a)
~ ~; ] ~;. .~ ,, ,~ 2 ~, ) 2~ ~ ?~ ~ ~ ; 2), )12 2] ~
~."
~:~
der(11)
der(22)
11
,~ ~ ,~ ~
22
Figure 4 a. High-resolution banding of derivative chromosomes 11 and 22 and their normal homologues from two different cells, b. Arrows indicate breakpoints identified in the translocation, t(ll;22)(p15.5;ql 1.23). and thus not thin actin or thick myosin filaments, as would be expected in tumors of rhabdomyoblastic origin [2]. Results of immunohistochemical stains have been inconsistent. Zanetti et al. concluded that the tumor was rhabdomyoblastic in origin because staining with antimyoglobin antisera was demonstrated [8]. However, Tsuneyoshi et al. noted no staining with antimyoglobin or antineurofilament [14]. He noted staining with anticytokeratin and antivimentin, both of which are expressed by intermediate filaments. Rutledge et al. verified Tsuneyoshi's work by demonstrating an absence of immunoperoxidase staining for myoglobin [15]. The failure to detect myoglobin by histochemical staining techniques or primitive sarcomeres by electron microscopy suggest that this tumor is not rhabdomyoblastic. Table 1
Properties of cell line
Cell line designation Tumor type Origin of cell line Electron microscopy Immunohistochemistry
Population doubling time (passage 22) Claning efficiency Tumorigenic in nude mice Karyotype
Tm87-16 Malignant rhabdoid tumor (retroperitoneal) Metastatic pleural effusion Intermediate filaments Positive: vimentin, S-100, EMA, and neuron-specific enolase Negative: desmin, leukocyte common antigen, cytokeratin, and alpha-fetoprotein 45 hours 4.4% Yes 46,XY,t(11;22)(p15.5;q11.23)
t(11;22) in a Rhabdoid Tumor Cell Line
37
In support of a neuroepithelial origin of this tumor, Blatt et al. reported staining with neuron-specific enolase [3]. We note staining of our tumor and cell line with antibodies to this protein. The cytoplasmic inclusions in rhabdoid tumors resemble those seen in amine-precursor-uptake-and-decarboxylation tumors (APUD tumors). Many of these tumors are thought to be of neuroepithelial origin [2, 16]. On electron microscopic examination, the cytoplasmic inclusions are characteristic intermediate filaments. The thick filaments and thin filaments characteristic of primitive sarcomeres or myoblastic differentiation are not seen. Our initial tumor sample and cell line demonstrate whorls of intermediate filaments and staining with neuron-specific enolase, supportive of a neuroepithelial origin for this tumor. Cytogenetic data on this diverse tumor are beginning to accumulate and consistent findings have yet to emerge. We propose that this may be due to the broad spectrum of tumors sharing the rhabdoid diagnosis. Recently, Biegel et al. noted m o n o s o m y 22 in two malignant rhabdoid tumors of the brain [17]. Douglass et al. [18] reported loss of one chromosome 22 and a partial long arm deletion of the homologous chromosome 22 in a rhabdoid tumor of the brain. The breakpoint, 22q11, was similar to that seen in our retroperitoneal rhabdoid tumor, 22q11.23. Cytogenetic abnormalities were present in three of the other nine rhabdoid tumors reported by Douglass et al. The abnormalities were varied and did not involve chromosome 22 [18]. We have demonstrated a consistent chromosome abnormality in our rhabdoid tumor cell line involving chromosomes 11 and 22, that is, 46,XY,t(11;22)(p15.5;q11.23). Thus, four rhabdoid tumors that have been analyzed cytogenetically exhibit abnormalities of chromosome 22. Deletion of material on chromosome 22 was seen in three rhabdoid tumors and is possible in our tumor if one postulates that exchange of DNA in our tumor translocation was not precisely reciprocal. An oncogene on the homologous 22 could have been unmasked. It is unlikely that the breakpoint cluster region (bcr) on chromosome 22 is involved because the breakpoint in our tumor is distal to bcr. Alternatively, the breakpoint on chromosome 11 in our tumor is at the site of the HRAS1 protoooncogene. The role of HRAS1 in this tumor remains to be elucidated, but it may be translocated to chromosome 22. A change in regulatory control might then take place, leading to tumorigenesis. Much remains to be understood about this chromosome translocation and its relationship to the development of the rhabdoid tumor. This cell line provides a model for study of the rhabdoid tumor and, by exhibiting precise breakpoints, it provides a unique clue to the pathogenesis of the tumor. The authors gratefully acknowledge the assistance of Dr. Hart Isaacs, Helen Ho, and Daisy Boquiren.
REFERENCES
1. Beckwith JB 11983): Wilm's tumor and other renal tumors of childhood: A selective review from the National Wilm's Tumor Study Pathology Center. Hum Pathol 14:481-492. 2. Haas JE, Palmer NF, Weinberg AG, Beckwith JB (1981): Ultrastructure of malignant rhabdoid tumor of the kidney. Hum Pathol 12:646-857. 3. Blatt J, Russo P, Taylor S (19681: Extrarenal rhabdoid sarcoma. Med Pediatr Oncol 14:221-226. 4. Small EJ, Gordon GJ, Dahms BB (1985): Malignant rhabdoid tumor of the heart in an infant. Cancer 55:2850-2853. 5. Biggs PJ, Garen PD, Powers JM, Garvin AJ (19871: Malignant rhabdoid tumor of the central nervous system. Hum Pathol 18:332-337. 8. Frierson HF, Mills SE, Innes DJ 119851: Malignant rhabdoid tumor of the pelvis. Cancer 55:1963-1967.
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P . S . Karnes et al.
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