Establishment and Characterization of a New Ewing's Sarcoma Cell Line Ken Kodama, Osamu Doi, Masahiko Higashiyama, Yoichi Mori, Takeshi Horai, Ryuhei Tateishi, Yasuaki Aoki, and Shinichi Misawa
ABSTRACT: A new human Ewing's sarcoma cell line (CADO-ESI) was established from the malignant pleurol effusion of a 19-year-old woman. These cells grew both anchorage dependently and anchorage independently. When cultured in bacteriologic dishes, they grew as tightly packed multicellular tumor spheroids; they were also capable of proliferating in soft agar. Flow cytometric DNA analysis demonstrated a nearly diploid DNA content (DNA index = 0.902). Chromosomal studies of cultured cells showed an isodicentric chromosome 8 in all examined cells, but t(11;22)(q24;q12), a translocation reported previously in Ewing's sarcoma, was not detected. Under normal culture conditions, no morpholagic evidence of neural differentiation was detected. In addition, immunocytochemical studies showed that vimentin was intensely positive, whereas neurofiloment (NF) and neuron-specific enolase (NSE) were weakly positive. Treatment with cyclic AMP (cAMP) induced pronounced morphologic evidence of neural differentiation and strong expression of NF in cultured cells. S-100 protein, glial fibrillary acidic protein (GFAP), desmin, cytokeratin, and epithelial membrane antigen were not detected immunohistochemically in either untreated or cAMP-treated cells, however. These data suggest that this cell line is derived from a highly undifferentiated neural cell with high chromosomal clonality, differentiating into neural features under certain conditions.
INTRODUCTION Ewing's sarcoma (ES} is a rather rare, small round-cell undifferentiated tumor of bone and occasionally of soft tissues that occurs in children and young adults. Several reports have described establishment of permanent cell lines from this tumor [1-8]. These cell lines are useful for elucidating their histogenesis and studying the sensitivity to anticancer drugs or radiation, but the histogenesis of ES remains controversial. Chromosomal abnormalities, i.e., a reciprocal t(ll;22)(q24;q12) and/or hyperdiploidy, have been reported by several groups of investigators [4, 9, 10]. We recently established a new cell line of ES, that possesses unique phenotypic characteristics. We report the cell characteristics, results of chromosomal analysis, immunocytochemical properties, and differentiating characteristics induced by agents known for their ability to induce terminal differentiation. From the Departments of Thoracic Surgery (K. K., O. D., M. H.), Cell Biology (Y. M.), Medicine (T. H.), Pathology (R. T.), and Orthopedic Surgery (Y. A.), The Center for Adult Diseases, Osaka; and 3rd Department of Medicine (S. M.), Kyoto Prefectural University of Medicine. Kyoto, Japan.
Address reprint requests to: Dr. Ken Kodama, Department of Thoracic Surgery, The Center for Adult Diseases, 3 Nakamichi, 1-chome, Higashinari-ku, Osaka 537, Japan. Received October 16, 1989; accepted January 3, 1991.
19 © 1991 Elsevier Science Publishing Co., Inc. 555 Avenue of the Americas, New York, NY 10010
Cancer Genet Cylogenet 57:19-30 (1991) 0165-4608/91/503.50
20
K. Kodama et al.
MATERIALS AND METHODS
Brief Clinical History of the Patient The patient, a 19-year-old Japanese woman, was admitted to our hospital in December 1986 with complaints of weight loss and a tumor in the right buttock. A destructive change in the ilium was demonstrated radiologically. ES was diagnosed on the basis of the results of tumor biopsy. Simultaneously, chest roentgenogram disclosed multiple lung metastases. After intensive chemotherapy, the patient underwent surgery in March 1987 for resection of the right ilium and the fourth and fifth lumbar and first through third sacral vertebral arches and centrum, followed by skin flap transplantation. Although the patient received postoperative chemotherapy intermittently until February 1988, the lung metastases gradually grew larger. Fourteen months after the operation, the patient died of respiratory failure resulting from lung metastases and malignant pleuritis.
Cell Line Establishment Our cell line, designated CADO-ES1, was derived from the malignant pleural effusion of the patient. These cells grew partly in suspension and partly attached to tissueculture flasks (Coming Glass Works, NY) in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) (GIBCO, Grand Island, NY) in a humidified atmosphere with 5% CO 2 at 37°C. The cells were subcultured by trypsinization with 0.25% trypsin (1 : 250; Difco Laboratories, Detroit, MI), and 0.02% EDTA in a calcium- and magnesium-free balanced salt solution. When these cells were placed in 100-ram plastic Petri dishes (Eiken, Tokyo, Japan) that had not been treated for cell attachment (henceforth called bacteriologic plates), they grew as anchorage-independent cell aggregates and formed multicellular tumor spheroids (MTS). Growth in semisolid medium was performed in RPMI 1640 medium supplemented with 10% HI-FCS in a 0.3% agarose-containing top layer over a 0.5% agarosecontaining bottom layer. The cultured cells were routinely tested for mycoplasma contamination and were always negative.
MORPHOLOGIC EXAMINATIONS Morphologic examination of growing cultures was performed using an inverted phasecontrast microscope (Nikon, Tokyo, Japan). Tumor tissues obtained by operation and autopsy and from nude mouse xenografts were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Electron microscopic examination of MTS and nude mouse xenografts was performed by fixation in a 2% glutaraldehyde-picric acid fixative, postfixation in OsO4, embedding in Epon, and staining with uranyl acetate-lead citrate.
Heterotransplantation Six-week-old female athymic mice (CD1 nu/nu) were purchased from Charles River Company, Atsuki, Japan. A suspension of 5 × 10 8 CADO-ES1 cells in 0.5 ml medium was injected subcutaneously (s.c.) to the backs of the mice. The cell inocula were allowed to grow until palpable tumors were formed. The animals were then sacrificed,
New Ewing's Sarcoma Cell Line
21
A
B Figure 1 Light microscopic appearances of primary site (A), and nude mouse xenograft (B) (H&E, original magnification × 66). the tumors were fixed by the methods described above, and light and electron microscopic examinations were performed.
Flow Cytometric Analysis Cell suspensions were prepared according to a method described previously [11]. Freshly harvested cells were fixed in 70% ice-cold ethanol. After washing with phosphate-buffered saline (PBS), the cell suspensions were processed for 20 min in a PBS
22
K. Kodama et al. solution containing 1 mg/ml RNase (Sigma, St. Louis, MO) at 37°C for 20 minutes. The samples were washed twice by centrifugation (2,000 rpm, 5 minutes) and incubated with freshly prepared 0.01% pepsin (Wako Chemicals, Osaka, Japan) at pH 1.5 at 37°C for 20 minutes. After the samples were stained with 50 ~g/ml propidium iodide (Sigma) and filtered through a 50-gm nylon mesh filter, they were analyzed with an FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA); 10,000 nuclei were read per sample. Data were analyzed using the Cellfit program (Cellfit-DNA software; Becton Dickinson). Human lymphocytes were used as staining controls for the normal diploid (DNA index = 1) DNA content.
Chromosomal Analysis Exponentially growing anchorage-dependent and anchorage-independent cells (MTS) from the seventh, 30th, and 55th passages were treated with colchicine at a concentration of 0.5 ~.g/ml in RPMI 1640 supplemented with 10% HI-FCS for 40 minutes at 37°C. The cells were harvested by trypsinization. After hypotonic treatment in 0.075 M KC1, the cells were fixed in Carnoy's solution at O°C before spreading on slides. G-banding of the chromosomes was obtained by trypsinization. Chromosome identification and karyotype designation were made according to International System for Human Cytogenetic Nomenclature [12].
Differentiation Experiment and Immunoperoxidase Staining CADO-ES1 cells were tested for their response to exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma) and cAMP (Sigma), which have the ability to induce terminal differentiation in other cell lines. TPA was used at concentrations of 25, 50, and 100 nM; cAMP was used at concentrations of 0.25, 0.625, and 2.5 mM [13, 14]. Untreated control cells and differentiated cells were grown in plastic flasks or in chamber slides. Throughout the experiments, the morphologic responses of the cells to the differentiating agents were documented by phase-contrast microscopy of viable, unfixed cells. After 6 days, the cells grown in plastic flasks were harvested, and an aliquot of the cells was placed on a slide glass, dried, and fixed in 10% neutral buffered formalin for 30 minutes. The cells grown in chamber slides were fixed directly in 10% neutral buffered formalin. Immunocytochemical observations were made by the avidin-biotin peroxidase complex (ABC) method. The monoclanal antibodies used were specific for neurofilament (NF), vimentin, glial fibrillary acidic protein (GFAP), all from Lipshow Immunon, Detroit, MI, and desmin and epithelial membrane antigen (EMA) (Dakopatts, Denmark). The polyclonal antibodies used were directed at S-100 protein (Lipshow), neuron-specific enolase (NSE) (Biogenex Laboratories, Dublin, CA) and pankeratin (Dako Corporation, Santa Barbara, CA). Antigen-antibody-peroxidase complex formation was detected with freshly prepared 3,3'-diaminobenzidine and hydrogen peroxide. The cells were counterstained with hematoxylin, washed in running water for 5 minutes, dehydrated, cleared, and mounted in synthetic medium. Control sections of the same samples were processed simultaneously. RESULTS
Light-Microscopic Findings The primary tumor of the ilium was composed of small, closely packed cells with scanty cytoplasm and indistinct cellular border (Fig. 1A). The tumor cells formed solid sheets, separated by thin vascular stroma into irregular nests. No rosettelike
New Ewing's Sarcoma Cell Line
23
Figure 2 Electronmicrograph of MTS after fourth passage shows small cells with cytoplasmic pools of glycogen and complex cytoplasmic processes.
structures were detected. The cells had small oval nuclei. The nuclei were vesicular and small round or oval, with distinct nuclear membranes. Some tumor cells showed prominent nucleoli. Numerous mitotic figures were observed. PAS stain showed positive granules in the tumor cytoplasm, which were eliminated by a diastase digestion procedure. The histologic appearance of the xenotransplanted tumor in nude mice closely resembled that of the primary tumor [Fig. 1B).
Electron Microscopic Findings Ultrastructural studies were performed on MTS after the fourth passage and on the xenotransplanted tumor. Ceils from MTS showed rounded nuclei with unmarginated chromatin and small nucleoli. The cytoplasm contained abundant lysosomes and mitochondria and short strands of rough endoplasmic reticulum. Many cells had cytoplasmic glycogen deposits. No intercellular junctions were noted, and cell-cell attachments consisted of simple apposition. Complex cytoplasmic processes were present (Fig. 2), but neurosecretory granules were not detected. The xenotransplanted tumor cells were very similar in appearance to MTS, but glycogen deposits and ribosomes were more prominent in tumor cells than in MTS. Inversely, lysosomes were prominent in MTS.
Tissue Culture CADO-ES1 showed both anchorage-dependent and anchorage-independent growth patterns (Fig. 3A and B). The cultures reached confluency quickly and were passed once a week. When placed in tissue culture flasks, these cells were small, plump, and
24
K. Kodama et al. round (Fig. 3A). When placed in bacteriologic plates, they grew in the form of very compact MTS, which were also passed every week (Fig. 3B). These cells were also capable of proliferating in soft agar.
Heterotransplantation Into Nude Mice Subcutaneous heterotransplantation of 5 × 106 cells from the tenth passage into five nude mice led to formation of palpable tumors in all animals within 10 days. The tumor doubling time was 3.8 days. No metastases to the lung, liver, lymph nodes, or brain were detected, but when the same number of cells was injected into the peritoneal cavity, neither abdominal tumors nor malignant ascites developed.
Flow Cytometric Analysis Figure 4 shows DNA distribution histograms obtained from the samples resolving the normal lymphocyte and tumor cell mixture. The coefficient of variation of G0/G1 was 4.2% for normal lymphocytes and 4.3% for the tumor cells. The tumor cells had nearly diploid DNA content as compared with normal lymphocytes, and the DNA index was 0.902. The percentage of cycling (S + G2/M cells was 39.5%.
Chromosomal Analysis Fifty mitotic cells from each of the seventh 30th, and 55th passages were photographed and karyotyped. The modal chromosome number was 47. An isodicentric chromosome 8, idic(8)(p11.2), was observed in every cell analyzed. Thus, the karyotype was 47,XX, + idic(8)(p11.2). (Fig. 5). The reciprocal t(11;22)(q24;q12) reported to be specific for Ewing's sarcoma was not found.
Differentiation and Immunohistochemical Findings Treatment with cAMP induced pronounced morphologic changes in cultured cells. The cells developed elongated processes and were interconnected by numerous neuritic processes (Fig. 6). The degree of morphologic differentiation toward neural cells was more prominent in treatment with cAMP than with TPA. The results of immunoreactivity in each group treated with or without the differentiating agents are shown in Table 1. Cultured cells in all three groups strongly expressed vimentin. The cytoplasms of all cells stained intensely and quite uniformly; the cytoplasms of some untreated cells stained positively for NF (Fig. 7A) and NSE. The treated cells eventually expressed NF strongly, particularly after treatment with cAMP (Fig. 7B), but the reactivity for NSE was similarly intense in untreated and treated groups. On the other hand, no cells stained positively for S-100 protein, GFAP, desmin, pankeratin, or EMA. DISCUSSION ES is a highly undifferentiated tumor with no specific morphologic characteristics other than glycogen deposits. Because of the lack of useful lineage markers, its histogenesis remains controversial. Differential diagnosis of poorly differentiated small cell tumors in children and young adults, which include ES, embryonal rhabdomyosarcoma, neuroblastoma, peripheral neuroectodermal tumors, small cell osteogenic sarcoma and lymphoma, is often difficult to make by light microscopy alone [15]. We established a new cell line of ES, designated CADO-ES1, and described its characteristics. Based on the results of both light-microscopic studies of the primary
25
New Ewing's Sarcoma Cell Line
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tumor and ultrastructural studies of MTS and nude mouse xenografts, we confirmed the diagnosis of ES for this cell line. Karyotype analyses of short-term cultures and established cell lines of ES have been made [3, 4, 9, 10]. As a result, Ewing cells are characterized by a specific t{ll;22}(q24;q12). The percentage of such reciprocal translocations in each cell line ranges between 21 and 100% [3, 10]. According to a recent review of chromosomal analyses of 85 unrelated cases of ES reported by Turc-Carel et al. [16], the standard
26 22a~
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Figure 4 DNA histogram. Arrow shows the G0/G1 peak of ES tumor cells after 50th passage. Arrowhead shows diploid peak of normal lymphocytes.
Figure 5 A karyotype of CADO-ES1 tumor ceils with 47 chromosomes. All cells examined showed idic(8)(pll.2) (arrow).
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Figure 6 CADO-ES1 cells treated with 2.5 mM cAMP. Phase-dense cell bodies are interconnected by numerous neuritic processes (original magnification x 100).
t(11;22)(q24;ql 2) proved to be a remarkably consistent event, present in 83% of cases. The same translocation is also observed in neuroepithelioma [17]. These data suggest that Ewing cells share karyotypic features with derivatives of the neuroectoderm, possibly indicating a related histogenesis, but our ES line CADO-ES1 had no such translocation. Turc-Carel et al. [3, 10] also reported the absence of this translocation in an established ES line, IARC-ES3. Unexpectedly, these two cell lines show the same growth pattern, characterized by formation of tightly packed floating clusters [5]. Why only these two lines grow as floating clusters is unclear and cannot be explained in terms of c h r o m o s o m a l abnormalities. The modal cell karyotype of the IARC-ES3 line is 48,XY, - 7, - 9, - 15, + 8, + der(1), + der(7), + der(9), + der(12) [10], whereas that of CADO-ES1 is 47,XX, + idic(8)(p11.2). Turc-Carel et al. [16] reported that only six (8%) of the 85 ES cases lacked cytologic e v i d e n c e of rearrangements i n v o l v i n g 11q24 and/or 22q12. Acco rd i n g to the review of Mugneret et al. [18] secondary ch r o m o so m al changes in addition to t(11;22)(q24;q12), trisomy 8 was consistently observed in half of the 43 cases of ES selected for analysis of numerical changes. Although intensive chemother-
Table 1
Cytochemical results
Working dilution
Untreated control ceils +TPA 100 nM +cAMP 2.5mM
NF
NSE
S-100
GFAP
Vimentin
Desmin
Pankeratin
EMA
1:100
1:1
1:400
1:400
1:400
1:100
1:1,000
1:100
+
+
-
-
+++
-
-
-
++ +++
+ +
-
-
++ + +++
-
-
-
Abbreviations: TPA, 12-O-tetradecanoylphorbol-13-acetate; NF, neurofilaments; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein; EMA, epithelial membrane antigen.
Strongly positive { + + + }, positive / + + ), weakly positive {+ ), and negative {- }.
28
K. Kodama et al.
4
A
B
Figure 7 Immunocytochemical staining of CADO-ES1 cells with neurofilaments (NF) (original magnification × 132]. (A) Some untreated cells show expression of NF (arrowhead). (B) Cells treated with 2.5 mM cAMP show strong expression of NF in the cytoplasm.
apy had been performed before the tissue culture materials were obtained, a unique c h r o m o s o m a l abnormality, idic(8)(p11.2), and a nearly d i p l o i d (hypodiploid) DNA pattern were noted in CADO-ES1. The idic(8) was consistently found in all mitotic cells at various periods of passage. Trisomy 8 is a frequent n o n r a n d o m secondary numerical change in hematologic malignancies [19, 20]. There have been no previous
New Ewing's Sarcoma Cell Line
29
reports regarding ES cell lines which, like CADO-ES1, have no chromosomal changes except a partial tetrasomy 8. On the other hand, trisomy 8 has been reported as a rare constitutional abnormality so far found only among liveborn trisomics in humans. An increased parental age has been reported to be of possible etiologic significance in the origin of trisomy 8 [21], and this extra autosome has been observed with or without mosaicism in various anomalies such as mental retardation and skeletal defects. Our patient had a normal birth and normal physical development, and no obvious malformations except slight mental retardation. Her father was 29 and mother was 26 years old at the time of her birth. Chromosomal studies on somatic cells had not been performed during her lifetime. Constitutional partial tetrasomy of chromosome 8 without serious physical or mental abnormalities has not been reported. The intermediate filament (IF) network is heterogeneous with regard to its constitutive proteins and can be biochemically and immunologically classified into five types: Keratin occurring in epithelial cells, vimentin in fibroblasts and mesenchymatous ceils, desmin in muscle cells, NF in neural cells, and GFAP in astrocytes. Because expression of IF proteins is retained after malignant transformations, immunotyping of IF has proven a valuable tool for diagnosis of human malignant tumors [5]. NSE, on the other hand, is now considered nonspecific; however, it is still a useful marker for neurons and shows positivity in neuroblastoma cells [22]. S-100 protein is also nonspecific and widely distributed in the nervous system and certain other tissues. EMA is used in diagnostic pathology for epithelial cell identification and characterization. Immunocytochemical studies on our cell line demonstrated that vimentin was strongly positive, whereas NF and NSE were weakly positive, but S-100 protein, GFAP, desmin, keratin, and EMA were all negative. After cAMP treatment, pronounced morphologic changes toward neural cells, characterized by development of elongated processes with varicosities, were detected in CADO-ES1. In direct proportion to the morphologic differentiation, NF became strongly expressed, but no significant differences were recognized between untreated and cAMP- or TPA-treated cells with regard to the reactivity with 7 other antibodies. The similarity of morphologic differentiation of ES cell lines induced by treatment with cAMP or TPA had been reported by Cavazzana et al. [13]. Dellagi et al. [5] reported that after TPA exposure undifferentiated Ewing cells may acquire an IF phenotype related to that of epithelial cells. Neither keratin nor EMA expression was detected in CADO-ES1, however, even though the cells had been treated with the same concentration of TPA reported by Dellagi et al. [5]. These findings suggest that CADO-ES1 represents a highly undifferentiated neural tumor which can, under certain circumstances in vitro, acquire a phenotype related to that of neural cells. REFERENCES
1. Dickman PS, Liotta LA, Triche TJ (1982): Ewing's sarcoma. Characterization in established cultures and evidence of its histogenesis. Lab Invest 47:375-382. 2. Siegel GP, Thorgeirsson UP, Russo RG, Wallace DM, Liotta LA, Berger SL (1982): Interferon enhancement of the invasive capacity of Ewing's sarcoma cells in vitro. Proc Natl Acad Sci USA 79:4064-4068. 3. Turc-Carel CT, Philip I, Berger MP, Philip T, Lenoir GM (1983): Chromosomal translocation in Ewing's sarcoma. N Engl J Med 309:497-498. 4. Maletz N, McMorrow LE, Greco MA, Wolman SR (1986): Ewing's sarcoma. Pathology, tissue culture, and cytogenetics. Cancer 58:252-257. 5. Dellagi K, Lipinski M, Paulin D, Portier MM, Lenoir GM, Brouet JC (1987): Characterization of intermediate filaments expressed by Ewing tumor cell lines. Cancer Res 47:1170-1173.
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K. K o d a m a et al.
6. Cavazzana AO, Miser JS, Jefferson J, Triche TJ (1987): Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 127:507-518. 7. van Valen F, Jurgen H, Winkelmann W, Keck E (1987): B-Adrenergic agonist- and prostaglandin-mediated regulation of cAMP levels in Ewing's sarcoma cells in culture. Biochem Biophys Res Commun 146:665-691. 8. McKeon C, Thiele CJ, Ross RA, Kwan M, Triche TJ, Miser JS, Israel MA (1988): Indistinguishable pattern of protooncogene expression in two distinct but closely related tumors: Ewing's sarcoma and neuroepithelioma. Cancer Res 48:4307-4311. 9. Aurias A, Rimbaut C, Buffe D, Dubousset J, Mazabraud A (1983): Chromosomal translocation in Ewing's sarcoma. N Engl J Med 309:496-497. 10. Turc-Carel C, Philip I, Berger M, Philip T, Lenoir GM {1964): Chromosome study of Ewing's sarcoma (ES) cell lines. Consistency of a reciprocal translocation t(ll;22)(q24:q12). Cancer Genet Cytogenet 12:1-19. 11. Volm M, Mattern J, Sonka J, Vogt-Schaden M, Wayss K (1985): DNA distribution in nonsmall cell lung carcinoma and its relationship to clinical behavior. Cytometry 6:345-356. 12. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harden DG, Klinger HP (eds.); published in collaboration with Cytogenet Cell Genet (Karger, Basel 1985): also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 13. Cavazzana AO, Jefferson J, Triche TJ (1987): Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 127:507-516. 14. Aguanno S, Bouche M, Adamo S, Molinaro M (1990): 12-O-tetradecanoylphorbol-13-acetateinduced differentiation of a human rhabdomyosarcoma cell line. Cancer Res 50:3377-3382. 15. Triche TJ, Askin FB, Kissane JM (1986): Neuroblastoma, Ewing's sarcoma and the differential diagnosis of small-, round-, blue-tumors. In: Finegold M, ed. Pathology of Neoplasia in Childhood and Adolescents, Vol. 18. W. B. Saunders, Philadelphia, pp. 145-195. 16. Turc-Carel C, Aurias A, Mugneret F, Lizard S, Sidaner I, Volk C, Thiery JP, Olschwang S, Philip I, Berger MP, Philip T, Lenoir GM, Mazabraud A (1986): Chromosomes in Ewing's sarcoma. I. An evaluation of 85 cases and remarkable consistency of t(ll;22)(q24;q12}. Cancer Genet Cytogenet 32:229-238. 17. Whang-Peng J, Triche TJ, Knutsen T, Miser J, Douglass EC, Israel MA (1984): Chromosome translocation in peripheral neuroepithelioma. N Engl J Med 311:564-585. 18. Mugneret F, Lizard S, Aurias A, Turc-CareL C (1968): Chromosomes in Ewing's sarcoma. II. Nonrandom additional changes, trisomy 8 and der(16)t(1;16). Cancer Genet Cytogenet 32:239-245. 19. First International Workshop on Chromosomes in Leukemia (1978): Chromosomes in Phpositive chronic granulocytic leukemia. Br J Haematol 39:305. 20. Heim S, Mitelman F (1986): Secondary chromosome aberration in the acute leukemias. Cancer Genet Cytogenet 22:331-338. 21. Caspersson T, Lindsten J, Zech L, Buckton KE, Price WH (1972) Four patients with trisomy 8 identified by the fluorescence and Giemsa banding techniques. J Med Genet 9:1-7. 22. Tsokos M, Linnoila RI, Chandra RS, Triche TJ (1984): Neuron-specific enolase in the diagnosis of neuroblastoma and other small round-cell tumors in children. Hum Patho115:575-584.
ABSTRACT: A new human Ewing's sarcoma cell line (CADO-ESI) was established from the malignant pleurol effusion of a 19-year-old woman. These cells grew both anchorage dependently and anchorage independently. When cultured in bacteriologic dishes, they grew as tightly packed multicellular tumor spheroids; they were also capable of proliferating in soft agar. Flow cytometric DNA analysis demonstrated a nearly diploid DNA content (DNA index = 0.902). Chromosomal studies of cultured cells showed an isodicentric chromosome 8 in all examined cells, but t(11;22)(q24;q12), a translocation reported previously in Ewing's sarcoma, was not detected. Under normal culture conditions, no morpholagic evidence of neural differentiation was detected. In addition, immunocytochemical studies showed that vimentin was intensely positive, whereas neurofiloment (NF) and neuron-specific enolase (NSE) were weakly positive. Treatment with cyclic AMP (cAMP) induced pronounced morphologic evidence of neural differentiation and strong expression of NF in cultured cells. S-100 protein, glial fibrillary acidic protein (GFAP), desmin, cytokeratin, and epithelial membrane antigen were not detected immunohistochemically in either untreated or cAMP-treated cells, however. These data suggest that this cell line is derived from a highly undifferentiated neural cell with high chromosomal clonality, differentiating into neural features under certain conditions.
INTRODUCTION Ewing's sarcoma (ES} is a rather rare, small round-cell undifferentiated tumor of bone and occasionally of soft tissues that occurs in children and young adults. Several reports have described establishment of permanent cell lines from this tumor [1-8]. These cell lines are useful for elucidating their histogenesis and studying the sensitivity to anticancer drugs or radiation, but the histogenesis of ES remains controversial. Chromosomal abnormalities, i.e., a reciprocal t(ll;22)(q24;q12) and/or hyperdiploidy, have been reported by several groups of investigators [4, 9, 10]. We recently established a new cell line of ES, that possesses unique phenotypic characteristics. We report the cell characteristics, results of chromosomal analysis, immunocytochemical properties, and differentiating characteristics induced by agents known for their ability to induce terminal differentiation. From the Departments of Thoracic Surgery (K. K., O. D., M. H.), Cell Biology (Y. M.), Medicine (T. H.), Pathology (R. T.), and Orthopedic Surgery (Y. A.), The Center for Adult Diseases, Osaka; and 3rd Department of Medicine (S. M.), Kyoto Prefectural University of Medicine. Kyoto, Japan.
Address reprint requests to: Dr. Ken Kodama, Department of Thoracic Surgery, The Center for Adult Diseases, 3 Nakamichi, 1-chome, Higashinari-ku, Osaka 537, Japan. Received October 16, 1989; accepted January 3, 1991.
19 © 1991 Elsevier Science Publishing Co., Inc. 555 Avenue of the Americas, New York, NY 10010
Cancer Genet Cylogenet 57:19-30 (1991) 0165-4608/91/503.50
20
K. Kodama et al.
MATERIALS AND METHODS
Brief Clinical History of the Patient The patient, a 19-year-old Japanese woman, was admitted to our hospital in December 1986 with complaints of weight loss and a tumor in the right buttock. A destructive change in the ilium was demonstrated radiologically. ES was diagnosed on the basis of the results of tumor biopsy. Simultaneously, chest roentgenogram disclosed multiple lung metastases. After intensive chemotherapy, the patient underwent surgery in March 1987 for resection of the right ilium and the fourth and fifth lumbar and first through third sacral vertebral arches and centrum, followed by skin flap transplantation. Although the patient received postoperative chemotherapy intermittently until February 1988, the lung metastases gradually grew larger. Fourteen months after the operation, the patient died of respiratory failure resulting from lung metastases and malignant pleuritis.
Cell Line Establishment Our cell line, designated CADO-ES1, was derived from the malignant pleural effusion of the patient. These cells grew partly in suspension and partly attached to tissueculture flasks (Coming Glass Works, NY) in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) (GIBCO, Grand Island, NY) in a humidified atmosphere with 5% CO 2 at 37°C. The cells were subcultured by trypsinization with 0.25% trypsin (1 : 250; Difco Laboratories, Detroit, MI), and 0.02% EDTA in a calcium- and magnesium-free balanced salt solution. When these cells were placed in 100-ram plastic Petri dishes (Eiken, Tokyo, Japan) that had not been treated for cell attachment (henceforth called bacteriologic plates), they grew as anchorage-independent cell aggregates and formed multicellular tumor spheroids (MTS). Growth in semisolid medium was performed in RPMI 1640 medium supplemented with 10% HI-FCS in a 0.3% agarose-containing top layer over a 0.5% agarosecontaining bottom layer. The cultured cells were routinely tested for mycoplasma contamination and were always negative.
MORPHOLOGIC EXAMINATIONS Morphologic examination of growing cultures was performed using an inverted phasecontrast microscope (Nikon, Tokyo, Japan). Tumor tissues obtained by operation and autopsy and from nude mouse xenografts were fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Electron microscopic examination of MTS and nude mouse xenografts was performed by fixation in a 2% glutaraldehyde-picric acid fixative, postfixation in OsO4, embedding in Epon, and staining with uranyl acetate-lead citrate.
Heterotransplantation Six-week-old female athymic mice (CD1 nu/nu) were purchased from Charles River Company, Atsuki, Japan. A suspension of 5 × 10 8 CADO-ES1 cells in 0.5 ml medium was injected subcutaneously (s.c.) to the backs of the mice. The cell inocula were allowed to grow until palpable tumors were formed. The animals were then sacrificed,
New Ewing's Sarcoma Cell Line
21
A
B Figure 1 Light microscopic appearances of primary site (A), and nude mouse xenograft (B) (H&E, original magnification × 66). the tumors were fixed by the methods described above, and light and electron microscopic examinations were performed.
Flow Cytometric Analysis Cell suspensions were prepared according to a method described previously [11]. Freshly harvested cells were fixed in 70% ice-cold ethanol. After washing with phosphate-buffered saline (PBS), the cell suspensions were processed for 20 min in a PBS
22
K. Kodama et al. solution containing 1 mg/ml RNase (Sigma, St. Louis, MO) at 37°C for 20 minutes. The samples were washed twice by centrifugation (2,000 rpm, 5 minutes) and incubated with freshly prepared 0.01% pepsin (Wako Chemicals, Osaka, Japan) at pH 1.5 at 37°C for 20 minutes. After the samples were stained with 50 ~g/ml propidium iodide (Sigma) and filtered through a 50-gm nylon mesh filter, they were analyzed with an FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA); 10,000 nuclei were read per sample. Data were analyzed using the Cellfit program (Cellfit-DNA software; Becton Dickinson). Human lymphocytes were used as staining controls for the normal diploid (DNA index = 1) DNA content.
Chromosomal Analysis Exponentially growing anchorage-dependent and anchorage-independent cells (MTS) from the seventh, 30th, and 55th passages were treated with colchicine at a concentration of 0.5 ~.g/ml in RPMI 1640 supplemented with 10% HI-FCS for 40 minutes at 37°C. The cells were harvested by trypsinization. After hypotonic treatment in 0.075 M KC1, the cells were fixed in Carnoy's solution at O°C before spreading on slides. G-banding of the chromosomes was obtained by trypsinization. Chromosome identification and karyotype designation were made according to International System for Human Cytogenetic Nomenclature [12].
Differentiation Experiment and Immunoperoxidase Staining CADO-ES1 cells were tested for their response to exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma) and cAMP (Sigma), which have the ability to induce terminal differentiation in other cell lines. TPA was used at concentrations of 25, 50, and 100 nM; cAMP was used at concentrations of 0.25, 0.625, and 2.5 mM [13, 14]. Untreated control cells and differentiated cells were grown in plastic flasks or in chamber slides. Throughout the experiments, the morphologic responses of the cells to the differentiating agents were documented by phase-contrast microscopy of viable, unfixed cells. After 6 days, the cells grown in plastic flasks were harvested, and an aliquot of the cells was placed on a slide glass, dried, and fixed in 10% neutral buffered formalin for 30 minutes. The cells grown in chamber slides were fixed directly in 10% neutral buffered formalin. Immunocytochemical observations were made by the avidin-biotin peroxidase complex (ABC) method. The monoclanal antibodies used were specific for neurofilament (NF), vimentin, glial fibrillary acidic protein (GFAP), all from Lipshow Immunon, Detroit, MI, and desmin and epithelial membrane antigen (EMA) (Dakopatts, Denmark). The polyclonal antibodies used were directed at S-100 protein (Lipshow), neuron-specific enolase (NSE) (Biogenex Laboratories, Dublin, CA) and pankeratin (Dako Corporation, Santa Barbara, CA). Antigen-antibody-peroxidase complex formation was detected with freshly prepared 3,3'-diaminobenzidine and hydrogen peroxide. The cells were counterstained with hematoxylin, washed in running water for 5 minutes, dehydrated, cleared, and mounted in synthetic medium. Control sections of the same samples were processed simultaneously. RESULTS
Light-Microscopic Findings The primary tumor of the ilium was composed of small, closely packed cells with scanty cytoplasm and indistinct cellular border (Fig. 1A). The tumor cells formed solid sheets, separated by thin vascular stroma into irregular nests. No rosettelike
New Ewing's Sarcoma Cell Line
23
Figure 2 Electronmicrograph of MTS after fourth passage shows small cells with cytoplasmic pools of glycogen and complex cytoplasmic processes.
structures were detected. The cells had small oval nuclei. The nuclei were vesicular and small round or oval, with distinct nuclear membranes. Some tumor cells showed prominent nucleoli. Numerous mitotic figures were observed. PAS stain showed positive granules in the tumor cytoplasm, which were eliminated by a diastase digestion procedure. The histologic appearance of the xenotransplanted tumor in nude mice closely resembled that of the primary tumor [Fig. 1B).
Electron Microscopic Findings Ultrastructural studies were performed on MTS after the fourth passage and on the xenotransplanted tumor. Ceils from MTS showed rounded nuclei with unmarginated chromatin and small nucleoli. The cytoplasm contained abundant lysosomes and mitochondria and short strands of rough endoplasmic reticulum. Many cells had cytoplasmic glycogen deposits. No intercellular junctions were noted, and cell-cell attachments consisted of simple apposition. Complex cytoplasmic processes were present (Fig. 2), but neurosecretory granules were not detected. The xenotransplanted tumor cells were very similar in appearance to MTS, but glycogen deposits and ribosomes were more prominent in tumor cells than in MTS. Inversely, lysosomes were prominent in MTS.
Tissue Culture CADO-ES1 showed both anchorage-dependent and anchorage-independent growth patterns (Fig. 3A and B). The cultures reached confluency quickly and were passed once a week. When placed in tissue culture flasks, these cells were small, plump, and
24
K. Kodama et al. round (Fig. 3A). When placed in bacteriologic plates, they grew in the form of very compact MTS, which were also passed every week (Fig. 3B). These cells were also capable of proliferating in soft agar.
Heterotransplantation Into Nude Mice Subcutaneous heterotransplantation of 5 × 106 cells from the tenth passage into five nude mice led to formation of palpable tumors in all animals within 10 days. The tumor doubling time was 3.8 days. No metastases to the lung, liver, lymph nodes, or brain were detected, but when the same number of cells was injected into the peritoneal cavity, neither abdominal tumors nor malignant ascites developed.
Flow Cytometric Analysis Figure 4 shows DNA distribution histograms obtained from the samples resolving the normal lymphocyte and tumor cell mixture. The coefficient of variation of G0/G1 was 4.2% for normal lymphocytes and 4.3% for the tumor cells. The tumor cells had nearly diploid DNA content as compared with normal lymphocytes, and the DNA index was 0.902. The percentage of cycling (S + G2/M cells was 39.5%.
Chromosomal Analysis Fifty mitotic cells from each of the seventh 30th, and 55th passages were photographed and karyotyped. The modal chromosome number was 47. An isodicentric chromosome 8, idic(8)(p11.2), was observed in every cell analyzed. Thus, the karyotype was 47,XX, + idic(8)(p11.2). (Fig. 5). The reciprocal t(11;22)(q24;q12) reported to be specific for Ewing's sarcoma was not found.
Differentiation and Immunohistochemical Findings Treatment with cAMP induced pronounced morphologic changes in cultured cells. The cells developed elongated processes and were interconnected by numerous neuritic processes (Fig. 6). The degree of morphologic differentiation toward neural cells was more prominent in treatment with cAMP than with TPA. The results of immunoreactivity in each group treated with or without the differentiating agents are shown in Table 1. Cultured cells in all three groups strongly expressed vimentin. The cytoplasms of all cells stained intensely and quite uniformly; the cytoplasms of some untreated cells stained positively for NF (Fig. 7A) and NSE. The treated cells eventually expressed NF strongly, particularly after treatment with cAMP (Fig. 7B), but the reactivity for NSE was similarly intense in untreated and treated groups. On the other hand, no cells stained positively for S-100 protein, GFAP, desmin, pankeratin, or EMA. DISCUSSION ES is a highly undifferentiated tumor with no specific morphologic characteristics other than glycogen deposits. Because of the lack of useful lineage markers, its histogenesis remains controversial. Differential diagnosis of poorly differentiated small cell tumors in children and young adults, which include ES, embryonal rhabdomyosarcoma, neuroblastoma, peripheral neuroectodermal tumors, small cell osteogenic sarcoma and lymphoma, is often difficult to make by light microscopy alone [15]. We established a new cell line of ES, designated CADO-ES1, and described its characteristics. Based on the results of both light-microscopic studies of the primary
25
New Ewing's Sarcoma Cell Line
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tumor and ultrastructural studies of MTS and nude mouse xenografts, we confirmed the diagnosis of ES for this cell line. Karyotype analyses of short-term cultures and established cell lines of ES have been made [3, 4, 9, 10]. As a result, Ewing cells are characterized by a specific t{ll;22}(q24;q12). The percentage of such reciprocal translocations in each cell line ranges between 21 and 100% [3, 10]. According to a recent review of chromosomal analyses of 85 unrelated cases of ES reported by Turc-Carel et al. [16], the standard
26 22a~
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Figure 4 DNA histogram. Arrow shows the G0/G1 peak of ES tumor cells after 50th passage. Arrowhead shows diploid peak of normal lymphocytes.
Figure 5 A karyotype of CADO-ES1 tumor ceils with 47 chromosomes. All cells examined showed idic(8)(pll.2) (arrow).
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27
New Ewing's Sarcoma Cell Line
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Figure 6 CADO-ES1 cells treated with 2.5 mM cAMP. Phase-dense cell bodies are interconnected by numerous neuritic processes (original magnification x 100).
t(11;22)(q24;ql 2) proved to be a remarkably consistent event, present in 83% of cases. The same translocation is also observed in neuroepithelioma [17]. These data suggest that Ewing cells share karyotypic features with derivatives of the neuroectoderm, possibly indicating a related histogenesis, but our ES line CADO-ES1 had no such translocation. Turc-Carel et al. [3, 10] also reported the absence of this translocation in an established ES line, IARC-ES3. Unexpectedly, these two cell lines show the same growth pattern, characterized by formation of tightly packed floating clusters [5]. Why only these two lines grow as floating clusters is unclear and cannot be explained in terms of c h r o m o s o m a l abnormalities. The modal cell karyotype of the IARC-ES3 line is 48,XY, - 7, - 9, - 15, + 8, + der(1), + der(7), + der(9), + der(12) [10], whereas that of CADO-ES1 is 47,XX, + idic(8)(p11.2). Turc-Carel et al. [16] reported that only six (8%) of the 85 ES cases lacked cytologic e v i d e n c e of rearrangements i n v o l v i n g 11q24 and/or 22q12. Acco rd i n g to the review of Mugneret et al. [18] secondary ch r o m o so m al changes in addition to t(11;22)(q24;q12), trisomy 8 was consistently observed in half of the 43 cases of ES selected for analysis of numerical changes. Although intensive chemother-
Table 1
Cytochemical results
Working dilution
Untreated control ceils +TPA 100 nM +cAMP 2.5mM
NF
NSE
S-100
GFAP
Vimentin
Desmin
Pankeratin
EMA
1:100
1:1
1:400
1:400
1:400
1:100
1:1,000
1:100
+
+
-
-
+++
-
-
-
++ +++
+ +
-
-
++ + +++
-
-
-
Abbreviations: TPA, 12-O-tetradecanoylphorbol-13-acetate; NF, neurofilaments; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein; EMA, epithelial membrane antigen.
Strongly positive { + + + }, positive / + + ), weakly positive {+ ), and negative {- }.
28
K. Kodama et al.
4
A
B
Figure 7 Immunocytochemical staining of CADO-ES1 cells with neurofilaments (NF) (original magnification × 132]. (A) Some untreated cells show expression of NF (arrowhead). (B) Cells treated with 2.5 mM cAMP show strong expression of NF in the cytoplasm.
apy had been performed before the tissue culture materials were obtained, a unique c h r o m o s o m a l abnormality, idic(8)(p11.2), and a nearly d i p l o i d (hypodiploid) DNA pattern were noted in CADO-ES1. The idic(8) was consistently found in all mitotic cells at various periods of passage. Trisomy 8 is a frequent n o n r a n d o m secondary numerical change in hematologic malignancies [19, 20]. There have been no previous
New Ewing's Sarcoma Cell Line
29
reports regarding ES cell lines which, like CADO-ES1, have no chromosomal changes except a partial tetrasomy 8. On the other hand, trisomy 8 has been reported as a rare constitutional abnormality so far found only among liveborn trisomics in humans. An increased parental age has been reported to be of possible etiologic significance in the origin of trisomy 8 [21], and this extra autosome has been observed with or without mosaicism in various anomalies such as mental retardation and skeletal defects. Our patient had a normal birth and normal physical development, and no obvious malformations except slight mental retardation. Her father was 29 and mother was 26 years old at the time of her birth. Chromosomal studies on somatic cells had not been performed during her lifetime. Constitutional partial tetrasomy of chromosome 8 without serious physical or mental abnormalities has not been reported. The intermediate filament (IF) network is heterogeneous with regard to its constitutive proteins and can be biochemically and immunologically classified into five types: Keratin occurring in epithelial cells, vimentin in fibroblasts and mesenchymatous ceils, desmin in muscle cells, NF in neural cells, and GFAP in astrocytes. Because expression of IF proteins is retained after malignant transformations, immunotyping of IF has proven a valuable tool for diagnosis of human malignant tumors [5]. NSE, on the other hand, is now considered nonspecific; however, it is still a useful marker for neurons and shows positivity in neuroblastoma cells [22]. S-100 protein is also nonspecific and widely distributed in the nervous system and certain other tissues. EMA is used in diagnostic pathology for epithelial cell identification and characterization. Immunocytochemical studies on our cell line demonstrated that vimentin was strongly positive, whereas NF and NSE were weakly positive, but S-100 protein, GFAP, desmin, keratin, and EMA were all negative. After cAMP treatment, pronounced morphologic changes toward neural cells, characterized by development of elongated processes with varicosities, were detected in CADO-ES1. In direct proportion to the morphologic differentiation, NF became strongly expressed, but no significant differences were recognized between untreated and cAMP- or TPA-treated cells with regard to the reactivity with 7 other antibodies. The similarity of morphologic differentiation of ES cell lines induced by treatment with cAMP or TPA had been reported by Cavazzana et al. [13]. Dellagi et al. [5] reported that after TPA exposure undifferentiated Ewing cells may acquire an IF phenotype related to that of epithelial cells. Neither keratin nor EMA expression was detected in CADO-ES1, however, even though the cells had been treated with the same concentration of TPA reported by Dellagi et al. [5]. These findings suggest that CADO-ES1 represents a highly undifferentiated neural tumor which can, under certain circumstances in vitro, acquire a phenotype related to that of neural cells. REFERENCES
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6. Cavazzana AO, Miser JS, Jefferson J, Triche TJ (1987): Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 127:507-518. 7. van Valen F, Jurgen H, Winkelmann W, Keck E (1987): B-Adrenergic agonist- and prostaglandin-mediated regulation of cAMP levels in Ewing's sarcoma cells in culture. Biochem Biophys Res Commun 146:665-691. 8. McKeon C, Thiele CJ, Ross RA, Kwan M, Triche TJ, Miser JS, Israel MA (1988): Indistinguishable pattern of protooncogene expression in two distinct but closely related tumors: Ewing's sarcoma and neuroepithelioma. Cancer Res 48:4307-4311. 9. Aurias A, Rimbaut C, Buffe D, Dubousset J, Mazabraud A (1983): Chromosomal translocation in Ewing's sarcoma. N Engl J Med 309:496-497. 10. Turc-Carel C, Philip I, Berger M, Philip T, Lenoir GM {1964): Chromosome study of Ewing's sarcoma (ES) cell lines. Consistency of a reciprocal translocation t(ll;22)(q24:q12). Cancer Genet Cytogenet 12:1-19. 11. Volm M, Mattern J, Sonka J, Vogt-Schaden M, Wayss K (1985): DNA distribution in nonsmall cell lung carcinoma and its relationship to clinical behavior. Cytometry 6:345-356. 12. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harden DG, Klinger HP (eds.); published in collaboration with Cytogenet Cell Genet (Karger, Basel 1985): also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 13. Cavazzana AO, Jefferson J, Triche TJ (1987): Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 127:507-516. 14. Aguanno S, Bouche M, Adamo S, Molinaro M (1990): 12-O-tetradecanoylphorbol-13-acetateinduced differentiation of a human rhabdomyosarcoma cell line. Cancer Res 50:3377-3382. 15. Triche TJ, Askin FB, Kissane JM (1986): Neuroblastoma, Ewing's sarcoma and the differential diagnosis of small-, round-, blue-tumors. In: Finegold M, ed. Pathology of Neoplasia in Childhood and Adolescents, Vol. 18. W. B. Saunders, Philadelphia, pp. 145-195. 16. Turc-Carel C, Aurias A, Mugneret F, Lizard S, Sidaner I, Volk C, Thiery JP, Olschwang S, Philip I, Berger MP, Philip T, Lenoir GM, Mazabraud A (1986): Chromosomes in Ewing's sarcoma. I. An evaluation of 85 cases and remarkable consistency of t(ll;22)(q24;q12}. Cancer Genet Cytogenet 32:229-238. 17. Whang-Peng J, Triche TJ, Knutsen T, Miser J, Douglass EC, Israel MA (1984): Chromosome translocation in peripheral neuroepithelioma. N Engl J Med 311:564-585. 18. Mugneret F, Lizard S, Aurias A, Turc-CareL C (1968): Chromosomes in Ewing's sarcoma. II. Nonrandom additional changes, trisomy 8 and der(16)t(1;16). Cancer Genet Cytogenet 32:239-245. 19. First International Workshop on Chromosomes in Leukemia (1978): Chromosomes in Phpositive chronic granulocytic leukemia. Br J Haematol 39:305. 20. Heim S, Mitelman F (1986): Secondary chromosome aberration in the acute leukemias. Cancer Genet Cytogenet 22:331-338. 21. Caspersson T, Lindsten J, Zech L, Buckton KE, Price WH (1972) Four patients with trisomy 8 identified by the fluorescence and Giemsa banding techniques. J Med Genet 9:1-7. 22. Tsokos M, Linnoila RI, Chandra RS, Triche TJ (1984): Neuron-specific enolase in the diagnosis of neuroblastoma and other small round-cell tumors in children. Hum Patho115:575-584.
