Establishment and Characterization of a Childhood Pre.B Acute Lymphoblastic Leukemia Cell Line, PER-278, with Chromosome Translocations t(1;19) and t(1;9) Ursula R. Kees, Robyn Lukeis, Jette Ford, Michael L. N. Willoughby, and O. Margaret Garson
ABSTRACT: Cell line PER-278 was established from a bone marrow sample of a lO-year-old boy diagnosed with pre-B acute lymphoblastic leukemia IALL). PER-278 cells show the pre-B phenotype, express cytoplasmic Ig, and exhibit two translocations: t(1;19)(q23;p13) and t(1;9)(q23;pt3). Assessment of the immunoglobulin rearrangements confirmed the clonal origin of cell line PER278, and comparison with the patient's leukemic cells showed an identical pattern: loci involved at the breakpoint on chromosome 1 code for the oncogene SKI and for the Fc receptor II and on chromosome 19 for the insulin receptor. The t(1 ;19) may contribute to the malignant transformation in leukemic cells of pre-B phenotype. INTRODUCTION C h r o m o s o m a l a b n o r m a l i t i e s h a v e i n d e p e n d e n t prognostic v a l u e in c h i l d h o o d acute l y m p h o b l a s t i c l e u k e m i a (ALL) [1-4]. Certain c h r o m o s o m a l t r a n s l o c a t i o n s h a v e b e e n l i n k e d to blast cells w i t h defined l y m p h o i d p h e n o t y p e s . A t r a n s l o c a t i o n i n v o l v i n g c h r o m o s o m e s 1 and 19 t(1;19)(q23;p13) is associated w i t h the pre-B p h e n o t y p e of ALL [5-8]. In a s t u d y of 410 patients at St. Jude Hospital, the t(1;19) was p r e s e n t in 5.1% of all patients and was the m o s t c o m m o n t r a n s l o c a t i o n [9]. We d e s c r i b e the e s t a b l i s h m e n t of a n e w pre-B ALL cell line PER-278 that c o n t a i n s a t(1;19) and an a d d i t i o n a l t(1;9), w i t h the b r e a k p o i n t s on c h r o m o s o m e 1 i d e n t i c a l at q23. MATERIALS A N D METHODS Clinical Data A 10-year-old w h i t e boy p r e s e n t e d in F e b r u a r y 1987 w i t h a 2 - w e e k history of aches and p a i n s in the knees and ankles. E x a m i n a t i o n s h o w e d pallor and o c c a s i o n a l bruises but no h e p a t o s p l e n o m e g a l y or l y m p h a d e n o p a t h y . H e m a t o l o g i c a l studies s h o w e d a From the Leukaemia Research Laboratory, Clinical Immunology Research Unit and Department of Haematology/Oncology, Princess Margaret Hospital, (U. R. K., J. F., M. L. N. W.), Perth, Western Australia and Department of Cytogenetics, St. Vincent's Hospital, Fitzroy, Victoria (R. L., O. M. G.) Australia. Address reprint requests to: Ursula R. Kees, Ph.D., Leukaemia Research Laboratory, Clinical Immunology Research Unit, Princess Margaret Hospital, GPO Box D184, Perth, Western Australia 6001. Received July 5, 1989; accepted September 18, 1989.
201 (('~1990 Elsevier Science Publishing (]o., Inc. 655 Avemle of the Americas, New York, NY 10010
Cancer Genet Cytogenet 46:201 208 (1990) ¢)165-4608/90/$03.50
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u.R. Kees et al. hemoglobin level of 71 g/L, and a white blood cell count of 9.3 × 109/L with 61% lymphoblasts, 13% neutrophils, and 22% lymphocytes. The platelet count was reduced at 4.2 × 109 L. A bone marrow (BM) examination confirmed ALL with 91% L1 cells according to the French-American-British (FAB) classification. Immunofluorescence with a panel of monoclonal antibodies (MoAbs) showed that the blast cells reacted with several antibodies specific for pre-B cells but did not stain with Tcell-specific antibodies (described later). The patient was treated with a protocol for patients with intermediate-risk ALL and has been in continuous remission, on maintenance therapy, for more than 2 years.
Establishment of Cell Line PER-278 Bone marrow cells obtained at the time of first diagnosis were collected in preservative-free heparin and separated on Ficoll-Hypaque (Pharmacia Fine Chemicals AB, Uppsala, Sweden). The culture conditions used to establish permanent cell lines were described previously [10]. The cells from this patient proliferated in medium supplemented with fetal calf serum and have been in continuous culture for >13 months. The cells grow as single cells with a doubling time of 62-68 hours. Attempts to clone PER-278 cells have not yet been successful. An agar culture technique repeatedly showed that the cell line is free of mycoplasma [11]. Epstein-Barr virus (EBV) nuclear antigens were detected according to the method of Reedman and Klein [121. Characterization of Cells
Morphology and cytochemical staining. Leukemic cells from the patient and PER278 cells were examined on May-Grtinwald-Giemsa-stained smears. Periodic acidSchiff (PAS), peroxidase, non-specific esterase, and acid phosphatase stains were performed by standard methods. The enzyme terminal deoxynucleotidyl transferase (TdT) was detected by indirect immunofluorescence using a rabbit anti-calf-TdT antibody and a fluorescein-isothiocyanate-coupled goat anti-rabbit IgG (Fab')2 (both reagents from Supertechs, Bethesda, MD).
Electron microscopy. PER-278 cells were fixed in suspension in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), pelleted and gelled in bovine serum albumin, dehydrated in ethanol, and embedded in epoxy resin. Sections were cut on a LKBultratome (Bromma, Sweden), stained with uranylacetate and lead citrate, and viewed with a Philips EM410 electron microscope (Eindhoven, The Netherlands).
Immunofluorescence studies. The MoAbs RFT-2, RFAL-1, and RFDR-1 were obtained from Dr. G. Janossy (The Royal Free Hospital, London, UK); FMC-56 was a gift from Dr. H. Zola (Flinders Medical Centre, Bedford Park, South Australia); HD-237 and HD-239 were provided by Dr. G. Moldenhauer (German Cancer Research Centre, Heidelberg, FRG); B-1 was purchased from Coulter Electronics, Brookvale, Australia, and the OKM-1 hybridoma cell line was obtained from the American Type Culture Collection, Rockville, MD. Immunofluorescence analysis Was performed as described previously [10]. Cells containing cytoplasmic Ig were assayed with a MoAb anti-IgM from Coulter Electronics.
Southern blot hybridization. DNA was isolated from PER-278 cells, leukemic cells from the patient, and normal peripheral blood mononuclear cells by standard phenol : chloroform extraction and ethanol precipitation. The conditions for enzyme
203
ALL Cell Line PER-278
¸
~. Figure I
Typical thin-section appearance of PER-278 ceils, including one in mitosis. Electronmicrograph × 5,400, reduced by 8"/o.
digestion and filter hybridization were described previously [131. The IgJi I probe consisted of a 2.4-kilobase (kb) Sau 3A fragment, and the CK probe was a 2.5-kb genomic EcoRI probe. To detect ~, gene rearrangements a 1.2-kb genomic Barn HI EcoRI fragment containing the C)~ 2 gene was used. (All three probes were provided by Dr. P. Leder, Harvard Medical School, Boston) [14-16[. A eDNA clone of the T-cell receptor (TCR)/3 chain was a gift from Dr. Tak Mak (University of Toronto, Toronto) [17J.
Cytogenetic analysis. Cells were transported in m e d i u m from Perth to Melbourne, and 10-ml aliquots were placed in Falcon flasks. These were incubated at least 17 hours. Several methods of harvesting cells were attempted; the most successful was fluorodeoxyuridine synchronization [18]. Trypsin G-banding was used to determine the karyotype.
RESULTS Cell line PER-278 was established from the patient's BM cells obtained at first examination (prior to therapy). Electronmicrographs (Fig. 1) show the salient ultrastructural features of the cultured cells to be in keeping with a l y m p h o b l a s t i c leukemic line of pre-B origin• They are rounded, with occasional short surface projections, and measure between 7 and 11 /~m in diameter. Relatively large blastic nuclei are indented, or more d e e p l y cleaved, and contain dispersed chromatin and one or two nucleoli
204
u . R . Kees et al. Table I
Characterization of PER-278 cells and patient's leukemic (:ells from BM
Analysis
Patient's {:ells
PER-278
+
+
C y t o c h e m i c a l stains PAS
Acid phosphatase Peroxidase N o n s p e c i f i c esterase Intracellular markers TdT clgM
+
+
NI}
+
hnmunofluorescence MoAb RH'-2 FMC-56 RFAL-1
CD" 7
H
9
94
92
5:]
11-33
48
B-1
10 11 19 20
HD-239 RFDR-1
22 -
6
53-.q6 12-19 3-14
88
51 96
OKM-1 HD-237
95 {:
Abbreviations: BM, bone marrow: PAS. periodic acid-Schiff; TdT, terminal {teoxynucleotidyl transferase; MoAb. monoclonal antibody; ND, not determined. ~' CD, cluster of differentiation, as defined at the Workshop on Leukocyte Differm~tialion Antigens in Boston [191. ~' l]ackground staining with unrelated MoAb was 2% which was subtracted. { Percentage of cells staining. Values for PER-278 represent the range deter mined in four experimenls performed in a 12 month period.
often adjacent to the nuclear envelope. Cytoplasmic details include abundant polysoines, variable short profiles of rough endoplasmic reticulum, and a scattering of lipidic droplets. Table 1 summarizes the characteristics of PER-278 cells in comparison with the patient's fresh leukemic cells from the diagnostic BM. Both cell populations stain with PAS and are positive for TdT, and PER-278 cells stain for cytoplasmic IgM. Surface marker analysis showed the same pattern for both cell populations: staining was obtained with MoAbs belonging to CD9, CD10, CD19, and RFDR-1, which recognizes polymorphic HLA-DR determinants. The cells did not react with CD7 and CDI 1 antibodies. The fresh leukemic cells did not stain with MoAbs B-1 {CD20) and HD239 (CD22), whereas a few PER-278 cells were positive with these MoAbs. Thus, both the cell line PER-278 and the patient's fresh leukemic cells express antigens that are present on pre-B ALL cells. Cell line PER-278 was negative for the EBV nuclear antigen, To characterize PER-278 cells further, w e performed Southern blot analysis to investigate the IgJH and TCR fi-chain configuration. The analysis showed that PER278 cells have one IgJn allele deleted and the other one is rearranged (Fig. 2) whereas the genes for TCR-fi, CK and CX are in germline configuration (not shown). With all four probes, the patient's fresh leukemic cells showed a pattern (Fig. 2) identical to the one detected for PER-278 cells. Thus the leukemic cells are monoclonal with respect to IgJH gene rearrangement. The cytogenetic analysis was performed on PER-278 cells that had been in culture
205
ALL Cell Line PER-278
A
B
C
16•~
-4[
Figure 2 Southern blot analysis of DNA samples from normal peripheral blood cells (A), patients's bone marrow cells (B), and PER-278 cells (C). DNA was analyzed as described in the Materials and Methods section. The samples were digested with restriction enzyme EcoRI and hybridized with the IgJHprobe. Size in kilobases of the germline fragment (dash). Arrows indicate rearrangements. for 19 and 24 weeks, respectively. Fifteen metaphases analyzed showed a single clone with the karyotype 4 6 , X Y , - 9 , - 19, + der(9)t(1;9)(q23;p13), + der(19)t(1;19)(q23;p13) (Fig. 3). Chromosome studies of the patient's fresh leukemic cells showed an abnormal p s e u d o d i p l o i d karyotype, but the metaphases were not of sufficient quality for Gbanding.
DISCUSSION Cell line PER-278 was established from a patient whose tumor cells can be assigned to stage III of the pre-B cell differentiation as defined by Nadler et al. [20]. In contrast to the fresh leukemic cells, a proportion of PER-278 cells expressed markers present
206
u . R . Kees et al.
1
2
3
5
4
/
.4
6
7
13
/
8
14
9
15
10
16
11
12
17
18
~O
19
20
21
22
Figure 3 Karyotype of PER-278 cells showing 46,XY, - 9, 19, + der(9)t(1;9)(q23;p13), +der(19)t(1;19)(q23;p13).
on stage IV cells, thus representing a marginally more differentiated cell. The configuration of Ig heavy- and light-chain genes was identical in the patient's leukemic cells and cell line PER-278, showing rearrangement and deletion of heavy-chain genes and all light-chain genes in germlike configuration. These findings are in agreement with p u b l i s h e d results on cells of p h e n o t y p i c stages Ill and IV [20, 21]. The t(1;19) present in the cell line was the most c o m m o n translocation occurring in a large series of ALL cases with c o m p l e t e l y banded chromosomes [9], and these patients had l e u k e m i a of pre-B type expressing clg. Cell line PER-278 expressed cIg whereas another cell line with the same translocation, RCH-ACV [22], did not express cIg. In a review of c h r o m o s o m e s in acute l y m p h o c y t i c leukemia, 10 of 13 patients with the t(1;19)(q23;p13) showed additional, mostly structural, abnormalities [23]. Both cell lines characterized with this translocation showed additional abnormalities: cell line RCH-ACV has trisomy 8 [22] and PER-278 cells have a translocation t(1;9). The translocation has been described occurring as either a balanced translocat i o n - - t ( 1 ; 1 9 ) ( q 2 3 ; p 1 3 ) - - o r in an unbalanced form, - 19, + der(19)t(1;19)(q23;p13). A study by Shikano et al [24] suggests that there may be no differences in the clinical and i m m u n o l o g i c a l characteristics between these two varieties, but that patients with either translocation have a poor prognosis [24]. The same conclusion was reached by Carroll et al. [6], whereas Michael et al. [5] c o n c l u d e d that this translocation may not
ALL Cell Line PER-278
207
always be associated with a poor prognosis. The patient from whom we established cell line PER-278 has been treated with a protocol for intermediate-risk patients that includes delayed intensification. He has been in c o n t i n u o u s remission for 2 years after diagnosis. I n v o l v e m e n t of chromosome 1 has been found in a variety of neoplasms [25], whereas the t(1;19) has been described in mainly lymphoid malignancies, both leukemia and lymphoma. Both breakpoints on chromosome 1 present in cell line PER-278 occurred at lq23, which has been reported to be the locus for the cellular oncogene SKI, mapped to region l q 2 2 - q 2 4 [26], and for the gene coding for the h u m a n Fc receptor IIa, assigned to 1q23-q24 [27]. Abnormalities of chromosome 19, both numerical and structural, are found in many different hematological malignancies, i n c l u d i n g myeloproliferative and myelodysplastic disorders as well as leukemia [28]. The hum a n i n s u l i n receptor gene which has structural homology to the epidermal growth factor (erb B) has been mapped to 19p13.2-p13.3 [29], which is the breakpoint involved in the t{1;19). Molecular analysis of breakpoint t(1;19) in cell line PER-278 is in progress. Abnormalities occurring at 9p are also frequently described in lymphoid malignancies and appear to be particularly associated with a lymphomatous type of ALL, either B or T cell. The ~- and/31-interferon genes have been mapped to 9p2122, and a recent study shows homozygous deletion of these genes in neoplastic cell lines c o n t a i n i n g partial deletion of 9p [30]. This cell line may also yield useful information regarding the i n v o l v e m e n t of these genes in translocation t(1;9).
This is publication No. 320 from the Clinical Immunology Research Unit. This work was supported by the Children's Leukaemia Research Fund of the Princess Margaret Children's Medical Research Foundation and a research grant from St. Vincent's Hospital. The authors are grateful to Drs. H. Zola, G. Janossy, and G. Moldenhauer for generous gifts of monoclonal antibodies. They thank Dr. J. Armstrong (Royal Perth Hospital, Australia) for electronmicroscopy analysis of the cell line, Dr. D. Moss (Queensland Institute of Medical Research, Brisbane, Australia) for Epstein-Barr virus tests, Pamela Ranford and Vaille Dawson for skilled technical assistance, and Sally Kelly for preparing the manuscript.
REFERENCES 1. Third International Workshop on Chromosomes in Leukemia 1980 (1981): Clinical significance of chromosomal abnormalities in acute lymphoblastic leukemia. Cancer Genet Cytogenet 4:111-142. 2. Third International Workshop on Chromosomes in Leukemia (1986): Chromosomal abnormalities identify high-risk and low-risk patients with acute lymphoblastic leukemia. Blood 68:320-322. 3. Williams DL, Harber J, Murphy SB, Look AT, Kalwinsky DK, Rivera G, Melvin SL, Stass S, Dahl GV (1986): Chromosomal translocations play a unique role in influencing prognosis in childhood lymphoblastic leukemia. Blood 68:205-212. 4. Michael PM, Garson OM, Ekert H, Tauro G, Rennie GC, Pilkington GR (1988): A prospective study of childhood acute lymphocytic leukemia: Hematologic, immunologic and cytogenetic correlation. Med Pediatr Oncol 14:153-161. 5. Michael PM, Levin MD, Garson OM (1984): Translocation 1;19--a new cytogenetic abnormality in acute lymphocytic leukemia. Cancer Genet Cytogenet 12:333-341. 6. Carroll AJ, Crist WM, Parmley RT, Roper M, Cooper MD, Finley WH (1984): Pre-B cell leukemia associated with chromosome translocation 1;19. Blood 63:721-724. 7. Williams DL, Look AT, Melvin SL, Roberson PK, Daht G, Flake T, Stass S (1984): New chromosomal translocations correlate with specifc immunophenotypes of childhood acute lymphoblastic leukemia. Cell 36:101-109. 8. Michael PM, Callen DF, Garson OM (1985): A review of the t(1;19) breakpoints in acute lymphocytic leukemia. Cancer Genet Cytogenet 17:79-80.
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9. Look AT (19881: The cytogenetics of childhood leukemia: Clinical and biologic implications. Pediatr Clin North Am 35:723-741. 10. Kees UR, Ford J, Price PJ, Meyer BF, Herrmann RP (1987): PER-117: A new h u m a n ALL cell line with an immature thymic phenotype. Leuk Res 11:489-498. 11. Schneider EL, Stanbridge EJ (1975): Comparison of methods for the detection of mycoplasma contamination of cell cultures: A review. In Vitro 11:20-34. 12. Reedman BM, Klein G (1973): Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer 11:499-520. 13. Kees UR (1988): Phorbol ester induces expression and function of interleukin-2 receptors on a h u m a n leukemic cell line with a T-cell precursor phenotype. Blood 72:1524-1529. 14. Ravetech JV, Siebenlist U, Korsmeyer S, Waldmann T, Leder P (1981): Structure of the h u m a n immunoglobulin ~, locus: Characterization of embryonic and rearranged J and D genes. Cell 27:583-591. 15. Hieter PA, Maizel JV, Leder P (1982): Evolution of h u m a n immunoglobulin kappa J region genes. J Biol Chem 257:1516-1522. 16. Hieter PA, Hollis GF, Korsmeyer SJ, Waldmann TA, Leder P (1981): Clustered arrangement of immunoglobulin X constant region genes in man. Nature 294:536-540. 17. Yanagi Y, Yoshikai Y, Leggett K, Clark SP, Aleksander I, Mak TW (1984): A h u m a n T cellspecific eDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nature 308:145-149. 18. Webber LM, Garson OM (1983): Fluordeoxyuridine synchronization of bone marrow cultures. Cancer Genet Cytogenet 8:123-132. 19. Reinherz EL, Haynes BF, Nadler LM, Bernstein ID (1986): Leukocyte Typing II, Vols. 1-3. Springer-Verlag, New York. 20. Nadler LM, Korsmeyer SJ, Anderson KC, Boyd AW, Slaughenhoupt B, Park E, Jensen J, Coral F, Mayer RJ, Sallon SE, Ritz J, Schlossman SF (1984): B cell origin of non-T cell acute lymphoblastic leukemia. I Clin Invest 74:332-340. 21. Korsmeyer SJ, Arnold A, Bakhshi A, Ravetch JV, Siebenlist U, Hieter PA, Sharrow SO, Le Bien TW, Kersey JH, Poplack DG, Leder P (1983): Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins. J Clin Invest 71:301-313. 22. Jack I, Seshadri R, Garson M, Michael P, Callen D, Zola H, Morley A (1986): RCH-ACV: A lymphoblastic leukemia cell line with chromosome translocation 1;19 and trisomy 8. Cancer Genet Cytogenet 19:261-269. 23. Hossfeld DK {1987): Chromosomes in acute lymphocytic leukemia. Cancer Genet Cytogenet 26:59-64. 24. Shikano T, Kaneko Y, Takazawa M, Ueno N, Ohkawa M, Fujimoto T (1986): Balanced and unbalanced 1;19 translocation-associated acute lymphoblastic leukemias. Cancer 58:2239-2243. 25. Mitelman F, Heim S (1988J: Consistent involvement of only 71 of the 329 chromosomal bands of the h u m a n genome in primary neoplasia-associated rearrangements. Cancer Res 48:7115-7119. 26. Chaganti RSK, Balazs I, Jhanwar SC, Murty VVVS, Koduru PRK, Grzeschik KH, Stavnezer E (1986): The cellular homologue of the transforming gene of SKV avian retrovirus maps to h u m a n chromosome region 1q22-q24. Cytogenet Cell Genet 43:181-186. 27. Sammartino L, Webber LM, Hogarth PM, McKenzie IFC, Garson OM (1988): Assignment of the gene coding for h u m a n FcRII (CD32) to bands q23q24 on chromosome 1. Immunogenetics 28:380-381. 28. Mitelman F (1985): Catalog of Chromosome Aberrations in Cancer, 2nd Ed. Alan R Liss, New York. 29. Yang-Feng TL, Francke U, Ullrich A (1985): Gene for h u m a n insulin receptor: Localization to site on chromosome 19 involved in pre-B-cell leukemia. Science 228:728-731. 30. Diaz MO, Ziemin S, Le Beau MM, Pitha P, Smith SD, Chilcote RR, Rowley JD (1988): Homozygous deletion of the a and/31 interferon genes in h u m a n leukemia and derived cell lines. Proc Natl Acad Sci USA 85:5259-5263.
ABSTRACT: Cell line PER-278 was established from a bone marrow sample of a lO-year-old boy diagnosed with pre-B acute lymphoblastic leukemia IALL). PER-278 cells show the pre-B phenotype, express cytoplasmic Ig, and exhibit two translocations: t(1;19)(q23;p13) and t(1;9)(q23;pt3). Assessment of the immunoglobulin rearrangements confirmed the clonal origin of cell line PER278, and comparison with the patient's leukemic cells showed an identical pattern: loci involved at the breakpoint on chromosome 1 code for the oncogene SKI and for the Fc receptor II and on chromosome 19 for the insulin receptor. The t(1 ;19) may contribute to the malignant transformation in leukemic cells of pre-B phenotype. INTRODUCTION C h r o m o s o m a l a b n o r m a l i t i e s h a v e i n d e p e n d e n t prognostic v a l u e in c h i l d h o o d acute l y m p h o b l a s t i c l e u k e m i a (ALL) [1-4]. Certain c h r o m o s o m a l t r a n s l o c a t i o n s h a v e b e e n l i n k e d to blast cells w i t h defined l y m p h o i d p h e n o t y p e s . A t r a n s l o c a t i o n i n v o l v i n g c h r o m o s o m e s 1 and 19 t(1;19)(q23;p13) is associated w i t h the pre-B p h e n o t y p e of ALL [5-8]. In a s t u d y of 410 patients at St. Jude Hospital, the t(1;19) was p r e s e n t in 5.1% of all patients and was the m o s t c o m m o n t r a n s l o c a t i o n [9]. We d e s c r i b e the e s t a b l i s h m e n t of a n e w pre-B ALL cell line PER-278 that c o n t a i n s a t(1;19) and an a d d i t i o n a l t(1;9), w i t h the b r e a k p o i n t s on c h r o m o s o m e 1 i d e n t i c a l at q23. MATERIALS A N D METHODS Clinical Data A 10-year-old w h i t e boy p r e s e n t e d in F e b r u a r y 1987 w i t h a 2 - w e e k history of aches and p a i n s in the knees and ankles. E x a m i n a t i o n s h o w e d pallor and o c c a s i o n a l bruises but no h e p a t o s p l e n o m e g a l y or l y m p h a d e n o p a t h y . H e m a t o l o g i c a l studies s h o w e d a From the Leukaemia Research Laboratory, Clinical Immunology Research Unit and Department of Haematology/Oncology, Princess Margaret Hospital, (U. R. K., J. F., M. L. N. W.), Perth, Western Australia and Department of Cytogenetics, St. Vincent's Hospital, Fitzroy, Victoria (R. L., O. M. G.) Australia. Address reprint requests to: Ursula R. Kees, Ph.D., Leukaemia Research Laboratory, Clinical Immunology Research Unit, Princess Margaret Hospital, GPO Box D184, Perth, Western Australia 6001. Received July 5, 1989; accepted September 18, 1989.
201 (('~1990 Elsevier Science Publishing (]o., Inc. 655 Avemle of the Americas, New York, NY 10010
Cancer Genet Cytogenet 46:201 208 (1990) ¢)165-4608/90/$03.50
202
u.R. Kees et al. hemoglobin level of 71 g/L, and a white blood cell count of 9.3 × 109/L with 61% lymphoblasts, 13% neutrophils, and 22% lymphocytes. The platelet count was reduced at 4.2 × 109 L. A bone marrow (BM) examination confirmed ALL with 91% L1 cells according to the French-American-British (FAB) classification. Immunofluorescence with a panel of monoclonal antibodies (MoAbs) showed that the blast cells reacted with several antibodies specific for pre-B cells but did not stain with Tcell-specific antibodies (described later). The patient was treated with a protocol for patients with intermediate-risk ALL and has been in continuous remission, on maintenance therapy, for more than 2 years.
Establishment of Cell Line PER-278 Bone marrow cells obtained at the time of first diagnosis were collected in preservative-free heparin and separated on Ficoll-Hypaque (Pharmacia Fine Chemicals AB, Uppsala, Sweden). The culture conditions used to establish permanent cell lines were described previously [10]. The cells from this patient proliferated in medium supplemented with fetal calf serum and have been in continuous culture for >13 months. The cells grow as single cells with a doubling time of 62-68 hours. Attempts to clone PER-278 cells have not yet been successful. An agar culture technique repeatedly showed that the cell line is free of mycoplasma [11]. Epstein-Barr virus (EBV) nuclear antigens were detected according to the method of Reedman and Klein [121. Characterization of Cells
Morphology and cytochemical staining. Leukemic cells from the patient and PER278 cells were examined on May-Grtinwald-Giemsa-stained smears. Periodic acidSchiff (PAS), peroxidase, non-specific esterase, and acid phosphatase stains were performed by standard methods. The enzyme terminal deoxynucleotidyl transferase (TdT) was detected by indirect immunofluorescence using a rabbit anti-calf-TdT antibody and a fluorescein-isothiocyanate-coupled goat anti-rabbit IgG (Fab')2 (both reagents from Supertechs, Bethesda, MD).
Electron microscopy. PER-278 cells were fixed in suspension in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), pelleted and gelled in bovine serum albumin, dehydrated in ethanol, and embedded in epoxy resin. Sections were cut on a LKBultratome (Bromma, Sweden), stained with uranylacetate and lead citrate, and viewed with a Philips EM410 electron microscope (Eindhoven, The Netherlands).
Immunofluorescence studies. The MoAbs RFT-2, RFAL-1, and RFDR-1 were obtained from Dr. G. Janossy (The Royal Free Hospital, London, UK); FMC-56 was a gift from Dr. H. Zola (Flinders Medical Centre, Bedford Park, South Australia); HD-237 and HD-239 were provided by Dr. G. Moldenhauer (German Cancer Research Centre, Heidelberg, FRG); B-1 was purchased from Coulter Electronics, Brookvale, Australia, and the OKM-1 hybridoma cell line was obtained from the American Type Culture Collection, Rockville, MD. Immunofluorescence analysis Was performed as described previously [10]. Cells containing cytoplasmic Ig were assayed with a MoAb anti-IgM from Coulter Electronics.
Southern blot hybridization. DNA was isolated from PER-278 cells, leukemic cells from the patient, and normal peripheral blood mononuclear cells by standard phenol : chloroform extraction and ethanol precipitation. The conditions for enzyme
203
ALL Cell Line PER-278
¸
~. Figure I
Typical thin-section appearance of PER-278 ceils, including one in mitosis. Electronmicrograph × 5,400, reduced by 8"/o.
digestion and filter hybridization were described previously [131. The IgJi I probe consisted of a 2.4-kilobase (kb) Sau 3A fragment, and the CK probe was a 2.5-kb genomic EcoRI probe. To detect ~, gene rearrangements a 1.2-kb genomic Barn HI EcoRI fragment containing the C)~ 2 gene was used. (All three probes were provided by Dr. P. Leder, Harvard Medical School, Boston) [14-16[. A eDNA clone of the T-cell receptor (TCR)/3 chain was a gift from Dr. Tak Mak (University of Toronto, Toronto) [17J.
Cytogenetic analysis. Cells were transported in m e d i u m from Perth to Melbourne, and 10-ml aliquots were placed in Falcon flasks. These were incubated at least 17 hours. Several methods of harvesting cells were attempted; the most successful was fluorodeoxyuridine synchronization [18]. Trypsin G-banding was used to determine the karyotype.
RESULTS Cell line PER-278 was established from the patient's BM cells obtained at first examination (prior to therapy). Electronmicrographs (Fig. 1) show the salient ultrastructural features of the cultured cells to be in keeping with a l y m p h o b l a s t i c leukemic line of pre-B origin• They are rounded, with occasional short surface projections, and measure between 7 and 11 /~m in diameter. Relatively large blastic nuclei are indented, or more d e e p l y cleaved, and contain dispersed chromatin and one or two nucleoli
204
u . R . Kees et al. Table I
Characterization of PER-278 cells and patient's leukemic (:ells from BM
Analysis
Patient's {:ells
PER-278
+
+
C y t o c h e m i c a l stains PAS
Acid phosphatase Peroxidase N o n s p e c i f i c esterase Intracellular markers TdT clgM
+
+
NI}
+
hnmunofluorescence MoAb RH'-2 FMC-56 RFAL-1
CD" 7
H
9
94
92
5:]
11-33
48
B-1
10 11 19 20
HD-239 RFDR-1
22 -
6
53-.q6 12-19 3-14
88
51 96
OKM-1 HD-237
95 {:
Abbreviations: BM, bone marrow: PAS. periodic acid-Schiff; TdT, terminal {teoxynucleotidyl transferase; MoAb. monoclonal antibody; ND, not determined. ~' CD, cluster of differentiation, as defined at the Workshop on Leukocyte Differm~tialion Antigens in Boston [191. ~' l]ackground staining with unrelated MoAb was 2% which was subtracted. { Percentage of cells staining. Values for PER-278 represent the range deter mined in four experimenls performed in a 12 month period.
often adjacent to the nuclear envelope. Cytoplasmic details include abundant polysoines, variable short profiles of rough endoplasmic reticulum, and a scattering of lipidic droplets. Table 1 summarizes the characteristics of PER-278 cells in comparison with the patient's fresh leukemic cells from the diagnostic BM. Both cell populations stain with PAS and are positive for TdT, and PER-278 cells stain for cytoplasmic IgM. Surface marker analysis showed the same pattern for both cell populations: staining was obtained with MoAbs belonging to CD9, CD10, CD19, and RFDR-1, which recognizes polymorphic HLA-DR determinants. The cells did not react with CD7 and CDI 1 antibodies. The fresh leukemic cells did not stain with MoAbs B-1 {CD20) and HD239 (CD22), whereas a few PER-278 cells were positive with these MoAbs. Thus, both the cell line PER-278 and the patient's fresh leukemic cells express antigens that are present on pre-B ALL cells. Cell line PER-278 was negative for the EBV nuclear antigen, To characterize PER-278 cells further, w e performed Southern blot analysis to investigate the IgJH and TCR fi-chain configuration. The analysis showed that PER278 cells have one IgJn allele deleted and the other one is rearranged (Fig. 2) whereas the genes for TCR-fi, CK and CX are in germline configuration (not shown). With all four probes, the patient's fresh leukemic cells showed a pattern (Fig. 2) identical to the one detected for PER-278 cells. Thus the leukemic cells are monoclonal with respect to IgJH gene rearrangement. The cytogenetic analysis was performed on PER-278 cells that had been in culture
205
ALL Cell Line PER-278
A
B
C
16•~
-4[
Figure 2 Southern blot analysis of DNA samples from normal peripheral blood cells (A), patients's bone marrow cells (B), and PER-278 cells (C). DNA was analyzed as described in the Materials and Methods section. The samples were digested with restriction enzyme EcoRI and hybridized with the IgJHprobe. Size in kilobases of the germline fragment (dash). Arrows indicate rearrangements. for 19 and 24 weeks, respectively. Fifteen metaphases analyzed showed a single clone with the karyotype 4 6 , X Y , - 9 , - 19, + der(9)t(1;9)(q23;p13), + der(19)t(1;19)(q23;p13) (Fig. 3). Chromosome studies of the patient's fresh leukemic cells showed an abnormal p s e u d o d i p l o i d karyotype, but the metaphases were not of sufficient quality for Gbanding.
DISCUSSION Cell line PER-278 was established from a patient whose tumor cells can be assigned to stage III of the pre-B cell differentiation as defined by Nadler et al. [20]. In contrast to the fresh leukemic cells, a proportion of PER-278 cells expressed markers present
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u . R . Kees et al.
1
2
3
5
4
/
.4
6
7
13
/
8
14
9
15
10
16
11
12
17
18
~O
19
20
21
22
Figure 3 Karyotype of PER-278 cells showing 46,XY, - 9, 19, + der(9)t(1;9)(q23;p13), +der(19)t(1;19)(q23;p13).
on stage IV cells, thus representing a marginally more differentiated cell. The configuration of Ig heavy- and light-chain genes was identical in the patient's leukemic cells and cell line PER-278, showing rearrangement and deletion of heavy-chain genes and all light-chain genes in germlike configuration. These findings are in agreement with p u b l i s h e d results on cells of p h e n o t y p i c stages Ill and IV [20, 21]. The t(1;19) present in the cell line was the most c o m m o n translocation occurring in a large series of ALL cases with c o m p l e t e l y banded chromosomes [9], and these patients had l e u k e m i a of pre-B type expressing clg. Cell line PER-278 expressed cIg whereas another cell line with the same translocation, RCH-ACV [22], did not express cIg. In a review of c h r o m o s o m e s in acute l y m p h o c y t i c leukemia, 10 of 13 patients with the t(1;19)(q23;p13) showed additional, mostly structural, abnormalities [23]. Both cell lines characterized with this translocation showed additional abnormalities: cell line RCH-ACV has trisomy 8 [22] and PER-278 cells have a translocation t(1;9). The translocation has been described occurring as either a balanced translocat i o n - - t ( 1 ; 1 9 ) ( q 2 3 ; p 1 3 ) - - o r in an unbalanced form, - 19, + der(19)t(1;19)(q23;p13). A study by Shikano et al [24] suggests that there may be no differences in the clinical and i m m u n o l o g i c a l characteristics between these two varieties, but that patients with either translocation have a poor prognosis [24]. The same conclusion was reached by Carroll et al. [6], whereas Michael et al. [5] c o n c l u d e d that this translocation may not
ALL Cell Line PER-278
207
always be associated with a poor prognosis. The patient from whom we established cell line PER-278 has been treated with a protocol for intermediate-risk patients that includes delayed intensification. He has been in c o n t i n u o u s remission for 2 years after diagnosis. I n v o l v e m e n t of chromosome 1 has been found in a variety of neoplasms [25], whereas the t(1;19) has been described in mainly lymphoid malignancies, both leukemia and lymphoma. Both breakpoints on chromosome 1 present in cell line PER-278 occurred at lq23, which has been reported to be the locus for the cellular oncogene SKI, mapped to region l q 2 2 - q 2 4 [26], and for the gene coding for the h u m a n Fc receptor IIa, assigned to 1q23-q24 [27]. Abnormalities of chromosome 19, both numerical and structural, are found in many different hematological malignancies, i n c l u d i n g myeloproliferative and myelodysplastic disorders as well as leukemia [28]. The hum a n i n s u l i n receptor gene which has structural homology to the epidermal growth factor (erb B) has been mapped to 19p13.2-p13.3 [29], which is the breakpoint involved in the t{1;19). Molecular analysis of breakpoint t(1;19) in cell line PER-278 is in progress. Abnormalities occurring at 9p are also frequently described in lymphoid malignancies and appear to be particularly associated with a lymphomatous type of ALL, either B or T cell. The ~- and/31-interferon genes have been mapped to 9p2122, and a recent study shows homozygous deletion of these genes in neoplastic cell lines c o n t a i n i n g partial deletion of 9p [30]. This cell line may also yield useful information regarding the i n v o l v e m e n t of these genes in translocation t(1;9).
This is publication No. 320 from the Clinical Immunology Research Unit. This work was supported by the Children's Leukaemia Research Fund of the Princess Margaret Children's Medical Research Foundation and a research grant from St. Vincent's Hospital. The authors are grateful to Drs. H. Zola, G. Janossy, and G. Moldenhauer for generous gifts of monoclonal antibodies. They thank Dr. J. Armstrong (Royal Perth Hospital, Australia) for electronmicroscopy analysis of the cell line, Dr. D. Moss (Queensland Institute of Medical Research, Brisbane, Australia) for Epstein-Barr virus tests, Pamela Ranford and Vaille Dawson for skilled technical assistance, and Sally Kelly for preparing the manuscript.
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