[CANCER RESEARCH 48, 2876-2879, May 15, 1988]Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia Cell Lineswithout Classical Breakpoint Cluster Region Rearrangement1Louie Naumovski, Rodman Morgan, Frederick Hecht, Michael P. Link, Bertil E. Glader, and Stephen D. Smith2Department of Pediatrics and Pathology, Stanford University School of Medicine, Stanford, California 94305 [L. N., M. P. L., B. E. G., S. D. S.J; Division ofHematology/Oncology, Children 's Hospital at Stanford, Palo Alto, California 94304 ¡M.P.L., B. E. G., S. D. S.J; and the Genetics Center and Cancer Center, Southwest BiomédicalResearch Institute, Scottsdale, Arizona 85251 [R. M., F. H.]ABSTRACTThe Philadelphia (Ph) chromosome translocation which is classicallyobserved in chronic myeloid leukemia ((.'Ml,) is sporadically found inacute lymphoblastic leukemia (ALL). In CML the translocation breakpoint on chromosome 22 is within the breakpoint cluster region, while inchildhood ALL, no detectable change in breakpoint cluster region isroutinely observed. In order to investigate the nature of this difference,we have established and characterized two cell lines from a child withPh positive ALL. The cell lines have retained the cytochemical stainingpattern, enzyme activity, monoclonal antibody profile, and immunoglob-ulin gene rearrangements of the child's malignant cells. The cell lineshad the same Ph translocation t(9;22) (q34;qll) as the child's malignantcells along with additional chromosome changes. Southern blot analysisshowed that the Ph translocation did not involve the 5.8-kilobase breakpoint cluster region segment characteristically seen in CML. The celllines reported here will be a valuable resource in ascertaining the biological significance of the Ph translocation seen in ALL.INTRODUCTIONCytogenetic studies have shown a characteristic translocationbetween chromosomes 9 and 22 leading to the Ph3 chromosomein greater than 90% of cases of CML (1,2). This translocationoccurs specifically between the c-abl gene on chromosome 9and a region known as ber on chromosome 22 (3). Cells containing this translocation produce a chimeric transcript andexpress a fusion protein with t\rosine kinase activity which ispossibly involved in the pathogenesis of CML (4-6).A Ph translocation is found in approximately 10% of childrenwith ALL and is cytogenetically indistinguishable from thatfound in CML (7). Several cases of Ph positive ALL have beenexamined for the characteristic rearrangement of ber. It hasbeen found that, while there is considerable heterogeneity ofthe chromosomal rearrangements, most chromosomal translocations found in childhood Ph positive ALL do not occur withinber (8, 9).We report here characterization of 2 cell lines derived froma child with Ph positive ALL. The cell lines possess the samecytochemical staining pattern, biochemical enzyme levels,monoclonal antibody profile, and immunoglobulin gene rearrangements as the patient's malignant cells. While the cellline's karyotype demonstrates the Ph (and other chromosomalalterations), neither the cell lines nor the patient's malignantcells have a rearrangement at her. Since the cell lines carry thePh, molecular studies of the genes located at the chromosomalReceived 8/20/87; revised 1/2/88; accepted 2/18/88.The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.'Supported by USPHS Grants CA-34233 and CA-41124 awarded by theNational Cancer Institute, Department of Health and Human Services.1Scholar of the Leukemia Society of America. To whom requests for reprintsshould be addressed, at Children's Hospital at Stanford, 520 Sand Hill Road,Palo Alto, CA 94304.3The abbreviations used are: Ph. Philadelphia, CML, chronic myelogenousleukemia; her. breakpoint cluster region; ALL, acute lymphoblastic leukemia;ADA, adenosine deaminase; NP, nucleoside phosphorylase; TdT, terminal deox-yribonucleotidyl transferase; EBV, Epstein-Barr virus.
translocation breakpoints may be useful in elucidating the nature of Ph positive ALL.MATERIALS AND METHODSCase Report. An 8-yr-old boy presented to the Children's Hospitalat Stanford in August 1983 with bone pain, fever, and anemia. Thebone marrow aspirate was hypercellular with 97% lymphoblasts, andthe cytochemical stains and immunophenotyping confirmed the diagnosis of acute lymphoblastic leukemia. A complete remission wasinduced with chemotherapy, but the child subsequently relapsed anddied with progressive leukemia 26 mo after diagnosis.Source of Malignant Cells. Malignant cells were collected from thefirst and second bone marrow relapse and separated into aliquots forcell culture experiments, immunophenotyping, enzyme studies, kary-otyping, and immunogenotyping. The protocol procedures were approved by the Medical Committee for the Use of Human Subjects inResearch at Stanford University, and informed consent was obtainedfrom the patient's parents.Establishment and Maintenance of the Cell Lines. The techniques forculturing lymphoblasts were a modification of our previously reportedmethods (10). Briefly, lymphoblasts were mixed with agar (0.3%),plated onto Petri dishes (which contained a feeder layer), and culturedat 37°Cin an incubator gassed with 5% O2, 6% CO2, and 89% N2.Characterization of Cell Antigens. Cell surface antigens were identified by the binding of monoclonal antibody as detected by indirectimmunofluorescence by fluorescence-activated cell sorting (11). Thepresence of cytoplasmic n was determined by a standard techniqueusing fluorescien-conjugated goat anti-human ¿iantibody (SouthernBiotech, Birmingham, AL) (12).Enzyme Analysis of Malignant Cells from Patient and Cell Lines.ADA, NP, and TdT activities were measured according to establishedmethods (13).Cytogenetic Analysis. The patient's bone marrow cells were studiedclini most iinally using techniques previously described (10). The karyotype of each cell line was analyzed after 7 mo in culture.Molecular Genetic Studies. High-molecular-weight DN A was isolatedfrom each cell line and bone marrow from the second relapse andtreated with restriction enzymes, and Southern analysis was done (14).The 3' end ber probe (Oncogene Science), which detects ber generearrangments in almost all cases of Ph-positive CML (3), and animmunoglobulin JH fragment, which detects rearrangements at theheavy-chain immunoglobulin genes (IS), were used as probes. DNAfrom the patient's marrow, the cell lines, and positive and negativecontrols were analyzed for the presence of the EBV genome using aprobe for the BamV fragment of EBV (16).RESULTSCell Culture and Cytochemical Stains. The bone marrow cellswere plated in agar as a single cell suspension, and after 3 wkin culture, multiple colonies were observed (cloning efficiency,0.002%). Colony cells were gradually weaned to suspensionculture and have been growing continuously for over 18 mo.The SUP-B13 cell line was generated from the patient's firstbone marrow relapse and has a doubling time of approximately35 h. The SUP-B15 cell line was grown from the patient'ssecond bone marrow relapse and has a doubling time of approximately 18 h.2876

Ph CHROMOSOME-POSITIVE ALL CELL LINESTable 1 Reactivity of monoclonal antibodies with the patient's malignant cellsand the SUP-BI3 and SUP-BIS cell linesAntibodies Bonemarrowcells SUP-B13 SUP-B1502MHLA-DRCALLALeu 12CBlOKT 9OKT 10Leu MIMy 7My 9SigclgLeu 1, 2, 3, 4, 5, 6, 9
+++V
ND*NDND
' ++++, >80% of cells stain positive; +++, 60 to 79% of cells stain positive;
-, <20% of cells stain positive; ++, 40 to 59% of cells stain positive; +, 20 to39% of cells stain positive.'' ND, not done; Sig, surface immunoglobulin; clg, cytoplasmic immunoglob-ulin.The cell lines as well as the patient's diagnostic bone marrowcells stained positive for acid phosphatase, while the periodicacid-Schiff, nonspecific esterase, chloroacetate esterase, andSudan black stains were negative. The morphology (French-American-British classification) of the bone marrow blast cellswas 82% LI and 18% L2, while both cell lines had exclusivelyL2 morphology.\f H3 Cellular Antigens. The cell lines were concordant with thepatient's tumor in 13 of 16 paired antigens tested (Table 1).The bone marrow and cell lines expressed multiple B-lineagemarkers including CALLA but lacked T-cell markers (Table 1).There was discordance between the patient's tumor and the celllines in the expression of CB1, OKT9, and OKT 10 antigens.Cells were immunologically classified as pre-B-cells because thecells expressed cytoplasmic ¿ibut lacked surface immunoglobulin (Table 1). Each cell line expressed some myeloid antigens;however, the cell lines differ in that SUP-B13 expressed LeuM 1and My9 but lacked My?, while SUP-B 15 expressed My? butlacked LeuM 1 and My9.Enzyme Analysis. The ADA level on the patient's bone marrow, SUP-B 13, and SUP-B 15 cells was 20, 17.1, and 17.5enzyme units/mg of protein, while NP levels were 0.35, 0.46,and 0.27 enzyme units/mg of protein, respectively. Previously,normal lymphocytes had been shown to possess ADA levels of5.4 ±1.2enzyme units/mg of protein with the NP level of 0.36±0.05 enzyme units/mg of protein (17). The normal NP andelevated levels of ADA are characteristic of immature B-lymph-oid malignancies (13, 17). In addition, the cells stained positively for TdT.Karyotype Analysis. Cytogenetic analysis on the patient'sbone marrow cells at the time of presentation showed: 46,XY(8 cells); 46,XY,t(9;22)(q34;qll) (2 cells). The karyotypes ofthe bone marrow cells at relapse showed: 46,XY (6 cells);ÜII II I»6789It *• |t13 14 15 ifli IIft if
* ft % •19 20
16fi«21 17«ft22 18I
MARKERSFig. 1. Karyotype of cell line SUP-B13 analyzed by G-banding: 46,XY,t(9;22)(q34;ql I)t(l;4)(p22;q33)der(14q), 16p+. The Philadelphia chromosome is indicatedby an arrow. This karyotype was also seen in the patient's bone marrow and the SUP-BIS cell line.2877