NK precursor phenotype

Leukemia Research 27 (2003) 165–171

Megakaryoblastic leukemia cell line MOLM-16 derived from minimally differentiated acute leukemia with myeloid/NK precursor phenotype Yoshinobu Matsuo a,∗ , Hans G. Drexler b , Kinuyo Kaneda c , Kensuke Kojima c , Yuji Ohtsuki d , Masamichi Hara e , Masaki Yasukawa f , Mitsune Tanimoto c , Kunzo Orita a a

Fujisaki Cell Center, Hayashibara Biochemical Labs Inc., Fujisaki, Okayama 702-8006, Japan DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig 38124, Germany c Department of Medicine II, Okayama University Medical School, Shikata-cho, Okayama 700-8558, Japan d Department of Pathology II, Kochi Medical School, Nankoku, Kochi 783-8505, Japan e Division of Hematology, Ehime Prefectural Central Hospital, Kasuga-cho, Matsuyama, Ehime 790-0024, Japan f First Department of Internal Medicine, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan b

Received 4 March 2002; accepted 30 April 2002

Abstract The megakaryoblastic leukemia cell line MOLM-16 was established at relapse from the peripheral blood of a 77-year-old Japanese woman with minimally differentiated acute myeloid leukemia (AML-M0). Immunophenotyping of the fresh leukemic cells revealed a myeloid/NK precursor phenotype being positive for CD7, CD13, CD33, CD34, and CD56. In addition, megakaryocyte-associated antigens CD41 and CD61 were found to be positive. The established cell line designated MOLM-16 was proliferatively responsive to the treatment with various cytokines including EPO, GM-CSF, IL-3, PIXY-321, and TPO. MOLM-16 revealed characteristics of the megakaryocytic lineage in terms of immunophenotyping being positive for CD9, CD31, CD36, CD41, CD61, CD62P, CD63, CD110, CD151, thrombospondin, von Willebrand factor (vWf), and fibrinogen. Electron microscopic analysis showed positivity for ultrastructural platelet peroxidase in the nuclear envelope. The karyotype analysis of MOLM-16 revealed various numerical and structural abnormalities including t(6;8)(q21;q24.3), t(9;18)(q13;q21) and marker chromosomes. The extensive immunological, cytogenetic and functional characterization of MOLM-16 suggests that this cell line may represent a scientifically significant in vitro model which could facilitate the evaluation of megakaryocytic differentiation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acute minimally differentiated leukemia; Myeloid/NK precursor leukemia; Megakaryoblastic cell line

1. Introduction Since leukemia represents a heterogeneous malignancy of the hematopoietic system in terms of cellular morphology, clinical presentation, disease course and response to treatment, several classifications have been proposed in the past which initially relied solely on morphological–cytochemical features, but recently also included immunophenotypical, cytogenetic and molecular genetic parameters. In 1976 and subsequently in 1985, the French–American–British (FAB) Cooperative Group defined standardized criteria to qualify acute leukemias as myeloid (AML) or lymphoid (ALL) [1,2]. There has been increasing interest for a new distinct entity, characterized by fewer than 3% myeloper∗

Corresponding author. Tel.: +81-86-276-8621; fax: +81-86-274-2150. E-mail address: [email protected] (Y. Matsuo).

oxidase (MPO) and/or Sudan black B (SBB)-positive blasts, which has been referred to as “minimally differentiated acute myeloid leukemia” or “AML-M0”. By definition, the diagnosis of AML-M0 requires less than 3% MPO positivity and/or SBB positivity, expression of myeloid-associated antigens and lack of expression of T/B lymphoid lineage-associated antigens. Thus, for the recognition of AML-M0 (and AML-M7) immunophenotypic evaluation is integrated in the FAB scheme as a part of the diagnostic procedure [3,4]. In this report, we describe the establishment of the novel leukemia cell line MOLM-16 derived from a patient with AML-M0 which carried megakaryoblastic characteristics and displayed a myeloid/NK precursor acute leukemia phenotype of CD7+ CD13+ CD33+ CD34+ CD56+ [5,6]. The cell line has mainly megakaryoblastic features.

0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 0 2 ) 0 0 0 8 1 - 4

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2. Materials and Methods 2.1. Patient A 77-year-old Japanese woman was referred to us on 7 October 1999, with a diagnosis of AML-M0. On physical examination, small lymph nodes were noted in the cervical region but hepatosplenomegaly was not observed. Her hemoglobin concentration was 4.9 g/dl, white blood cell count was 5.8 × 109 /l with 16% blasts, 64% neutrophils, 6% monocytes and 14% lymphocytes; platelet count was 2.4 × 109 /l. A peripheral blood sample showed 80% MPO cytochemical staining-negative blasts with an irregular nucleus with fine chromatin, inconspicuous nucleoli and moderately basophilic cytoplasm. Azurophilic granules or Auer bodies were not recognized (Fig. 1A). Immunophenotyping of freshly isolated leukemia cells from the peripheral blood

revealed that the cells expressed a phenotype of CD7+ CD13+ CD33+ CD34+ CD56+ (Table 1). Positivity for megakaryocyte-associated antigens CD41 and CD61 was 13 and 28%, respectively. Cytogenetic analysis of bone marrow cells showed the following karyotype: 46, XX, add(3)(q27), add(9)(p11), −14, −18, add(19)(p13), −21, +3mar in 16/20 and 46, XX in 4/20 metaphases analyzed. She failed to achieve complete remission during chemotherapy for AML including bephenoyl cytosine arabinoside, daunomycin and 6-mercaptopurine, and died on 14 January 2000. 2.2. Cell line establishment During relapse, after chemotherapy, on 21 December 1999, a heparinized peripheral blood specimen, obtained with informed consent, was provided by Ehime Prefectural Central Hospital, Internal Medicine. Mononuclear cells were isolated by Ficoll-Hypaque gradient density centrifugation. The interface containing mononuclear cells was harvested and washed twice with fresh RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) culture medium. Mononuclear cells were suspended in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FBS: Invitrogen, Tokyo, Japan) and antibiotics, 100 U/ml penicillin and 50 ␮g/ml streptomycin, with 20% 5637 culture supernatant (5637 CM) [7,8]; cells were incubated in a 24-well-culture plate (Corning, Corning, NY) at 37 ◦ C in a humidified 5% CO2 atmosphere. The culture was then fed once a week by replacing one-half to one-third of the volume of the culture contents with fresh 5637 CM during the subsequent 12 weeks. In the 13th week, a slow yet sustained proliferation of the cultured cells was noted which was then designated as MOLM-16. The 5637 CM was continuously used as the source of growth factors for maintaining constant proliferation. The cell line was found to be free of mycoplasma infection using polymerase chain reaction (PCR) and DAPI staining. We attempted to characterize the cells as detailed as possible, as outlined previously [9]. 2.3. Morphological studies

Fig. 1. Cellular morphology of primary and cultured cells. Leukemia cells at diagnosis were characterized by a high nucleo-cytoplasmic ratio, an irregular nucleus with fine chromatin, inconspicuous nucleoli and a non-granular basophilic cytoplasm (A); the established MOLM-16 cells (B) have an irregular nucleus with a single nucleolus and a non-granular deeply basophilic cytoplasm containing a well-developed Golgi apparatus, vacuoles and occasionally extending protrusions (May–Grünwald–Giemsa staining; original magnification: 310×).

Microscopic observation was performed on cytospin smears of the established cell line after May–Grünwald– Giemsa staining. Cytochemical staining for MPO was also performed using cytospin smears and standard methodology. Conventional ultrastructural studies and ultrastructural platelet peroxidase (PPO) reaction were performed as described previously [10,11]. 2.4. Immunophenotyping Immunophenotyping of fresh leukemic blasts and of the cell line was performed using the antibodies listed in Table 1. Experiments were performed between three to five times.

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The sources of these reagents and the methods used have been described in detail elsewhere [12–14].

Table 1 Immunophenotyping of primary leukemia cells and MOLM-16 CD#

Antibody

Primary leukemia cells

167

MOLM-16

2.5. Cytogenetic preparation and analysis

T/NK-Ags CD2 CD3 CD4 CD7 CD8 CD16 CD56 CD57 CD94 CD158A CD158B CD161

NU-TER NU-T3 NU-TH/I 3A1 NU-TS/C Leu11b NKH-1 Leu7 HP-3B1 EB6 GU83 NKRP1A

6 <1 70 81 <1 12 57 4 5 5 4 7

0 0 5 80 0 0 0 0 0 0 0 0

B-Ags CD10 CD19 CD22 CD79A

NU-N1 B4 RFB-4 HM-57

<1 <5 <1 <1

0 0 0 0

My-Ags CD13 CD14 CD33 MPOa

MCS-2 MY-4 MY-9 AM-9

79 <5 75 (90)

100 0 100 0 (0)

Plt-Ags CD9 CD31 CD36 CD41 CD42b CD42d CD61 CD62L CD62P CD63 CD107A CD107B CD110 CD151 Thrombospondina vWfa Fibrinogena

Tp82 10G9 5F1 VIPL-I AN51 FMC25 VIPL-II Dreg56 CLB/Thrombo6 H5C6 H5G11 CD3 6E10 11B P12 F8/86 MAB121

n.t. n.t. n.t. 13 n.t. n.t. 28 4 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t.

20 100 100 80 0 0 80 0 5 100 0 0 0 100 0 0 0

Other-Ags CD25 CD34 CD47 CD71 CD117 CD119 CD235A HLA-DRa TdTa

Tac MY10 BRIC126 B3/25 1C1 GIR-208 GA-R2 NU-Ia Anti-TdT

<1 79 n.t. 55 73 n.t. n.t. 40 <5

20 100 100 100 100 100 60 0 0

Standard cytogenetic harvesting, preparation and staining procedures were used throughout. Briefly, mitotic cells were harvested by colcemid arrest (0.004 ␮M for 1.5 h) and, after hypotonic treatment with 0.075 M KCl and 0.9% sodium citrate (3:1 for 20 min), fixed in chilled methanol:acetic acid (5:2). Trypsin G-banding was performed on slides after aging for 10 days. Slides were incubated briefly (10–15 s) in trypsin solution and stained for 3 min in 5% Wright and 5% Giemsa in Sorensen’s buffer (pH 6.8) [15]. Analysis was performed using a Nikon Optiphot-2 microscope (Nikon, Tokyo, Japan) connected to an image analysis system (CytoVision Version 3.52, Applied Imaging, Joel Datum, Tokyo, Japan) equipped with a high-resolution laser printer (Mitsubishi S3600-30, Fukuyama, Japan). The consensus karyotypes were derived by analysis of 20 cells. 2.6. Epstein–Barr virus infection Epstein–Barr virus (EBV) infection was determined by PCR using an EBV nuclear antigen (EBNA-1) specific primer set as described previously [16]. (100) (0) (0) (100) (0) (80)

2.7. Mycoplasma detection Detection of mycoplasma infection was carried out by the PCR technique using specific primer sets and DNA staining as described previously [16,17].

(20)

2.8. DNA fingerprinting

(20) (20) (50)

Genomic DNAs were isolated from the established cell lines. The DNAs were analyzed using a multilocus probe, designated [gtg]5 (digoxigenin conjugated [gtg]5 probe, Kurabo, Osaka, Japan) and the short tandem repeat loci specific primer sets for D5S818, D7S820, D13S317, and D16S539 as described in detail previously [18,19].

The values shown are the percent positive cells analyzed by immunofluorescence microscopy. For MOLM-16, the values shown are the means of percent positive cells in multiple tests. The value given in parenthesis represent percent immunofluorescence positive cells for cytoplasmic antigen expression of CD41, CD42b, CD42d, CD61, CD62L, CD62P, CD110, thrombospondin, vWf, fibrinogen, and MPO; n.t.: not tested; T/NK-Ags: T-cell/NK-cell-associated antigens; B-Ags: B-cellassociated antigens; My-Ags: myeloid/monocytic-associated antigens; Plt-Ags: platelet/megakaryocyte-associated antigens. a MPO, thrombospondin, vWf, fibrinogen, HLA-DR and TdT have not been CD clustered.

2.9. Proliferation assay The proliferation assay was carried out as follows. In brief, the proliferative response of cells to the various effectors was examined by cell counting in hematocytometers or standard 3 H-thymidine incorporation and ␤-scintillation counting (LKB 1209 Rackbeta, Pharmacia, Freiburg, Germany). The cells were seeded in triplicate in 100 ␮l medium in flat-bottomed 96-well plates and incubated in the absence or presence of cytokines; for the last 4 h of the 52 h-incubation period, 1 ␮Ci [methyl-3 H]thymidine (Amersham, Freiburg, Germany) was added to each well. Cells were seeded at 6.25 × 104 cells/ml. As the cells are

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constitutively dependent on externally added growth factors and normally grown in medium containing such factors, the cells were starved overnight in serum-free medium (SFM; Invitrogen, Karlsruhe, Germany) and were washed extensively immediately prior to the experiments. The results expressed as percentages of control were calculated by dividing the counts per minute (cpm) in the 3 H-thymidine uptake assay of the growth factor-containing wells by the cpm of the media alone control wells. Experiments were performed at least three times for each cytokine. Further details of this type of cytokine experiments have been described previously [8,20]. 2.10. RT-PCR Reverse transcription-PCR (RT-PCR) was utilized for platelet-derived growth factor (PDGF) A chain and von Willebrand factor (vWf) gene expressions in constitutive MOLM-16 cells and MOLM-16 cells stimulated with 10−7 M 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma, St. Louis, MO, USA) following the methods described previously [14]. Amplification of the hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene was included as a control.

in defined medium (70% medium, 20% FBS, 10% DMSO), stored in liquid nitrogen, thawed again (with viabilities of more than 70%) and successfully reconstituted. The data presented here were obtained from cells that had been in culture for 6 months and did not change significantly during further culturing. The cell line was found to be free from mycoplasma and EBV infection. MOLM-16 cells have a nucleus of irregular shape, a non-granular deeply basophilic cytoplasm with occasional cytoplasmic protrusions (Fig. 1B). Electron microscopic photographs of MOLM-16 cells show a prominent nucleolus and a wide cytoplasm with a distinct endoplasmic reticulum. The ultrastructural detection of platelet peroxidase was positive in the nuclear envelope. No demarcation membranes were evident (Fig. 2). Hence, the cell line MOLM-16 could be arrested at an immature differentiation stage of the megakaryoblastic lineage. This cell line was also proliferatively responsive to the treatment with EPO, GM-CSF, IL-3, PIXY-321, and TPO. TGF-␤ and TNF-␣ had reproducibly strong inhibitory effects on MOLM-16 growth as shown in Fig. 3. The following cytokines which are not listed in Fig. 3 did not have any significant proliferation-inducing or inhibiting effects at

3. Results and discussion We describe here the establishment and characterization of the new growth factor-dependent leukemia cell line MOLM-16 derived from the peripheral blood of a patient with AML-M0 carrying the myeloid/NK precursor leukemia phenotype CD7+ CD13+ CD33+ CD34+ CD56+ and lacking expression of specific T/B lymphoid lineage-associated antigens. The peripheral blood leukemia cells of the patient showed a relatively abundant basophilic cytoplasm with an irregular nucleus displaying fine chromatin and an inconspicuous nucleolus. Azurophilic granules and Auer bodies were not detected. Peripheral blood mononuclear cells obtained from the patient were cultured as described in the Section 2. In the 13th week of culture, a slow yet sustained proliferation of the cultured cells was noted, and the cells were subcultured. The cells proliferated consistently as free-floating cells in suspension. This continuous cell line was designated MOLM-16. The cells were maintained with RPMI 1640 medium supplemented with 10% FBS and 20% culture supernatant of 5637 cell line which contains several cytokines including GM-CSF, IL-3, IL-6, SCF and others (constitutively expressed transcripts were found for G-CSF, GM-CSF, IL-1␣, IL-1␤, IL-6, IL-7, IL-8, TNF-␣, TNF-␤ at the RT-PCR level) [7]. The cell line has since been maintained permanently in 5637 CM; the cells have a doubling time of about 4–5 days. The cell line was grown continuously for more than 12 months. The maximum cell density is about 2.7 × 106 cells/ml. The cells can be cryopreserved

Fig. 2. Electron microscopic photographs of MOLM-16 cells. (A) A representative MOLM-16 cell exhibiting an irregular nucleus with a prominent nucleolus, containing linear rough endoplasmic reticulum, scattered mitochondria and abundant ribosomes in the cytoplasm, extending thick bulbous cytoplasmic process (arrow) (original magnification: 10,000×); no demarcation membranes are evident. (B) Platelet peroxidase activity was positive in the nuclear envelope (arrowhead, PPO reaction without counterstain; original magnification: 19,000×).

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Fig. 3. Proliferative response of MOLM-16 cells to cytokines. Cells were incubated with the following conditioned media (CM) or recombinant cytokines: erythropoietin (EPO, 5 U/ml); 5637 CM (20% v/v from bladder carcinoma cell line 5637); PIXY-321 (fusion protein of IL-3 and GM-CSF, 10 ng/ml); KU1919 CM (20% v/v from bladder carcinoma cell line KU1919); granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/ml); basic fibroblast growth factor (bFGF, 50 ng/ml); thrombopoietin (TPO, 100 U/ml); interleukin-3 (IL-3, 10 ng/ml); oncostatin M (OSM, 10 ng/ml); stem cell factor (SCF, 100 ng/ml); IL-1␣ (10 ng/ml); interferon-␥ (IFN-␥, 50 ng/ml); IL-13 (10 ng/ml); nerve growth factor (NGF, 10 ng/ml); IL-15 (10 ng/ml); IL-5 (10 ng/ml); granulocyte-CSF (G-CSF, 10 ng/ml); transforming growth factor-␤1 (TGF-␤1, 50 ng/ml); tumor necrosis factor-␣ (TNF-␣, 10 ng/ml).

all: FLT3 ligand, IFN-␤, IFN-␥, insulin-like growth factor-I (IGF-I), IL-2, IL-4, IL-6, IL-7, IL-9, leukemia inhibitory factor (LIF), macrophage-CSF (M-CSF), TNF-␤. The immunophenotyping of the MOLM-16 cells is summarized in Table 1and reveals a typical megakaryoblastic immunomarker profile being positive for megakaryocyteassociated antigens as follows: CD9, CD31, CD36, CD41, CD61, CD62P, CD63, CD110, CD151, thrombospondin, vWf, and fibrinogen. The erythroid lineage-associated CD47 and CD235A and the myeloid-associated CD13 and CD33 were also found to be positive. Various antigens expressed regularly on immature cells, namely CD7, CD34, CD117 and CD119, were also positive. The other antigens detected on the primary leukemic blasts, CD56, HLA-DR and MPO, were negative on the cell line. RT-PCR detected specific bands for vWf and PDGF (Fig. 4). Chromosome analysis was carried out by the standard G-banding technique producing the following representative karyotype of MOLM-16:44, XX, add(4)(q12), −5, add(6)(q21), del(6) (q?13q21), der(8)t(6;8)(q21;q24.3), −9, dup(14)(q?22q?22), add(16)(q13), der(18) t(9;18)(q13;q21), der(19)add(19)(p13.1), add(19) (q?13.4), −20, −21, +mar1, +mar2 (Fig. 5). Thus, MOLM-16 has complex numerical and structural chromosomal abnormalities including the two unique translocations t(6;8)(q21;q24.3) and t(9;18)(q13;q21). These translocation have not yet been reported previously and their pathological significance remains unknown. The

derivation of MOLM-16 from the primary leukemia cells was authenticated using DNA fingerprinting. Immunophenotyping of the fresh MPO-negative leukemia blasts showed these cells to be positive for the plateletassociated antigens CD41 and CD61. It is conceivable that MOLM-16 is clonally derived from this subset of megakaryoblastic malignant cells present in the primary leukemia specimen.

Fig. 4. Expression of vWf, PDGF and HGPRT genes by MOLM-16 cells. Lane 1, MOLM-16; lane 2, MOLM-16 treated with TPA; lane 3, MEG-01 megakaryoblastic cell line treated with TPA; lane 4, CCRF-CEM T-cell leukemia cell line; lane 5, H2 O.

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Fig. 5. Karyotype analysis. Chromosome analysis was performed by the G-banding technique. The stemline karyotype is described in Section 3. Structural aberrations are indicated by arrows.

The immunomarker profile of the primary specimen (CD7+ CD16− CD13+ CD33+ CD34+ CD56+ ) is compatible with what has been described as myeloid/NK precursor leukemia [5,6,21]. Cytogenetic abnormalities affecting chromosome 7 or chromosome 3p appear to be recurrent in this disease [22]. However, both the fresh leukemia cells and the MOLM-16 cells did not have such aberrations. Since myeloid/NK precursor acute leukemia has been reported as a distinct subtype of AML-M0 [23], further investigation is needed to elucidate to what extent myeloid leukemia and myeloid/NK precursor leukemia do indeed overlap. The present study demonstrated also that further clarification of the differential diagnosis between AML-M0 and AML-M7 is warranted. In summary, the megakaryoblastic leukemia cell line MOLM-16 was established from a patient with AML-M0 carrying a CD7+ CD13+ CD33+ CD34+ CD56+ phenotype. The MOLM-16 cell line should be a useful tool for the investigation of a variety of pathobiological aspects of acute leukemia.

Acknowledgements Y. Matsuo provided the concept, design, assembled and analyzed the data, drafted and revised the manuscript. H.G. Drexler contributed to the interpretation of the data, provided critical input for the revision, and gave administrative

support. K. Kaneda provided study materials and assisted with data analysis. K. Kojima provided study materials and technical support. Y. Ohtsuki, M. Hara, and M. Yasukawa assisted with data analysis. M. Tanimoto provided technical support. K. Orita obtained the necessary funding and gave final approval. References [1] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposals for the classification of the acute leukaemias: French–American–British (FAB) Cooperative Group. Br J Haematol 1976;33:451–8. [2] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French–American–British Cooperative Group. Ann Int Med 1985;103:620–5. [3] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposal for the recognition of minimally differentiated acute myeloid leukaemia (AML-M0). Br J Haematol 1991;78:325–9. [4] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Criteria for the diagnosis of acute leukemia of megakaryocyte lineage (M7): a report of the French– American–British Cooperative Group. Ann Int Med 1985;103: 460–2. [5] Scott AA, Head DR, Kopecky KJ, Appelbaum FR, Theil KS, Grever MR, et al. HLA-DR− , CD33+ , CD56+ , CD16− myeloid/NK cell acute leukemia: a previously unrecognized form of acute leukemia potentially misdiagnosed as French–American–British acute myeloid leukemia-M3. Blood 1994;84:244–55.

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