Leukemia (1997) 11, 1469–1477 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23) Y Matsuo1, RAF MacLeod2, CC Uphoff2, HG Drexler2, C Nishizaki1, Y Katayama3, G Kimura3, N Fujii3, E Omoto3, M Harada3 and K Orita1 1Fujisaki Cell Center, Hayashibara Biochemical Labs, Inc., Okayama; 3Department of Medicine II, Okayama University Medical School, Okayama, Japan and 2DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany We describe two new human leukemia cell lines, MOLM-13 and and MOLM-14, established from this patient at relapse, in MOLM-14, established from the peripheral blood of a patient which RT-PCR and molecular cytogenetic analysis revealed at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines MLL-AF9 fusion resulting from a micro-insertion which had express monocyte-specific esterase (MSE) and MLL-AF9 fusion been overlooked at diagnosis. mRNA. Gene fusion is associated with a minute chromosomal Continuous cell lines provide permanent sources of insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are materials for researchers in several areas,5 but may be of parti- the first cell lines with, and represent the third reported case cular interest for studies on the molecular changes involving of, MLL gene rearrangement arising via chromosomal insertion. recurrent breakpoints. Leukemia–lymphoma cell lines have Both cell lines carry trisomy 8 which was also present during been instrumental in the molecular study of many break- the MDS phase, as well as the most frequent trisomies associa- 6 ted with t(9;11), ie, +6, +13, +19 variously present in different points. subclones. Despite having these features in common, differ- ences in antigen expression were noted between the two cell 1 2 2 1 lines: that of MOLM-13 being CD34 , CD13 , CD14 , CD15 , Case report CD331; whereas MOLM-14 was CD41, CD131, CD141, CD151, 1 CD33 . Differentiation to macrophage-like morphology could 4 be induced in both cell lines after stimulation with INF-g alone, The clinical details have been reported in detail elsewhere. or in combination with TNF-a, which treatment also induced or Briefly, a 20-year-old male patient was readmitted to Oka- upregulated, expression of certain myelomonocyte-associated yama University Hospital with fever and swelling of the lymph antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. nodes in May 1995. In February 1995 he had received a trans- Together, these data confirm that both cell lines are likely to fusion of red blood cells and antibiotic treatment combined be novel in vitro models for studying monocytic differentiation with granulocyte colony-stimulating factor (G-CSF) for hypo- and leukemogenesis. Keywords: cell lines; AML-M5a; interclonal phenotypic hetero- cellular MDS (refractory anemia with excess of blasts: RAEB) geneity; insertion; MLL-AF9 after which trilineage hematopoietic recovery was attained without detectable blast infiltration. At diagnosis of MDS, interface cytogenetic and RT-PCR analyses, respectively, Introduction showed trisomy 8 and absence of AF9-MLL rearrangement in the bone marrow (BM). On readmission with florid leukemia, 3 A subtle, reciprocal translocation exchanging the terminal his peripheral white blood cell count was 13200/mm , with 56% leukemic blasts. His hemoglobin concentration was short and long arm segments of chromosomes 9 and 11, 3 respectively t(9;11)(p21-22;q23), is associated with acute 15.0 g/dl, and his platelet count was 22 000/mm . The bone myeloid leukemia (AML)-M5,1 particularly the M5a subtype.2 marrow aspiration showed 92% leukemic blasts, and the mor- phological diagnosis was made as AML-M5a. Cytogenetic Ascertainment may be difficult in suboptimal preparations + and, despite being regarded as the ‘standard’ cytogenetic analysis was interpreted as: 47, XY, 8, t(9;11)(p22;q23). change in acute monoblastic leukemia, its overall incidence Immunophenotyping analysis of fresh leukemia blasts and pattern of associations remain uncertain.2 A recent study revealed no significant expression of CD antigens associated comparing AML-M1 and -M5 patients, analyzed simul- with the myelo–monocytic lineage. CD34 was 30% positive taneously by reverse transcriptase-polymerase chain reaction and non-lineage-associated HLA-DR was found to be positive (RT-PCR), Southern blotting and fluorescence in situ hybridiz- at 70%; whereas those indicative of lymphoid lineage includ- ation (FISH) with an MLL-specific yeast artificial chromosome ing CD3, CD4, CD8, CD10, CD19 were absent or weakly (YAC) probe, suggests the overall incidence of MLL rearrange- expressed. Myeloperoxidase activity was weakly detected on ment in AML-M5 may be as high as 60%.3 It was apparent in the fresh leukemic blasts. Despite receiving chemotherapy, he that study that approximately half the cases with MLL succumbed to rapidly progressive leukemia on 15 August rearrangement went undetected by cytogenetic methods, 1995. including a cryptic t(6;11)(q27;q23) resulting from a cyto- genetically invisible insertion juxtaposing AF6 and MLL. A case of AML-M5a with de novo MLL-AF9 fusion evolving Materials and methods from MDS with trisomy 8 present at all phases has recently been described.4 We describe a pair of cell lines, MOLM-13 Cell culture During relapse, after chemotherapy, a heparinized peripheral Correspondence: Y Matsuo, Fujisaki Cell Center, Hayashibara blood specimen, obtained with informed consent, was pro- Biochemical Labs, Inc., 675-1 Fujisaki, Okayama 702, Japan vided by Okayama University Hospital, Second Department Received 3 April 1997; accepted 19 May 1997 of Medicine. Mononuclear cells were isolated by Ficoll– New AML-M5a cell lines with ins(11;9)(q23;p22p23) Y Matsuo et al 1470 Hypaque density centrifugation. The interface containing anti-human immunoglobulins (Igs) specific for k and l light mononuclear cells was harvested and washed twice with fresh chains and for a, d, g and m heavy chains (Cappel Labora- RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) culture tories, West Chester, PA, USA), rabbit anti-terminal deoxynu- medium. Mononuclear cells were suspended in RPMI 1640 cleotidyl transferase (TdT) (P-L Biochemicals, Milwaukee, WI, medium supplemented with 10% fetal calf serum (FCS: USA), monoclonal antibodies (mAbs) CD3 (NU-T3), CD4 GIBCO, Grand Island, NY, USA) and antibiotics, 100 U/ml (NU-Th/i), CD8 (NU-Ts/c), CD10 (NU-N1), CD20 (NU-B2), penicillin and 50 mg/ml streptomycin without any other CD13 (MCS-2), NU-Ia for HLA-class II (Nichirei, Tokyo, growth factors, and were incubated in two plastic culture Japan), CD19 (B4), CD33 (MY9), CD34 (MY10) (Coulter ° flasks at 37 C in a humidified 5% CO2 atmosphere. Each cul- Immunology, Hialeah, FL, USA). All mAbs were used in an ture was then fed once a week by replacing one half to one indirect immunofluorescence (IF) test with FITC-conjugated third of the volume of the culture contents with fresh nutrient goat anti-mouse IgG or IgM (Cappel Laboratories). In addition medium during the subsequent 2 weeks. In the third week, a to the immunophenotyping of the fresh leukemia cells, the slow yet sustained proliferation of cultured cells was noted following mAbs were used to type the established cell lines: in both flasks which were then designated as MOLM-13 and CD11a (SPV-L7; purchased from Nichirei); CD9 (BA-2; gift MOLM-14. from Dr TW LeBien, University of Minnesota, Minneapolis, The cell lines were found to be free of mycoplasma infec- MN, USA); CD14 (MY4; purchased from Coulter tion using standard broth-agar cultivation and DAPI staining. Immunology); CD15 (LeuM1; purchased from Becton Dickin- son, Mountain View, CA, USA); CD120A and CD120B (MR1- 4 and MR2-1, respectively; a gift from Dr WA Buurman, Uni- Morphological studies versity of Limburg, Maastricht, The Netherlands); CD115 Cytospin smears of the MOLM-13 and MOLM-14 cells, (D171; obtained through Dr T Kishimoto, Osaka University, stained with Wright–Giemsa, were used for cytochemical Japan via the 6th International Workshop on Human Leuko- myeloperoxidase (POX) staining. cyte Differentiation Antigens (HLDA); and all others were obtained through Dr S Shaw (National Institute of Health, Bethesda, MD, USA) (via the 5th International Workshop on Immunophenotyping HLDA). Immunofluorescent-positive cells were determined by fluorescent microscopy (Nikon, Tokyo, Japan). Immunophenotyping of fresh leukemia blasts from the peri- pheral blood was performed using the following antibodies (Table 1): fluorescein isothiocyanate (FITC)-conjugated goat Isoelectric focusing and isoenzyme staining Table 1 Marker profiles of MOLM-13 and MOLM-14 Enzyme extraction, separation by isoelectric focusing (IEF) and (% immunofluorescence positive visualization of esterase isoenzymes have been described in cells) detail elsewhere.7 In short, enzymes were extracted by repeated cycles of freezing and thawing and were solubilized Fresh leukemia MOLM-13 MOLM-14 by addition of Triton X-100 (Serva, Heidelberg, Germany). Ali- cells quots of enzyme-containing supernatants (referring to equal numbers of cells per sample) were separated on horizontal TdT ,100polyacrylamide thin-layer gels composed of 4.8% CD4 NU-TH/I , 10 100 acrylamide/bisacrylamide and ampholytes of pH range 2–11 CD9 BA-2 nt 5 80 (Servalyt; Serva) using an LKB Multiphor System