Leukemia (1998) 12, 907–911 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Recombinant human megakaryocyte growth and development factor (MGDF) increases the numbers of megakaryocyte progenitor cells to normal values in long- term bone marrow cultures of patients with AML in first remission C Kasper1, A Schwarzer1, EA De Wynter1, J Chang1, TM Dexter1, D Ryder2 and NG Testa1 1Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, and 2Department of Medical Statistics, Christie Hospital, Manchester, UK The megakaryopoietic potential in the bone marrow (BM) of Materials and methods patients in first remission after treatment for acute myelogen- ous leukaemia (AML) was investigated using long-term bone marrow cultures (LTC) stimulated with megakaryocyte growth Patients and development factor (MGDF). The baseline number of megakaryocyte colony-forming cells (Meg-CFC) was very low. BM samples were collected with informed consent from 13 However, there was a 10 to 100-fold increase of Meg-CFC in patients with AML in first complete remission. Patients’ cultures treated with 10 ng/ml MGDF with mean numbers within characteristics are shown in Table 1. the normal range for the first 4 weeks of culture with a 24-fold As induction and consolidation treatment patients received increase in their cumulative numbers. Similarly, a 12-fold two to three cycles of DAT chemotherapy, containing dauno- increase in the numbers of megakaryocytes (MKs) was found 2 by CD61 immunostaining. These effects were lost at the dose rubicin (60 mg/m i.v. days 1–3), cytosine arabinoside of 100 ng/ml. In contrast, the cumulative mean numbers of Meg- (200 mg/m2 continuous infusion, days 1–7) and thioguanin CFC in the control cultures from normal bone marrow (NBM) (200 mg/m2, days 1–7), followed by up to two cycles of inter- were not significantly different from those in cultures treated mediate dose cytosine arabinoside (1.5 g/m2 at 12 h intervals, with 10 or 100 ng/ml MGDF. These results demonstrate that days 1–3). Two patients also received high-dose busulfan MGDF stimulates megakaryocytopoiesis in patients with AML in first remission, restoring the Meg-CFC compartment to (16 mg/kg body weight) with peripheral blood progenitor cell normal values, a result with potential clinical implications for rescue prior to the collection of the bone marrow sample, and their treatment with autologous transplantation. one patient an allogenic BM transplantation. Keywords: megakaryocyte growth and development factor (MGDF); thrombopoietin; colony assay; long-term bone marrow culture (LTC); AML Long-term cultures (LTC) BM samples were obtained after informed consent from heal- Introduction thy donors (NBM) for BM transplantation or AML patients (AMLBM). Cells were separated from the red cells by gravity Megakaryocyte growth and development factor (MGDF) or sedimentation in 0.1% methylcellulose for 45–60 min at room thrombopoietin, the ligand of the c-mpl receptor, has been temperature, and washed twice in Iscove’s modified Dulbec- purified and cloned by several groups independently.1–6 co’s medium (IMDM, Gibco BRL) plus 2% fetal calf serum MGDF has potent effects on the generation, proliferation and (FCS, Gibco BRL, Life Technologies, Paisley, UK). 1.5–2 × 106 differentiation of megakaryocyte (MK) progenitor cells in vitro buffy coat cells/ml diluted in 10 ml LTC medium that con- and in vivo.7–10 tained 80% IMDM, 10% FCS, 10% horse serum (HS, Sera Lab- − Patients treated for acute myelogenous leukemia (AML) who oratories, Crawley Down, UK), and 5 × 10 7 M hydrocortisone achieved first remission show severe and persistent haemato- were inoculated into tissue culture flasks (Falcon, Becton poietic damage manifested by substantially decreased num- Dickinson, Franklin Lakes, NJ, USA), gassed with 5% CO2 in bers of clonogenic progenitor cells and of stem cells.11 AML air and incubated at 33°C.14 Rh MGDF (a gift from Amgen patients in first or subsequent remissions may be treated with Corporation, Thousand Oaks, CA, USA) was added to the LTC autologous transplantation following high-dose chemo- at two concentrations, 10 or 100 ng/ml. The cultures were therapy.12,13 Severe and prolonged thrombocytopenia is usu- maintained by weekly feeding replacing half of the growth ally observed after this treatment. medium with fresh medium and MGDF; control cultures with- The effect of MGDF on long-term cultures (LTC) derived out MGDF were performed concurrently. The non-adherent from patients with AML in first complete remission was inves- cells removed at feeding were used for the assays described tigated in order to assess their megakaryopoietic potential and below. response to stimulation. The results indicate that MGDF has the ability to increase the numbers of MK progenitors (Meg- + CFC) and CD61 cells from a very low baseline to within Clonogenic assays normal values. Cells (105) were plated in a mixture containing 30% FCS, 1% deionized bovine serum albumin (BSA), 10% conditioned medium from the bladder carcinoma cell line 5637 (as a source of growth factors), 2 U erythropoietin (Boehringer Mannheim, West Lothian, UK) and 1.35% methylcellulose Correspondence: C Kasper, Department of Internal Medicine (Cancer Research), West German Tumor Center, University of Essen, Hufe- (Sigma, St Louis, MO, USA). Cultures were plated in triplicate landstr. 55, 45122 Essen, Germany; Fax: 0201 723 5924 and incubated at 37°C for 14 days in a fully humidified atmo- Received 3 October 1997; accepted 30 January 1998 sphere of 5% CO2,5%O2 in nitrogen. Colonies consisting of MGDF stimulates megakaryocytopoiesis in first remission AML C Kasper et al 908 Table 1 Patients’ characteristics No. Age Sex FAB Cytogenetics No. of chemotherapy Time between CR prior to BM sample and BM sample DAT ID Ara-C (month) 1 53 M M2 t(8;21) 3 2 6 2 42 M M5b ND 2 1 2 3 54 M M1 ND 2 1 6 4 36 F M2 normal 3 2 8 5 48 M M4 del (9) 1 0 0 662MM2−7205 7 63 M M2 normal 2 0 0 8 58 F M0 normal 3 2 8 9a 26 M M2 normal 2 2 8 10 32 M M4 t(8;21), t(4;10) 3 0 3 11 20 M M2 t(3;5), del (9) 3 0 3 12b 28 M M2 ND 2 0 106 13a 23 M M2 ND 2 1 56 aPatients studied two (No. 9) and 50 months (No. 13) after autologous transplantation with mobilized peripheral blood progenitor cells. bThis patient was studied 101 month after allogeneic bone marrow transplantation. FAB, French–American–British classification; ID Ara-C, intermediate-dose cytosine arabinoside; CR, complete remission; BM, bone marrow; ND, not documented. 50 translucent cells or more were counted as GM-CFC- Results derived colonies using an inverted microscope. To assay Meg-CFC 1–2 × 105 cells were plated in a mixture In NBM cultures, Meg-CFC were detected for 7 weeks. How- − of 30% pretested human aplastic anaemia serum, 5 × 10 5 M ever, they were detected only until week 5 in the cultures 2-mercaptoethanol, 5% conditioned medium from phyto- from patients with AML (Figure 1). In those cultures only very haemagglutinin-stimulated leukocytes (PHA-LCM) and 1.35% methylcellulose. Cultures were plated and incubated as described above. We used the established criteria for defining MK colonies: colonies of at least five conspicuously large cells with translucent ‘crystal’ clear cytoplasm and a well-delin- eated cell border of high refractility were scored as Meg- CFC.14 Their megakaryocytic nature was confirmed by picking some colonies for CD61 immunostaining. All results were cal- culated as total colony-forming cells in the supernatant (SN) per culture; the data in Figures 1–3 are expressed as mean ± s.e.m. Cell morphology Cytospins were prepared with 5 × 104 cells for Pappenheim stain and CD61 immunostaining (DAKO, Glostrup, Denmark) as previously described,15 to determine the number of megakaryocytic cells. Statistics Formal statistical analysis was confined to the MK profiles. It was felt that the cumulative output in weeks 1–4 captured the aspect of primary interest as it encloses the time of acute regeneration after chemotherapy and also covers the time pre- viously used for autologous transplants with cultured AMLBM cells.16 On occasion, however, when data were missing for some of the weeks, the observed cumulative output continued to be right censored for such individuals (ie it is assumed that the actual output is known only to be larger than that Figure 1 The upper panel shows the numbers of Meg-CFCs per observed). Right censored data may be analysed with ‘sur- í vival’ methods. The statistical analysis was done with a Cox culture in the supernatant of AML for untreated control cultures ( , n = 13), or treated with 10 ng/ml (b, n = 8) or 100 ng/ml MGDF (̆, regression model with appropriate adjustment for the corre- n = 7). The lower panel shows data from NBM cultures for untreated 17 lation of repeated observations for the same individual. All (n = 13), or treated with 10 ng/ml (n = 6) or 100 ng/ml MGDF (n = tests performed in this framework were Wald type tests. 9). Data are expressed as mean numbers of Meg-CFC ± s.e.m. MGDF stimulates megakaryocytopoiesis in first remission AML C Kasper et al 909 low levels of Meg-CFC were seen during the first 4 weeks of nificance was lost in the cultures treated with 100 ng/ml culture. The output of Meg-CFC in untreated AML cultures MGDF (P = 0.19). Treatment of NBM with MGDF had no sti- was at least one log lower than in NBM cultures (P = 0.001).