In Vitro Growth of Erythropoietic Progenitor Cells in Long-%rm Remission of Acute Leukemia

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1 International Journal of Cell Cloning 4: (1986) In Vitro Growth of Erythropoietic Progenitor Cells in Long-%rm Remission of Acute Leukemia C. Peschel, G. Konwalinka, D. Geissler, H. Bmunsteiner Department of Internal Medicine, University of Innsbruck, Austria Key Words. CFU-e BFU-e * Acute leukemia * Long-term remission Abstract. In vitro growth of CFU-e and BFU-e in bone marrow and of circulating BFU-e in a group of adult long-term survivors of acute leukemia has been evaluated. Six patients with acute nonlymphoblastic and three patients with acute lymphoblastic leukemia in first continuous remission for more than four years (range 4-12 years) and without maintenance therapy for at least one year were studied. BFU-e and CFU-e growth in patients bone marrow was not statistically different from a control group of 12 healthy adult volunteers. However, proliferation of BFU-e in peripheral blood of patients was significantly reduced (p < 0.001). This growth pattern was found in both lymphoblastic and myeloblastic leukemia. Introduction The prognostic significance of the growth pattern in the CFU-gm assay [l, 21 in acute leukemia has been recently confirmed in large series [3,4]. The remission induction of acute leukemia is associated with the appearance of normal colony formation of bone marrow myeloid progenitor cells. However, some abnormalities of CFU-gm proliferation have been observed to persist in remission of acute leukemia by several groups [3,5-81. In a recent study on long-term survivors of acute leukemia, we described increased numbers of more mature myeloid progenitor cells (cluster-forming cells, CFU-gm day 7), whereas examination of circulating CFU-gm revealed reduced colony formation [7]. Although a large ~ Correspondence: Christian Peschel, Laboratory of Immunology, Bldg. 10, Room lln311, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD Received February 20, 1985; accepted December 2, Press, Inc.

2 Erythropoiesis in Long-Term Remission of AL 187 number of studies deal with in vitro growth of myeloid progenitor cells, only a few groups have investigated erythroid progenitors in acute leukemia at presentation and during remission [%lo]. Therefore, we examined in vitro growth of late and early erythroid progenitor cells (CFU-e, BFU-e) in the bone marrow and of circulating BFU-e in a group of adult long-term survivors of acute leukemia. Materials and Methods Patients Six patients with acute nonlymphoblastic leukemia (ANLL) and three patients with acute lymphoblastic leukemia (ALL) were studied in long-term remission. The diagnosis was based on morphological and cytochemical criteria. The patients were treated with different combination chemotherapies in routine use for remission induction and maintenance therapy. Six patients were also included in a recent study on CFU-gm proliferation [7]. All patients were in first continuous remission for more than four years (range 4-12 years) and had not received maintenance therapy for more than one year. Cell Separation Heparinized bone marrow and peripheral blood samples were collected and layered over Lymphoprep (density glml). Interface cells were depleted of adherent cells by plastic adherence. Cell suspensions at a concentration of 1 X lo6 cells/ml were incubated in Petri dishes (100 X 20 mm; Falcon Labware, Oxnard, CA) for 30 min. Nonadherent cells were gently decanted and incubated for a further 90 min. After the second incubation period, nonadherent cells were referred to as monocyte-depleted and contained less than 2 % esterase-positive cells. Erythroid Progenitor Cell Assays Erythroid progenitor cells (CFU-e, BFU-e) were stimulated in a miniaturized culture system as previously described [ll]. Aliquots of 50 p1 containing 2 x 104 nonadherent bone marrow cells or 8 x 10' nonadherent peripheral blood cells were pipetted into 96-well micro-titer culture plates (Nunclon microwell plates, flat bottom; Nunc Roskilde, Denmark). After solidification, 50 p1 of a liquid overlayer containing erythropoietin (Epo step III; Connaught Medical Research Laboratory, Willowdale, Ontario, Canada), deionized and delipidated bovine serum albumin (BSA; Behring Werke Hoechst, Vienna, Austria) and transferrin saturated with FeCI3 (Behring Werke Hoechst, Vienna, Austria) were added. CFU-e and BFU-e were stimulated with Epo 0.3 and 1.2 U/ml, respectively. All cultures contained a final concentration of BSA 1% and transferrin 0.04%. After 7 days (CFU-e) or 14 days (BFU-e) of incubation, agar cultures were processed as previously described [12] by fixation with 2.5 % glutaraldehyde, washing in distilled water and mounting on microscopic slides. After drying on a warm plate, the whole agar layer was stained with May-Grunwald/Giemsa and"scored. CFU-e were defined as hemoglobinized aggregates containing no more than 65 cells at day 7 of incubation. BFU-e were scored at day 14 as single or multiple colonies of greater than 65 cells containing hemoglobin.

3 Peschel/Konwalinka/Geissler/Braunsteiner 188 Statistical Analysis All cultures were performed in quadruplicate. Results of different experiments are expressed as mean f SD. Student s t test was used to examine the differences in culture results between the patients group and the normal control group. Results Standard values for CFU-e and BFU-e numbers in normal bone marrow were 105 f 45 (mean * SD) and 53 f 31, respectively, as assessed in I2 healthy adult volunteers. The mean number of CFU-e in nonadherent bone marrow cells of the whole patient group was 141 f 82, 117 f 66 for patients in remission after ANLL and 188 f 104 for patients in remission after ALL (Fig. IA). These differences reached no statistical significance (p > 0.1). In vitro growth of BFU-e in patients bone marrow was also within the normal range (61 f 31, p > 0.5). The BFU-e values for patients in remission from ANLL (62 f 39) and ALL (59 f 53) exhibited similar proliferation capacity (Fig. lb). In contrast to the findings in patients bone marrow, the growth pattern of circulating BFU-e showed a clear-cut abnormality. The number of BFU-e in the peripheral blood of 16 normals was 28 f 15 colonies/8 X lo4 seeded cells. Examination of circulating erythroid progenitors in the patients group revealed that the number of BFU-e was significantly reduced (5 f 2,p < 0.001). This reduction was independent of the kind of leukemia existing at presentation (ANLL 5 f 1, ALL 4 f 3). Discussion Lmg-term remission from acute leukemia achieved with routine chemotherapy regimens is found in about 5 %-lo% of adult patients [13]. Therefore, our small group of long-term survivors from acute leukemia (up to I2 years) represents a highly selected population. Even in these patients a defective growth pattern of erythropoietic progenitor cells resembling the proliferation pattern previously described for myeloid precursor cells [7] could be demonstrated. Whereas the proliferation of bone marrow BFU-e was within the normal range, the number of circulating BFU-e in the patients peripheral blood showed a marked reduction. This was a constant finding in patients in remission from both ALL and ANLL. It is unlikely that the highly reduced BFU-e number in peripheral blood is due to a lack of endogenous burst-promoting activity rather than a reduced progenitor cell number. In our hands the depletion of adherent cells does not influence the number and size of burst colonies in normal patients (unpublished observation). Recently we were able to demonstrate a reduction of BFU-e in nor-

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5 Peschel/Konwalinka/Geissler/Braunsteiner 190 unlikely that the impaired proliferation capacity of peripheral blood progenitor cells is caused by a persistent defective hematopietic clone after remission induction because both ALL and ANLL patients are affected. Furthermore, an intermittent growth pattern, typical for acute myelogenous leukemia at presentation, has recently been observed in patients with ALL in remission, whereas the ANLL growth pattern of CFU-gm in ALL has never been observed [3]. The greatly reduced number of circulating progenitor cells might be explained by a disturbance of the egress mechanism for committed stem cells in the peripheral blood. This observation may contribute to the investigation of harmful effects on the equilibrium of hematopoiesis, which are caused by previous infiltration of leukemic cells or high-dose chemotherapy. Acknowledgment This work was supported by the "Fonds zur Foerderung der wissenschaftlichen Forschung" Projektnummer P References Moore MAS, Spitzer G, Williams N, Metcalf D, Buckley S. Agar culture studies in 127 cases of untreated acute leukemia: the prognostic value of reclassification according to in vitro growth characteristics. Blood 1974;44:1-18. Spitzer G, Dicke KA, Gehan EA, et al. A simplified in vitro classification for prognosis in adult leukemia: the application of in vitro results in remission predictive models. Blood 1976;48: Mertelsmann R, Moore MAS, Broxmeyer HE, Cinincione C, Clarkson B. Diagnostic and prognostic significance of the CFU-c assay in acute nonlymphoblastic leukemia. Cancer Res 1981;41: Keating MJ, Smith TL, Gehan EA, et al. Factors related to length of complete remission in acute leukemia. Cancer 1980;45: Tebbi K, Rubin S, Cowan DH, McCulloch, EA. A comparison of granulopoiesis in culture from blood and bone marrow cells of nonleukemic individuals and patients with acute leukemia. Blood 1976;48: Gustavsson A, Olofsson T, Olsson I. Oscillations of marrow culture growth in acute myeloid leukemia during remission induction and remission. Acta Haematol 1979;62: Peschel C, Konwalinka G, Geissler D, et al. Studies of myelopoiesis in vitro on blood and bone marrow cells of patients with acute leukemia in long-term remission. Leuk Res 1983;7: Urabe A, Murphy MJ Jr, Haghbin M, Gee TS. Erythroid progenitors (BFU-e, CFU-e) in acute leukemia. J Clin Pathol 1979;32: Lan S, McCulloch EA, Till JE. Cytodifferentiation in the acute myeloblastic leukemias in man. JNCI 1978;60: Eridani S, Sawyer B, Batten E. Haemopoietic patterns of acute leukaemia in remission. CFU-e and CFU-gm colony formation. Acta Haematol 1983;70:11-18.

6 Erythropoiesis in bng-term Remission of AL Konwalinka G, Peschel C, Boyd J, et al. A miniaturized agar system for clonal growth of erythropoietic progenitor cells. Exp Hematol 1984;12: Konwalinka G, Geissler D, Peschel C, et al. A micro-agar culture system for cloning human erythropoietic progenitor cells in vitro. Exp Hematol 1982;10: Whittaker JA, Reizenstein P, Callender ST, et al. Long survival in acute myelogenous leukemia: an international collaborative study. Br J Med 1981;282: Peschel C, Konwalinka G, Geissler D, et al. Studies on differentiation of committed hemopoietic progenitor cells with monoclonal antibodies directed against myeloid differentiation antigens. Exp Hematol 1985;13:l Ferrero D, Broxmeyer HE, Pagliardi GI, et al. Antigenically distinct subpopulations of myeloid progenitor cells (CFU-gm) in human peripheral blood and marrow. Proc Natl Acad Sci USA 1983;80: Linch DC, Boyle D, Beverley PCL. T cell and monocyte requirements for erythropoiesis. Acta Haematol 1982;