Bone Marrow Transplantation, (1997) 20, 465–471 1997 Stockton Press All rights reserved 0268–3369/97 $12.00 Effect of chemotherapy for acute myelogenous leukemia on hematopoietic and fibroblast marrow progenitors C Carlo-Stella1, A Tabilio2, E Regazzi1, D Garau1, R La Tagliata3, S Trasarti3, C Andrizzi3, M Vignetti3 and G Meloni3 1Department of Hematology, University of Parma; 2Department of Hematology, University of Perugia; and 3Department of Cellular Biotechnologies and Hematology, ‘La Sapienza’ University, Rome, Italy Summary: Keywords: hematopoietic progenitors; microenviron- mental progenitors; acute myelogenous leukemia; chemo- Since reduced marrow cellularity and prolonged pancy- therapy; LTC-IC; hematopoietic engraftment topenia following autologous bone marrow transplan- tation (ABMT) have been frequently observed in patients with acute myelogenous leukemia (AML) included in the AML10 GIMEMA/EORTC trial, the The structural integrity of the hematopoietic system is question was raised to what extent hematopoietic and maintained by a relatively small population of self- microenvironmental progenitor cells were involved in renewing stem cells which can differentiate to produce pro- these patients. Marrow hematopoietic progenitors were genitors committed to terminal maturation.1 The develop- investigated by a short-term methylcellulose assay ment of hematopoietic cells in vivo occurs in intimate quantitating multipotent CFU-Mix, erythroid BFU-E association with a heterogeneous population of mesenchy- and granulocyte–macrophage CFU-GM, as well as a mal, connective tissue type cells and their associated long-term assay quantitating long-term culture- biosynthetic products, which constitute the stromal tissue initiating cells (LTC-IC). The marrow microenviron- of the bone marrow.2 Stromal cells of the hematopoietic ment was studied by evaluating the incidence of fibro- microenvironment include fibroblasts, endothelial cells, blastoid progenitors (CFU-F) and the capacity of stro- adipocytes, and macrophages.2 Based on a number of stud- mal layers to support allogeneic hematopoietic ies,3 the existence of self-renewing stromal stem cells with progenitors. As compared to normal controls (n = 57), multilineage differentiation capacity and capable of gener- AML patients (n = 26) showed a statistically significant ating progenitors with restricted development potential, reduction of the mean (± s.e.m.) number of CFU-Mix including fibroblast, osteoblast and chondrocyte progeni- (5.3 ± 0.6 vs 0.8 ± 0.2, P < 0.0001), BFU-E (68 ± 5 vs tors, has been hypothesized.4–6 20 ± 4, P < 0.0001), CFU-GM (198 ± 11 vs 144 ± 15, P Standard- and high-dose therapies currently used for the < 0.008), and LTC-IC (302 ± 46 vs 50 ± 8, P < 0.001). treatment of hematological and nonhematological malig- The mean (± s.e.m.) incidence of marrow CFU-F was nancies induce transient or permanent damage of hemato- not significantly reduced as compared to normal con- poietic and stromal progenitor cell compartments.7,8 trols (48 ± 6 vs 52 ± 7, P < 0.73). Seventeen AML stro- Despite such chemotherapy-induced defective progenitor mal layers were tested for their capacity to support the cell growth, the reinfusion of autologous marrow can recon- growth of allogeneic hematopoietic progenitors. Seven stitute the hematopoietic system, even in acute myelogen- samples failed to support any progenitor cell growth, ous leukemia (AML) patients treated with remission induc- seven had a significantly lower supportive activity as tion regimens exerting a significant marrow toxicity.9,10 compared to normal stromal layers (13 ± 5 vs 249 ± 56, Recently, therapeutic trials have been conducted in AML P < 0.002), whereas three cultures could not be ana- patients which were aimed at evaluating the efficacy of lyzed due to contamination. In conclusion, induction induction and consolidation regimens containing new anti- and consolidation regimens used in AML patients of the leukemic drugs, such as idarubicin or mitoxantrone.11 AML AML10 protocol induce a markedly defective in vitro patients included in the AML10 GIMEMA/EORTC pilot growth of primitive hematopoietic progenitors and a study receiving idarubicin, cytosine arabinoside, and etopo- severe functional defect of marrow stroma. The associ- side (ICE) as induction therapy and mitoxantrone and cyto- ation of hematopoietic with microenvironmental dam- sine arabinoside (NOVIA) as consolidation therapy, have age might play a key role in the delayed hematopoietic been reported to have reduced marrow cellularity, often regeneration observed following ABMT in patients of preventing marrow harvest and dramatically reducing the the AML10 trial. feasibility of autologous bone marrow transplantation (ABMT).12,13 In addition, as compared to historical controls treated with the conventional daunorubicin and cytosine 14 Correspondence: Dr C Carlo-Stella, Cattedra di Ematologia, Universita` di arabinoside (D3A7) protocol, patients autografted with Parma, Via Gramsci, 14, I-43100 Parma, Italy marrow harvested following the ICE/NOVIA regimens, Received 21 February 1997; accepted 26 May 1997 have experienced a significantly prolonged pancytopenia Bone marrow damage after chemotherapy for AML C Carlo-Stella et al 466 associated with increased transplant-related mortality. apy or at marrow harvest, respectively. Normal values for Reduced marrow cellularity and poor graft function were bone marrow progenitor cell growth were provided from also observed in patients included in the AML10 57 healthy subjects (38 males, 19 females) with a median randomized trial who received the same intercalating drug age of 42 years (range, 20–67). (idarubicin, mitoxantrone or daunorubicin) during remission induction and consolidation therapy.13 These Cell separation procedures clinical findings prompted us to hypothesize that adminis- tration of chemotherapy regimens of the AML10 protocols After informed consent was given, bone marrow was could result in severe toxic effects involving both hemato- obtained by aspiration from the posterior iliac crest and poietic and microenvironmental progenitor cell compart- mononuclear cells (MNC) were separated by centrifugation ments. (400 g, 30 min, 4°C) on a Ficoll–Hypaque gradient (density It was, therefore, the aim of the present study to investi- = 1.077 g/ml). Interface cells were washed and suspended gate marrow hematopoietic and microenvironmental pro- in RPMI-1640 (Gibco, Grand Island, NY, USA) sup- genitors in AML patients of the AML10 pilot or ran- plemented with 20% fetal bovine serum (FBS; Hyclone, domized trials. Hematopoietic progenitors were studied by Logan, UT, USA). means of a short-term methylcellulose assay allowing us to quantitate multipotent (CFU-Mix), erythroid (BFU-E) and CFU-Mix, BFU-E and CFU-GM assay granulocyte–macrophage (CFU-GM) progenitors as well as a long-term assay which allows us to quantitate primitive The assay for multilineage colony-forming units (CFU- progenitors capable of initiating and sustaining hematopo- Mix), erythroid bursts (BFU-E), and granulocyte– iesis in long-term culture (long-term culture initiating cells, macrophage colony-forming units (CFU-GM) was carried LTC-IC).15 The marrow microenvironment was studied by out as described elsewhere.18 Briefly, 5 × 104 MNC were quantitating marrow fibroblast colony-forming cells (CFU- plated in 35-mm Petri dishes in 1-ml aliquots of Iscove’s F) and by evaluating the capacity of stromal layers to modified Dulbecco’s medium (IMDM; Seromed, Berlin, support the growth of allogeneic normal hematopoietic Germany) containing: 30% FBS (Stem Cell Technologies, progenitors.16 Vancouver, Canada); 10−4 m 2-mercaptoethanol (Gibco), and 1.1% (w/v) methylcellulose. Cultures were stimulated with interleukin-3 (10 ng/ml; Sandoz, Basel, Switzerland), Materials and methods granulocyte colony-stimulating factor (10 ng/ml, Amgen, Thousand Oaks, CA, USA), granulocyte–macrophage Patients colony-stimulating factor (10 ng/ml; Sandoz) and erythro- poietin (3 U/ml; Amgen). Progenitor cell growth was evalu- ° Twenty-six patients (12 males, 14 females) with a median ated after incubation (37 C, 5% CO2) for 14–18 days in a age of 45 years (range, 14 to 59) and a diagnosis of AML humidified atmosphere. Four dishes were set up for each were included in this study. The diagnosis of AML was individual data point per experiment. CFU-Mix defined as based on the criteria of the French–American–British containing at least erythroid and granulocytic cells, BFU- (FAB) Cooperative Group.17 The study population included E with >500 cells and CFU-GM with >40 cells were patients with different FAB subtypes (M1 (n = 3), M2 (n scored from the same dish. = 9), M4 (n = 8), M5 (n = 5), and M6 (n = 1)). All patients had been treated at the Department of Hematology, ‘La Long-term culture-initiating cell (LTC-IC) assay Sapienza’ University, Rome, according to different remission induction and consolidation protocols. Nine The long-term culture-initiating cell (LTC-IC) assay was patients enrolled in the AML10 GIMEMA/EORTC pilot performed according to a previously described tech- study received the ICE (idarubicin, cytosine arabinoside, nique.15,19 Briefly, MNC were resuspended in complete etoposide) regimen as remission induction therapy and the medium consisting of alpha-medium (Gibco) supplemented NOVIA (mitoxantrone, cytosine arabinoside) regimen as with fetal bovine serum (12.5%), horse serum (12.5%), l- consolidation therapy, whereas the remaining 17 patients glutamine (4 mm), 2-mercaptoethanol