Peripheral Blood Stem Cell (PBSC) Mobilization with Chemotherapy Followed by Sequential IL-3 and G-CSF Administration in Extensively Pretreated Patients

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Peripheral blood stem cell (PBSC) mobilization with chemotherapy followed by sequential IL-3 and G-CSF administration in extensively pretreated patients

K Kolbe1, C Peschel1, B Rupilius1, D Despre´s1, K Burger2, I Sklenar2,LFa¨rber2, C Huber1 and HG Derigs1

1Division of Hematology, III. Department of Medicine, Johannes Gutenberg University, Mainz; and 2Novartis AG, Nu¨rnberg, Germany

Summary:

Extensive pretreatment has been identified as a signifi- Myeloablative therapy with autologous peripheral blood cant risk factor for failure of sufficient PBSC mobiliz- stem cell (PBSC) rescue has become a widely used treat- ation. From published data and our own experience we ment modality for a variety of advanced malignancies. The defined pretreatment variables which render patients at use of PBSC as a stem cell source leads to more rapid risk for not collecting at least 2.5 × 106 CD34-positive engraftment and may also have less tumor cell contami- cells per kg bodyweight (BW). These variables were pre- nation compared to bone marrow.1–3 The number of CD34- vious unsuccessful PBSC mobilization trial, previous positive cells per kilogram body weight (BW) infused has large field radiotherapy, four or more cycles of myelo- been shown to correlate with neutrophil and platelet recov- suppressive chemotherapy regimens, and combinations ery kinetics. The reinfusion of more than 2.5 × 106 CD34- of extended field radiotherapy plus chemotherapy. positive cells/kg BW ensures an early and sustained hema- Based on these inclusion criteria we treated 19 patients topoietic recovery after myeloablative therapy.4,5 with disease-specific conventional-dose chemotherapy Mobilization protocols utilize hematopoietic growth fac- followed by sequential subcutaneous administration of tors such as granulocyte colony-stimulating factor (G-CSF) IL-3 (5 ␮g/kg BW) for 5 consecutive days and G-CSF or granulocyte–macrophage colony-stimulating factor (10 ␮g/kg) until PBSC collection or neutrophil recovery. (GM-CSF) administered during steady-state hematopoiesis Patients were 10 males and nine females with a median or after cytotoxic chemotherapy, to increase the number of age of 43 years. Diagnoses were non-Hodgkin’s lym- circulating hematopoietic progenitor cells. However, col- -multiple myel- lection of sufficient CD34-positive cells may not be poss ,2 ؍ Hodgkin’s disease n ,5 ؍ phoma n and testicular can- ible in all patients. It has been shown that pretreatment with 4 ؍ AML n ,4 ؍ CML n ,2 ؍ oma n Twelve patients had prior unsuccessful trial cytotoxic agents or radiotherapy adversely affects the yield .2 ؍ cer n of PBSC mobilization with chemotherapy followed by of CD34-positive cells.6–8 On the other hand, age, sex, dis- G-CSF. Except for mobilization chemotherapy-related ease status and bone marrow involvement did not signifi- neutropenic fever, no major toxicities (WHO grade у2) cantly influence mobilization results in lymphoma patients.9 were observed. Growth factors were well tolerated. Col- There is preclinical and preliminary clinical evidence lection of at least 2.5 ؋ 106 CD34-positive cells per kg that the combination of G-CSF or GM-CSF with inter- BW was possible in 11 out of 19 patients (58%). In five leukin-3 (IL-3) acts synergistically to mobilize PBSC in out of 12 patients with a previous unsuccessful trial of steady-state conditions or after cytotoxic therapy.10 Geissler PBSC mobilization, the study regimen mobilized suf- 11 ficient CD34-positive cells. Nine patients went on to et al described in six extensively pretreated lymphoma high-dose chemotherapy followed by autologous PBSC patients the successful mobilization of PBSC by sequential transplantation. Prompt hematologic recovery was seen IL-3 (7 days) and G-CSF administration. In this series no in all of them. In conclusion, the sequential adminis- mobilization chemotherapy was given. Two patients had an tration of IL-3 followed by G-CSF after conventional- unsuccessful trial of PBSC mobilization with G-CSF 11 12 dose chemotherapy allows successful PBSC collection in alone. D’Hondt et al reported on the safe and successful the majority of extensively pretreated patients. administration of IL-3 combined with G-CSF for PBSC Keywords: PBSC; IL-3; G-CSF; transplantation; mobil- mobilization. ization In the present study, we evaluated the sequential administration of mobilization chemotherapy followed by IL-3 and G-CSF for efficiency of PBSC mobilization in extensively pretreated patients or in patients who had an unsuc- Correspondence: Dr HG Derigs, Division of Hematology, III. Department cessful trial of PBSC collection in the past. of Medicine, Johannes Gutenberg-Universita¨t, Langenbeckstr. 1, D-55101 Mainz, Germany Received 27 March 1997; accepted 27 August 1997 PBSC mobilization with IL-3 and G-CSF in extensively pretreated patients K Kolbe et al 1028 Materials and methods CS3000 plus; Baxter Deutschland, Unterschleiβheim, Germany). Leukaphereses were performed daily until a Patient population minimum of 2.5 × 106/kg BW CD34-positive cells were collected. Apheresis procedures were stopped if the CD34- From August 1994 until August 1996, 19 patients, all positive population in the peripheral blood dropped below treated at the University Hospital Mainz, were enrolled into 5/␮l. The cells were cryopreserved in cell culture medium, a prospective pilot study of sequential IL-3 and G-CSF which contained heparin 10 U/ml, dimethylsulfoxide 10%, administration after chemotherapy for PBSC collection. penicillin 50 U/ml and streptomycin 0.05 mg/ml. The cell Inclusion criteria for patients entering the study were one or suspensions were frozen at a controlled rate in a computer- more of the following: (1) previous large field radiotherapy; controlled freezing chamber (Cryoson, Scho¨llkrippen, (2) more than four previous myelosuppressive chemo- Germany) and stored in liquid nitrogen at −196°C until therapy cycles; (3) previous combined extended field radio- reinfusion. therapy and chemotherapy; and (4) previous unsuccessful In the present study PBSC were mobilized by disease- trial of PBSC mobilization. Characteristics of the patients specific chemotherapeutic treatment followed by daily sub- are shown in Table 1. All patients gave informed consent cutaneous administration of 5 ␮g/kg BW IL-3 (Sandoz, before entering the study. Nu¨rnberg, Germany) for 5 days followed by daily subcutaneous injections of 10 ␮g/kg BW G-CSF until completion Mobilization procedure and PBSC collection of leukapheresis. Progenitor harvest was started and performed as mentioned above. Twelve out of 19 patients had an unsuccessful mobilization trial in the past, consisting of disease-specific chemotherapeutic treatment followed by daily subcutaneous adminis- Conditioning regimen and PBSC reinfusion tration of 5–10 ␮g/kg BW G-CSF (Filgrastim; Amgen, Thousand Oaks, CA, USA) until completion of leukapher- The pretransplant conditioning regimens consisted of total esis (Table 2). WBC was monitored daily during hematopo- body irradiation (TBI; 14.4 Gy in 12 doses over 4 days) ietic recovery and CD34-positive cells were evaluated daily and cyclophosphamide (120 mg/kg) in one patient; busul- after passing the WBC nadir and recovering to a WBC of fan (16 mg/kg) and cyclophosphamide (120 mg/kg) in one 1.0 × 109/l. Leukapheresis procedures were generally per- patient; carmustin (300 mg/m2), melphalan (140 mg/m2), formed if a CD34-positive cell population of у5/␮l was cytosine arabinoside (1600 mg/m2), etoposide (400 mg/kg) detected by flow cytometry. Leukapheresis was performed in two patients, mephalan (200 mg/kg) in two patients, car- with a continuous flow blood cell separator (Fenwall mustin (600 mg/m2), cyclophosphamide (90 mg/kg), cyto-

Table 1 Pretreatment characteristics of patients mobilized with sequential administration of IL-3 and G-CSF

Patient No. Sex Age Diagnosis Remission, status Previous cytotoxic therapy Previous mobilization attempts

1 M 51 MM PD MP (10) VAD (4), HDCy, XRT L1–5 + sacrum 1 2 M 25 HD 1. Rel. dose increased COPP/ABVD (4) 0 3 F 41 AML CR DAV (2) 1 4 F 54 lg NHL 2. PR COP (?), PmMi (2), XRT retrop.? 0 5 F 53 hg NHL 2. CR CHOP (4), XRT inv. Y 0 6 F 34 AML CR AraC, Ida (1), AraC, Amsa (1) 1 7 M 34 hg NHL 2. CR CHOEP (6), XRT Mantel ﬁeld 0 8 M 59 MM PR VAD (6), XRT cerv. + thor. spine, sacrum 0 9 M 51 Seminoma PR PEB (2), PEI (1), XRT retrop. 1 10 M 45 hg NHL PR BMF B-NHL (6), IEV (1) 1 11 M 35 AML CR AraC, Ida (1), AraC, Amsa (1) 1 12 F 43 lg NHL 2. CR CHOEP (4), XRT total nodal 2 13 F 44 HD 1. Rel. COPP/ABVD (2), XRT total nodal 0 14 M 25 CML CP IFN, Hydrea, Ida, AraC 1 15 F 36 CML BC IFN, Hydrea, Ida, AraC 1 16 M 57 CML CP IFN, Hydrea, Ida, AraC 1 17 F 30 CML AP IFN, Hydrea, Ida, AraC 1 18 F 57 AML 3. Rel DAV (2), DA (2), AV (3), HAM (1) 1 19 M 46 NSGCT 2. Rel. PBE (4), VIP (3) 0

Sex: M = male; F = female. Diagnosis: MM = multiple myeloma; HD = Hodgkin’s disease; AML = acute myeloid leukemia; NHL = non-Hodgkin’s lymphoma; lg = low grade; hg = high grade; CML = chronic myeloid leukemia. Remission status: PD = progressive disease; Rel. = relapse; PR = partial remission; CR = complete remission; CP = chronic phase; BC = blast crisis. Cytotoxic therapy: XRT = radiation therapy; MP = melphalan, prednisone; VAD = vincristine, adriamycin, dexamethasone; HDCy = high-dose cyclophosphamide; COPP/ABVD = cyclophosphamide, vincristine, procarbacine, prednisone, adriamycin, bleomycin, vinblastine, dacarbacine; DA = daunorubicin, cytosine arabinoside; AV = etoposide, cytosine arabinoside; DAV = daunorubicin, cytosine arabinoside, etoposide; HAM = high-dose cytosine arabinoside, mitoxantrone; COP = cyclophosphamide, vincristine, prednisone; PmMi = prednimustine, mitoxantrone; AraC = cytosine arabinoside; Ida = idarubicine; Amsa = amsacrine; CHOP = cyclophosphamide, adriamycin, vincristine, prednisone; CHOEP = cyclophosphamide, adriamycin, vincristine, etoposide, prednisone; PEB = cisplatinum, etoposide, bleomycin; PEI = cisplatin, etoposide, ifosfamide; IEV = ifosfamide, epiorubicin, etoposide; IFN = interferon-␣. PBSC mobilization with IL-3 and G-CSF in extensively pretreated patients K Kolbe et al 1029 Table 2 Characteristics of patients with unsuccessful mobilization trial in the past

Patient No. Mobilization chemotherapy G-CSF dose/duration Maximum number of Time interval between ﬁrst (ﬁrst trial) of treatment CD34-positive cells per and second mobilization ␮l peripheral blood attempt (months)

1 HDCy 5␮g/kg; 8 days 0 3 3 DAV 5 ␮g/kg; 12 days 0 1.5 6 AraC/Amsa 10 ␮g/kg; 15 days 0 1.5 9 PEI 8 ␮g/kg; 8 days 4 1 10 IEV 8 ␮g/kg; 13 days 0 1 11 AraC/Amsa 10 ␮g/kg; 5 days 0 1 12 CHOEP 5 ␮g/kg; 10 days 2 3 14 Ida/AraC 10 ␮g/kg; 27 days 0 3 15 Ida/AraC 10 ␮g/kg; 26 days 5 2.5 16 Ida/AraC 10 ␮g/kg; 12 days 4 4.5 17 Ida/AraC 10 ␮g/kg; 23 days 11 3 18 HAM 5 ␮g/kg; 17 days 12 12

Cytotoxic therapy: HDCy = high-dose cyclophosphamide; DAV = daunorubicin, cytosine arabinoside, etoposide; HAM = high-dose cytosine arabinoside, mitoxantrone; AraC = cytosine arabinoside; Ida = idarubicine; Amsa = amsacrine; CHOEP = cyclophosphamide, adriamycin, vincristine, etoposide, prednisone; PEI = cisplatin, etoposide, ifosfamide; IEV = ifosfamide, epirubicin, etoposide. sine arabinoside (24 g/m2), etoposide (1600 mg/kg) in three Colony growth was evaluated in semisolid medium as patients; and carboplatin (2100 mg/m2), etoposide previously described.13 In brief, mononuclear cells from (2250 mg/m2) in one patient. Forty-eight hours after the last leukapheresis products were separated by Ficoll gradient dose of chemotherapy, all of the cryopreserved apheresis centrifugation. Cells were suspended at a density of 5 × 103, products were thawed rapidly at the bedside and were rein- 104 and 2 × 104 in culture medium containing 0.3% agar in fused via an indwelling central venous catheter. All patients a volume of 0.25 ml and plated in multiwell tissue culture received prophylactic oral antibiotic therapy with ofloxacin. plates (Nunc, Wiesbaden, Germany). After solidification of Antibiotic combination therapy was administered for fever the agar layer, a liquid overlayer of culture medium was greater than 38.3°C, and amphotericin B was added for added to the culture plates containing hematopoietic growth documented fungal infection or persistent fever. A platelet factors. The final concentration of growth factors was as count greater than 20 × 109/l was maintained by single- follows: G-CSF 200 ng/ml; IL-3 50 ng/ml. At the end of a donor platelet transfusions, and packed red blood cells were 14-day incubation period, the agar cultures were fixated given when the hemoglobin was less than 8.5 g/dl. All with glutaraldehyde 2.5%, washed with distilled water for blood products were leukocyte depleted and irradiated. 4 h, transferred on to microscopic slides, dried at 40°C, stained with May–Gru¨nwald–Giemsa, and permanently mounted. Colony numbers were evaluated in such slides by Measurement of progenitor cell content light microscopy. The frequency of CFUs was calculated The percentage of cells that expressed the CD34 antigen as follows: frequency = (CFU count/seeding density). To was determined in whole blood or in a sample of the leu- evaluate the total number of collected CFUs/kg BW the kapheresis product by direct immunofluorescence. Twenty seeding density dependent optimal rate of CFUs was ␮l of the sample was incubated for 15 min at 4°C with divided by the sample Ficoll recovery multiplied by the fluorescence-labeled monoclonal antibodies against CD34 total white cell count in the leukapheresis products divided and CD45 (Immunotech, Hamburg, Germany). After lysis by BW. of the erythrocytes with isomolar ammonium chloride buffer for 10 min, the cells were resuspended in phosphate- buffered saline (PBS) that contained 0.2% (w/v) bovine Statistical analysis serum albumin (BSA). The cells were analyzed by flow The clinical and laboratory data of patients were analyzed cytometry (Coulter, Krefeld, Germany). We used a for- according to standard statistical methods. Pearson corre- ward-scatter vs CD45 fluorescence dot plot to discriminate lation was calculated between CD34-positive cells and CFU between the smallest lymphohematopoietic cell population content of leukapheresis products (GraphPad Software, San and erythrocyte debris. The CD34-positive cell population Diego, CA, USA). A significance level of P Ͻ 0.05 was was analyzed in a fluorescence-1 vs side-scatter character- chosen. istic (SSC) dot plot. Only cells with lymphoid or lymphob- lastoid characteristics (low SSC) were counted as CD34- positive cells. The rate of CD34-positive cells was calcu- Results lated as follows: rate = (CD34-positive cells/CD45-positive cells). To evaluate the total number of collected CD34-posi- Toxicity tive cells/kg BW the frequency of CD34-positive cells was multiplied by the total white cell count in the leukapheresis The observed side-effects of the cytokine administration products divided by BW. during the mobilization regimen are shown in Table 3. The PBSC mobilization with IL-3 and G-CSF in extensively pretreated patients K Kolbe et al 1030 Table 3 Frequency of adverse events of the mobilization regimen with 10000 sequential administration of chemotherapy, IL-3 and G-CSF.

Severity of adverse events (WHO) 1000 /kg BW)

1234 4

100 Fever 2 5 2 Headache 1 Nausea 2 Skeletal pain 2 10

adverse effects were mainly due to chemotherapy-induced Colony-forming units (10 myelotoxicity. Fever was seen in 47% of the patients, mostly WHO grade 1 or 2. A minority of patients suffered from headache, nausea and skeletal pain. Rashes did not occur. 1 10 100 Three febrile patients had microbiologically documented infections. Coagulase negative staphylococci could be CD34-positive cells (106/kg BW) identified in the blood cultures of two patients. One patient Figure 1 Correlation between CD34-positive cells and CFU-GM (᭿) developed an aspergillus pneumonia during the neutropenic and BFU-E (̆) in leukapheresis products of patients mobilized by chemo- period. She was treated with a prolonged course of ampho- therapy followed by IL-3 and G-CSF administration. Colony assays were tericin B and was subsequently successfully transplanted performed as described in Materials and methods. without reactivation of fungal infection. One patient developed a pectangina during a neutropenic fever episode. Table 4 shows the mobilization results according to patient characteristics. All patients with extensive previous chemotherapy and a significant number of patients with PBSC collection previous radiotherapy mobilized sufficient CD34-positive Nineteen patients with a variety of hematologic malignancies were entered into the mobilization study. In 14 of Table 4 Success of PBSC mobilization by sequential administration them a CD34-positive cell population of у5/␮l was of chemotherapy, IL-3, and G-CSF according to diagnosis, disease status, detected in the peripheral blood by flow cytometry during and pretreatment hematopoietic reconstitution. Eleven patients (58% of the total population) had a successful stem cell mobilization n Collection of Collection of Ͼ2.5 × 106/kg Ͻ2.5 × 106/kg × 6 + + with Ͼ2.5 10 CD34-positive cells/kg BW (median CD34 cells CD34 cells 4.5 × 106/kg). This subpopulation received growth factors at a median of 11 days and underwent a median of two Patients 19 11 8 apheresis procedures. Three of 14 patients received growth Male 10 8 2 factors for a median of 11 days and underwent a median Female 9 3 6 of three leukaphereses without mobilizing the minimum Diagnosis of 2.5 × 106 CD34-positive cells/kg BW (median Multiple myeloma 2 1 1 0.2 × 106/kg). In five patients, a population of у5/␮l CD34- High-grade NHL 3 3 0 Low-grade NHL 2 0 2 positive cells could never be detected in the peripheral Hodgkin’s disease 2 2 0 blood during a median of 20 days of cytokine adminis- Testicular cancer 2 2 0 tration. AML 4 1 3 The median number of CFU–GM collected in apheresis CML 4 2 2 procedures was 44.9 (n = 10; range 10.8–2528) × 104/kg. Disease status The median number of BFU-E mobilized was 3.5 (range Complete remission 6 3 3 × 4 Partial remission 5 5 0 0–81.5) 10 /kg. There was a good correlation between the Relapse 4 2 2 results in mobilization of CD34-positive cells and colony- CML chronic phase 3 2 1 forming cells as shown in Figure 1. The correlation coef- CML blast crisis 1 0 1 ficient (r) between leukapheresis content in CD34-positive Pretreatment cells and CFU-GM was 0.91 and correlation between Large field radiation 6 4 2 CD34-positive cells and BFU-E was 0.96. Ͼ6 myelosuppressive 3 3 0 Two of the four CML patients mobilized more than chemotherapy Radiation and 8 5 3 × 6 2.5 10 /kg CD34-positive cells. Cytogenetic analysis of chemotherapy leukapheresis products revealed only Ph-positive meta- Previous unsuccessful 12 5 7 phases. The only mobilized AML patient had no clonal mobilization trial marker to check for leukemic contamination. PBSC mobilization with IL-3 and G-CSF in extensively pretreated patients K Kolbe et al 1031 cells. Twelve patients had undergone an unsuccessful mobi- genitor mobilization by the sequential administration of IL- lization trial previously. The study protocol was able to 3 and GM-CSF compared to GM-CSF alone.14,15 Brugger mobilize sufficient CD34-positive cells for stem cell trans- et al16 reported the use of chemotherapy followed by GM- plantation in five of these patients. In addition to three leu- CSF with or without IL-3 for PBSC mobilization. They kemia patients, there was one patient with a germ cell found a significantly better CD34-positive cell collection in tumor and one patient with a high-grade NHL in this group. mildly to moderately pretreated patients who received the combination of IL-3 and GM-CSF compared to GM-CSF alone. D’Hondt et al12 reported on the successful mobiliz- Engraftment data ation of eight patients who received sequential or simultaneous IL-3 and G-CSF following chemotherapy. Geissler So far, nine patients, including one patient with CML and et al11 used IL-3 and G-CSF vs G-CSF alone without pre- one patient with AML, received high-dose chemotherapy ceding chemotherapy for PBSC mobilization in extensively followed by rescue with the collected PBSC. The median pretreated patients in an intrapatient comparison. They number of days to recover WBC Ͼ1000/␮l was 11 (range could demonstrate a significant better PBSC mobilization 9–15). Median number of days to platelet recovery after sequential IL-3 and G-CSF administration compared Ͼ20 000/␮l was 12 (range 9–27). The short time of bone with G-CSF alone. marrow recovery was reflected by the low need for RBC The cytokine combination of IL-3 and G-CSF was well (median 4, range 2–10 units packed RBC) or platelet tolerated in our patients and adverse reactions of the mobil- (median 6, range 2–9 single donor apheresis products) ization regimen were mainly chemotherapy related. No transfusions. The CML patient recovered with Ph-positive patient interrupted the mobilization treatment. As has been hematopoiesis. The AML patient had a relapse 2 months described by D’Hondt and others, the combination of IL-3 after autografting. and G-CSF seems to be better tolerated than the combination of IL-3 and GM-CSF.12,17 The sequential administration of IL-3 and G-CSF following chemotherapy allowed, in the majority of extensively pretreated patients investigated, the successful mobilization Discussion of sufficient PBSC, as shown by CD34-positive cell count. A number of patients with a previous unsuccessful trial of Collection of sufficient PBSC for safe rescue after mye- PBSC collection, were able to mobilize sufficient CD34- loablative therapy in heavily pretreated patients poses a positive cells for autografting, using IL-3 and otherwise major clinical problem. Based on literature data, the follow- nearly identical mobilization procedures. Engraftment in ing pretreatment conditions seem to pose an increased risk nine patients was rapid and sustained after high-dose ther- for insufficient PBSC mobilization: previous large field apy. These data imply that sufficient numbers of committed irradiation; previous repeated myeloaplasiogenic therapy; as well as early progenitor cells are mobilized by the regi- and combined previous chemotherapy and radiotherapy.4,7,9 men used. In an attempt to confirm these risk factors we performed a In the recent past, other cytokine combinations have been retrospective analysis of PBSC mobilization outcome at our tested for PBSC mobilization in heavily pretreated patients. center in 91 consecutive patients with a wide variety of 18 diagnoses, pretreatment and disease status. Sixty-eight per- Winter et al described a synergistic effect of sequential × 6 administration of GM-CSF and G-CSF on PBSC mobiliz- cent of patients mobilized more than 2.5 10 CD34-posi- 19 tive cells/kg BW by use of standard mobilization pro- ation. Moskowitz et al reported on the use of the recombi- cedures (chemotherapy + G-CSF 5 ␮g/kg BW s.c.). nant human stem cell factor (rhSCF) (5–15 ␮g/kg/day) and Univariate logistic regression models revealed active and Filgrastim (10 ␮g/kg/day) compared to Filgrastim alone for progressive disease status as the only statistically signifi- mobilization of PBSC in relapsed NHL patients. Heavily × 6 cant characteristic for mobilization failure (data pretreated patients had a median 2.9 (0.09–13.3) 10 /kg unpublished). Because of the heterogeneity and the limited BW CD34-positive cells after rhSCF plus Filgrastim number of cases, no other variables showed a statistically (n = 16) vs 1.4 (0.03–3.4) × 106/kg BW after Filgrastim significant association with the probability of a successful alone (n = 5).19 A prospective randomized clinical trial mobilization. However, none of five patients who comparing these two cytokine regimens after mobilization underwent large field radiotherapy (mantle field, inverted y chemotherapy in heavily pretreated NHL patients is cur- field, large field abdominal irradiation) and only one of rently ongoing. seven patients with more than two previous myelosuppress- In conclusion, the mobilization regimen with chemo- ive salvage chemotherapy cycles (Dexa-BEAM, IEV, therapy followed by sequential administration of IL-3 and HAM, VIP, PEB) reached the minimum number of G-CSF was safe and well tolerated. The mobilization of 2.5 × 106 CD34-positive cells/kg BW, thus suggesting that more than 2.5 × 106/kg CD34-positive cells was possible these factors may play a role in the successful mobilization in a significant number of patients, even after a previous of PBSC. unsuccessful trial of stem cell mobilization. Engraftment We started a trial to collect sufficient PBSC in heavily after PBSC transplantation was rapid and stable. In our pretreated patients, using IL-3 in addition to G-CSF after view, the data warrant the randomized evaluation of an IL- chemotherapy. Interleukin 3 has been used before for PBSC 3 and G-CSF containing mobilization regimen compared to mobilization. Different groups reported the improved pro- G-CSF alone in extensively pretreated patients. PBSC mobilization with IL-3 and G-CSF in extensively pretreated patients K Kolbe et al 1032 Acknowledgements 9 Haas R, Mohle R, Fruhauf S et al. 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