Recombinant human granulocyte and granulocyte ... - Nature

Bone Marrow Transplantation, (1998) 22, 625–630  1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt

Recombinant human granulocyte and granulocyte–macrophage colony-stimulating factor (G-CSF and GM-CSF) administered following cytotoxic chemotherapy have a similar ability to mobilize peripheral blood stem cells S Hohaus1, H Martin2, B Wassmann2, G Egerer1, U Haus3, L Fa¨rber3, KJ Burger3, H Goldschmidt1, D Hoelzer2 and R Haas1,4 1

Department of Internal Medicine V, University of Heidelberg; 2Department of Internal Medicine, University of Frankfurt; 3Novartis Pharma GmbH, Nu¨rnberg; and 4Clinical Cooperation Unit, Molecular Hematology and Oncology, German Cancer Research Center, Heidelberg, Germany

Summary: The availability of hematopoietic growth factors has greatly facilitated the mobilization and collection of peripheral blood stem cells (PBSC). It was the aim of this double-blind study to compare the PBSC-mobilizing efficacy of recombinant human G-CSF and GMCSF when administered post-chemotherapy. Twenty-six patients with relapsed Hodgkin’s disease were included in the study. Their median age was 31 years (range, 22– 59) and 14 patients were males and 12 were females. Patients were pretreated with a median of eight cycles of cytotoxic chemotherapy, while 18 patients had undergone extended field irradiation. The patients received dexamethasone 24 mg days 1–7, melphalan 30 mg/m2 day 3, BCNU 60 mg/m2 day 3, etoposide 75 mg/m2 days 4–7, Ara-C 100 mg/m2 twice daily days 4–7 (DexaBEAM). Twelve patients were randomized to receive 5 ␮g/kg/day G-CSF and 14 patients to receive 5 ␮g/kg/day GM-CSF, both administered subcutaneously starting on day 1 after the end of Dexa-BEAM. Primary endpoints of the study were the number of CD34+ cells harvested per kg body weight on the occasion of six consecutive leukaphereses and the time needed for hematological reconstitution following autografting. Twenty-one patients completed PBSC collection, and six patients of the G-CSF group and nine of the GM-CSF group were autografted. No difference was observed with respect to the median yield of CFU-GM and CD34+ cells: 32.5 × 104/kg vs 31.3 × 104/kg CFUGM, and 7.6 × 106/kg vs 5.6 × 106/kg CD34+ cells, for GCSF and GM-CSF, respectively (U test, P = 0.837 and 0.696). High-dose chemotherapy consisted of cyclophosphamide 1.7 g/m2 days 1–4, BCNU 150 mg/m2 days 1– 4, etoposide 400 mg/m2 days 1–4. All patients transplanted with more than 5 × 106 CD34+ cells/kg had a rapid platelet recovery (20 × 109/l) between 6 and 11 days and neutrophil recovery (0.5 × 109/l) between 9 and

Correspondence: Dr S Hohaus, Department of Internal Medicine V, University of Heidelberg, Hospitalstr. 3, 69115 Heidelberg, Germany Received 9 April 1998; accepted 9 June 1998

16 days, while patients transplanted with less than 5 × 106/kg had a delayed reconstitution, regardless of the kind of growth factor used for PBSC mobilization. In conclusion, our data indicate that in patients with Hodgkin’s disease G-CSF and GM-CSF given after salvage chemotherapy appear to be not different in their ability to mobilize PBSC resulting in a similar time needed for hematological reconstitution when autografted following high-dose therapy. Keywords: G-CSF; GM-CSF; mobilization; peripheral blood stem cells; autologous transplantation

Hematopoietic growth factors have greatly facilitated the mobilization and collection of peripheral blood stem cells (PBSC) for autologous and allogeneic transplantation following high-dose therapy. Cytotoxic chemotherapy is associated with an increase in the concentration of circulating hematopoietic progenitor cells during marrow recovery, while the addition of hematopoietic growth factors postchemotherapy further increases the rebound level of the progenitor cells.1,2 G-CSF and GM-CSF were first introduced to shorten the period of severe neutropenia and are now commonly used for the mobilization of PBSC.3 In normal healthy volunteers, both G-CSF and GM-CSF administered at a dose of 5 ␮g/kg/day during steady-state hematopoiesis resulted in the collection of 33.6- and 35.6-fold greater numbers of CFU-GM when compared with harvesting performed without mobilization.4 Evaluating the PBSC-mobilizing ability of both factors, Lane et al5 observed a significantly greater concentration of circulating CD34+ cells in normal volunteers on the 5th day of G-CSF treatment in comparison with GM-CSF (61 vs 3 CD34+ cells/␮l). Both growth factors were given during steadystate hematopoiesis at a dose of 10 ␮g/kg/day. Bolwell et al6 compared the hematological recovery after transplantation of G-CSF- and GM-CSF-mobilized PBSC. They suggested that hematopoietic recovery was faster when G-CSFmobilized PBSC, bone marrow and G-CSF were used posttransplantation in comparison with GM-CSF-mobilized PBSC, bone marrow and GM-CSF post-transplantation.6 In this study, the contribution of the cytokine-mobilized PBSC

G-CSF and GM-CSF similarly mobilize PBSC S Hohaus et al

626

is difficult to evaluate since GM-CSF or G-CSF were also administered following transplantation which may affect the speed of hematological recovery differently. In a prospective study on 22 patients with multiple myeloma Demuynck et al7 found no significant differences in the numbers of circulating progenitors between patients receiving G-CSF or GM-CSF after single-dose cyclophosphamide. It was the purpose of this prospective randomized study to compare the efficacy of GM-CSF and G-CSF in mobilizing PBSC when given following cytotoxic chemotherapy in patients with relapsed Hodgkin’s disease. In addition, the time needed for hematological reconstitution following transplantation of GM-CSF- and G-CSF-mobilized PBSC was examined. Patients and methods Between August 1992 and December 1994, 26 patients with relapsed Hodgkin’s disease were enrolled in the study. Patients’ characteristics including the type and amount of previous cytotoxic therapy are given in Table 1. The study was conducted under the guidelines of the Joint Ethical Committee of the University of Heidelberg. Each patient gave his informed consent to participate in this study. Cytotoxic chemotherapy consisted of dexamethasone, 24 mg p.o. in divided doses daily on days 1–7, melphalan 30 mg/m2 on day 3, carmustine 60 mg/m2 on day 3, etoposide 75 mg/m2 on days 4–7 (total dose 300 mg/m2), cytosinearabinoside 100 mg/m2 twice daily on days 4–7 (total dose 800 mg/m2) (DexaBEAM). The patients were randomized to receive either 5 ␮g/kg/day G-CSF (filgrastim, Neupogen; Amgen, Munich, Germany) or 5 ␮g/kg/day GM-CSF (molgramostim, Leucomax; Novartis Pharma, Nu¨rnberg, Germany) subcutaneously. The study medication was blinded by the pharmacist. The growth factors were given 1 day after the end of cytotoxic chemotherapy until the last day of leukapheresis.

Table 1

Patient characteristics GM-CSF n = 14

Male Female Age median range Karnofsky median range Pretreatment chemotherapy Regimens median range Cycles median range Radiotherapy (extended field) BM involvement

10 4

G-CSF n = 12 4 8

30.5 24–59

32 22–58

100 80–100

100 80–100

3 2–4

2 0–4

7.25 3–18 7 3

8 0–16 11 3

PBSC collection began when the CD34+ cell count reached at least 2 × 106/l, and six leukaphereses were performed in each patient. The minimal requirement for a sufficient autograft was a total number of 4 × 108 nuclear cells (TNC)/kg body weight. Patients responding to DexaBEAM received a second cycle of DexaBEAM and proceeded to PBSC-supported high-dose therapy provided that a complete or partial remission had been achieved. High-dose therapy consisted of cyclophosphamide 1.7 g/m2 on days 1–4 (total dose 6.8 g/m2), carmustine 150 mg/m2 on days 1–4 (total dose 600 mg/m2), and etoposide 400 mg/m2 on days 1–4 (total dose 1600 mg/m2) (CBV). No growth factors were administered after high-dose therapy. Primary endpoints of the study were the number of CFUGM and CD34+ cells harvested per kg body weight on the occasion of six consecutive leukaphereses and the time needed following autografting to reach a stable unsupported platelet count of 20 × 109/l and a neutrophil count of 0.5 × 109/l. Secondary endpoints of the study were the time from start of cytotoxic chemotherapy to the end of PBSC collection and the total number of platelet transfusions given after ABSCT. In addition, the incidence of infections and days of fever greater than 38.5°C, and the days of hospitalization after ABSCT were recorded. Immunofluorescence staining and flow cytometry For immunofluorescence analysis, 1 × 106 mononuclear cells (MNC) of the leukapheresis product or 20–50 ␮l of whole blood (EDTA) were incubated for 30 min at 4°C using the fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (moAb) CD34 (HPCA-2) (Becton Dickinson, Heidelberg, Germany). Isotype-identical antibodies served as controls: IgG1, IgG2a (FITC/PE-conjugated, Becton Dickinson, Heidelberg, Germany). The cells were analyzed with a Becton Dickinson FACScan as previously described.8 Clonogenic assay for hematopoietic progenitor cells The concentration of hematopoietic progenitor cells in peripheral blood was assessed using a semisolid clonogenic culture assay (Stem Cell Technologies, Vancouver, Canada), as previously described.8 Statistics The yield of CFU-GM and CD34+ cells collected on the occasion of six consecutive leukaphereses was compared by means of the Mann–Whitney U test. The time needed for hematological reconstitution was compared between the two groups by the Wilcoxon’s test of equality of survival curves over scale.

Results PBSC collection Twenty-six patients with refractory or relapsed Hodgkin’s disease were included in this double-blind prospective ran-


Toxicity The incidence and severity of adverse events did not differ between the two groups of patients (Table 2). Fever and bone pain were the most common side-effects with fever being more pronounced in the GM-CSF group. Still, adverse events in the group of patients treated with GMCSF resulted more often in a discontinuation of the study (four patients vs one patient). These adverse events were judged to be cytokine-related in only two of these patients who received GM-CSF (pain WHO two; pulmonary distress WHO three, 10 min after first administration of GMCSF), while musculoskeletal pain in one patient and an episode of acute psychosis with disturbance of consciousness in another patient were considered to be cytokine-unrelated adverse events. Other reasons for discontinuation in the GCSF group were insufficient PBSC mobilization in three patients and progressive disease in two patients, while one patient of the GM-CSF group wanted to continue the therapy in another hospital. As a result, there were six patients in the G-CSF group and nine patients in the GM-CSF group who proceeded to PBSC-supported high-dose therapy and completed the study. Hematological reconstitution following PBSC-supported high-dose therapy The median number of CD34+ cells and CFU-GM autografted per kg body weight tended to be greater in the GCSF group (10.0 × 106 vs 6.1 × 106 CD34+ cells/kg and 54.6 × 104/kg vs 35.6 × 104/kg). Reconstitution of neutrophils to counts of 0.5 × 109/l was observed within 10–19 days and was independent of the growth factor used for mobilization (median, 11.5 days (G-CSF) vs 13 days (GM-

a

627

CFU-GM (× 105/kg)

100

10

1

0.1

G-CSF

b

GM-CSF

100

CD34+ cells (×106/kg)

domized study to compare the PBSC-mobilizing efficacy of G-CSF and GM-CSF when given following cytotoxic chemotherapy. Twelve patients were randomized to receive G-CSF, while GM-CSF was given in 14 patients. In both groups of patients, leukaphereses were commenced 18 days following the first day of cytotoxic chemotherapy (median, range 16–28 days (G-CSF) and 16–22 days (GM-CSF)). In patients who received G-CSF, white blood cell counts tended to be greater on the days of leukapheresis than in patients treated with GM-CSF. The difference between the two groups was most pronounced on the day of the second leukapheresis with a median WBC of 26 × 109/l (G-CSF) vs 5 × 109/l (GM-CSF). The greater WBC in patients of the G-CSF group was related to an increased proportion of neutrophils (76 vs 26%). On the other hand, platelet counts during the collection period tended to be greater in patients of the GM-CSF group. The envisaged number of six leukaphereses could be performed in nine patients of the G-CSF group (75%) and in 12 patients of the GM-CSF group (85%). The total number of nucleated cells, CFU-GM and CD34+ cells harvested was not different between the patients of both groups (Figure 1). The median time from the start of cytotoxic chemotherapy to the end of PBSC collection was 24 and 25 days for patients who received G-CSF and GM-CSF, respectively (range 23–42 days (G-CSF) and 19–30 days (GM-CSF)).

10

1

0.1

G-CSF

GM-CSF +

Figure 1 Yield of CFU-GM (a) and CD34 cells (b) per kg body weight by six leukaphereses in nine patients receiving G-CSF (䊊) and 12 patients receiving GM-CSF (䉬) following DexaBEAM chemotherapy. The bars indicate the median values: 32.5 × 104/kg vs 31.3 × 104/kg CFU-GM, and 7.6 × 106/kg vs 5.6 × 106/kg CD34+ cells, for G-CSF and GM-CSF, respectively (U test, P = 0.837 and 0.696).

CSF)). The two patients with the longest time needed for neutrophil recovery (17 and 19 days) received a small number of CD34+ cells (1.9 and 0.8 × 106 CD34+ cells/kg, respectively). The time needed for platelet reconstitution was strongly related to the number of CD34+ cells autografted, irrespective of the factor used for mobilization. All patients who received more than 5 × 106 CD34+ cells/kg had unsupported platelet counts of 20.0 × 109/l between 6 and 11 days. On the other hand, in patients autografted with less than 5 × 106 CD34+ cells/kg the time needed for platelet reconstitution was between 20 and 32 days. The toxicity associated with the CBV high-dose therapy did not differ between the two groups of patients. For instance, the median days of fever of greater than 38.5°C was similar (3 days (G-CSF) vs 2.5 days (GM-CSF)), as well as the duration of hospitalization post-transplantation (21 days (G-


Table 2 Adverse events during G-CSF or GM-CSF-supported DexaBEAM

a

G-CSF (n = 12 GM-CSF (n = 14 patients) WHO grade patients) WHO grade 2

3

4

1

2

3

4

1 2 1 2 − − 1 − − 1

3 1 2 − 2 − − 1 − −

− − 2 − − − 1a − − −

− − − − − − − − − −

− 1 − − 2 − − 1 − −

5 1 3b − 1 − 2 − − −

1 − − − − 1a − − 1a −

− − − − − − − − − −

CD34+ cells (× 106/kg)

Fever Nausea, vomiting Musculoskeletal pain Mucositis Cutaneous Pulmonary Abdominal Heart CNS Flu-like symptoms

1

25

20

15

10

5

0 0

a

Resulting in discontinuation of the study. In two of three patients resulting in discontinuation of the study.

5

10

b

b

CSF) vs 19 days (GM-CSF)), and the number of platelet transfusions (4 (G-CSF) vs 5 (GM-CSF)). Blood counts performed 3 months following PBSC-supported high-dose therapy showed almost normal neutrophil counts in all patients with values between 1.4 × 109/l and 15.6 × 109/l, while the platelet count was still below 100 × 109/l in three patients of the G-CSF group and in two patients of the GM-CSF group. Discussion To our knowledge, this is the first double-blind randomized prospective study comparing the PBSC-mobilizing effect of G-CSF and GM-CSF when administered following cytotoxic chemotherapy. Patients with relapsed or refractory Hodgkin’s disease were included to receive DexaBEAM as salvage therapy. DexaBEAM is an effective salvage treatment resulting in a response rate of 60% in patients with Hodgkin’s disease who failed to respond to multidrug cytotoxic chemotherapy.9 Although DexaBEAM contains relatively myelotoxic compounds such as carmustine and melphalan, the regimen still permits mobilization of a sufficient number of hematopoietic progenitor cells in the majority of patients.2,10 In a previous report, we observed a better mobilization of PBSC when GM-CSF was given following DexaBEAM instead of DexaBEAM alone.2 In the study presented, we administered G-CSF and GM-CSF at a dose of 5 ␮g/kg/day until the last leukapheresis was performed. The hematopoietic growth factors G-CSF and GM-CSF are widely used for the mobilization of hematopoietic progenitor and stem cells.3 Both cytokines also affect the function of mature blood cells. G-CSF preferentially acts in a lineage-restricted fashion on the colony-forming unit granulocyte (CFU-G) and mature neutrophils, while GM-CSF is a pleiotropic cytokine with a broad spectrum of target cells.3,11–14 For instance, stimulation of macrophages with GM-CSF results in an increase of antimicrobial and antitumor effects.12,13 It also supports the maturation and function of dendritic cells, which are involved in the induction of primary T cell immune responses.14 We have shown that

15

20

25

30

35

25

30

35

Days 25

20

CD34+ cells (× 106/kg)

628

15

10

5

0 0

5

10

15

20

Days Figure 2 Hematological reconstitution after high-dose CBV therapy supported with G-CSF (䊊) or GM-CSF (䉬) post-chemotherapy mobilized peripheral blood stem cells. (a) Time to achieve a neutrophil count of ⭓0.5 × 109/l, (b) time to achieve an unsupported platelet count of ⭓20 × 109/l.

administration of GM-CSF post-chemotherapy results in an increase of T cells expressing the low-affinity receptor for IL-2 (CD25), which was associated with increased serum levels of sCD25.15,16 These immune-modulatory effects with induction of secondary cytokines might also explain some of the differences between both growth factors with respect to their side-effects. Fever and musculoskeletal pain were the most common adverse effects during cytokine administration. These side-effects might be more severe during steady-state administration or during the phase of leukocyte recovery when target cells for secondary cytokine production are abundantly present. The incidence and severity of adverse events observed during G-CSF- and GM-CSF-supported chemotherapy, were similar and mild. Still, adverse events in the GM-CSF group led more often to a discontinuation of the study. A similar incidence of adverse events was observed in a


placebo-controlled multi-center trial by Greenberg et al.17 The patients of his study received GM-CSF following PBSC transplantation. More patients in the GM-CSF group discontinued treatment due to adverse events. At the time when this study began, there were only scarce data on a relationship between the number of CD34+ cells autografted and the speed of hematological reconstitution. Therefore, a constant number of six leukaphereses was performed to collect a total number of nucleated cells of greater than 4 × 108/kg body weight. This threshold number was based on the results of an earlier study performed in patients with Hodgkin’s disease,2 who had an extensive cytotoxic pretreatment. The yield of CD34+ cells varied greatly between 0.41 and 22.1 × 106/kg body weight and was not related to the kind of growth factor used for mobilization. Twenty-five of 26 patients included in our study were extensively pretreated and had previously received a median number of 7.25 and 8 cycles of cytotoxic chemotherapy (G-CSF and GM-CSF group). In addition, 18 of the 26 patients had undergone extended field radiotherapy. The higher proportion of irradiated patients might have negatively influenced the collection yield in the G-CSF group. In a later study, including 61 patients with malignant lymphoma, we found that each cycle of chemotherapy resulted in an average decrease of 0.2 × 106 CD34+ cells/kg per leukapheresis in non-irradiated patients, while large field radiotherapy reduced the collection efficiency by 1.8 × 106/kg CD34+ cells.18 Other studies also showed the adverse effect of previous cytotoxic treatment on PBSC mobilization.19–23 Following high-dose therapy, autografting of G-CSF- or GM-CSF-mobilized PBSC resulted in a rapid hematological reconstitution, provided that more than 5 × 106 CD34+ cells/kg were transplanted. The relationship between the number of CD34+ cells autografted and the time needed for hematological reconstitution has been confirmed in a large number of studies.8,18,24,25 In a larger group of patients, we have defined the threshold to 2.5 × 106 CD34+ cells/kg.18 No difference in the speed of hematological reconstitution was noted between patients receiving G-CSF- or GM-CSFmobilized PBSC. These data indicate that CD34+ cells mobilized by either G-CSF or GM-CSF into peripheral blood have a similar engraftment potency and proliferative capacity. These results are not unexpected, since the expression of lineage- and differentiation-associated antigens on CD34+ cells such as CD38 and CD33 is similar on PBSC harvested following G-CSF- and GM-CSF-supported cytotoxic chemotherapy.7,26 In contrast, the proportion of more primitive CD34+/CD38− progenitor cells in peripheral blood appears to be greater during steady-state administration of GM-CSF in comparison to G-CSF administration.27 Whether this translates into a more rapid hematological recovery has not been demonstrated yet. Obviously, accessory cells contained in the leukapheresis products harvested following mobilization using GM-CSF or G-CSF post-chemotherapy are either similar or, if different, without apparent effect on engraftment and hematopoietic recovery. Successful transplantation of selected CD34+ cells supports the idea that accessory cells have no signifi-

cant effect on the hematopoietic recovery after high-dose therapy with autologous PBSC support.28 In conclusion, our data indicate that in patients with Hodgkin’s disease G-CSF and GM-CSF given after salvage chemotherapy appear to be no different in their ability to mobilize PBSC, resulting in a similar time needed for hematological reconstitution when autografted following highdose therapy. Still, a significant difference might have been missed due to the relatively small number of patients studied. Acknowledgements We would like to thank Helmut Bachmann for statistical analysis and Ursula Scheidler for expert secretarial assistance. We are grateful to Margit Pfo¨rsich, Evi Holdermann, Kirsten Flentje and Lena Volk for technical assistance.

References 1 Gianni AM, Siena S, Bregni M et al. Granulocyte–macrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 1989; I: 580–584. 2 Haas R, Hohaus S, Egerer G et al. Recombinant human granulocyte–macrophage colony-stimulating factor (rhGM-CSF) subsequent to chemotherapy improves collection of blood stem cells for autografting in patients not eligible for bone marrow harvest. Bone Marrow Transplant 1992; 9: 459–465. 3 Haas R, Murea S. The role of granulocyte colony-stimulating factor in mobilization and transplantation of peripheral blood progenitor and stem cells. Cytok Mol Therapy 1995; 1: 249– 270. 4 Winter JN, Lazarus HM, Rademaker A et al. Phase I/II study of combined granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor administration for the mobilization of hematopoietic progenitor cells. J Clin Oncol 1996; 14: 277–286. 5 Lane TA, Law P, Maruyama M et al. Harvesting and enrichment of hematopoietic progenitor cells mobilized into the peripheral blood of normal donors by granulocyte–macrophage colony-stimulating factor (GM-CSF) or G-CSF: potential role in allogeneic marrow transplantation. Blood 1995; 85: 275– 282. 6 Bolwell BJ, Goormastic M, Yanssens T et al. Comparison of G-CSF with GM-CSF for mobilizing peripheral blood progenitor cells and for enhancing marrow recovery after autologous bone marrow transplant. Bone Marrow Transplant 1994; 14: 913–918. 7 Demuynck H, Delforge M, Verhoef G et al. Comparative study of peripheral blood progenitor cell collection in patients with multiple myeloma after single-dose cyclophosphamide combined with rhGM-CSF or rhG-CSF. Br J Haematol 1995; 90: 384–392. 8 Hohaus S, Goldschmidt H, Ehrhardt R, Haas R. Successful autografting following myeloablative conditioning therapy with blood stem cells mobilized by chemotherapy plus rhGCSF. Exp Hematol 1993; 21: 508–514. 9 Pfreundschuh MG, Rueffer U, Lathan B et al. Dexa-BEAM in patients with Hodgkin’s disease refractory to multidrug chemotherapy regimens: a trial of the German Hodgkin’s Disease Study Group. J Clin Oncol 1994; 12: 580–586. 10 Dreger P, Marquardt P, Haferlach T et al. Effective mobilis-

629


630

11 12

13

14

15

16

17

18

19

ation of peripheral blood progenitor cells with ‘Dexa-BEAM’ and G-CSF: timing of harvesting and composition of the leukapheresis product. Br J Cancer 1993; 68: 950–957. Gasson JC. Molecular physiology of granulocyte–macrophage colony-stimulating factor. Blood 1991; 77: 1131–1145. Belosevic M, Davis CE, Meltzer MS, Nancy CA. Regulation of activated macrophage antimicrobial activities. Identification of lymphokines that cooperate with IFN-gamma for induction of resistance to infection. J Immunol 1988; 141: 890–896. Cannistra SA, Vellenga E, Groshek P et al. Human granulocyte–macrophage colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor-dependent mechanism. Blood 1988; 71: 672–676. Heufler C, Koch F, Schuler G. Granulocyte–macrophage colony-stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells. J Exp Med 1988; 167: 100–105. Ho AD, Haas R, Wulf G et al. Activation of lymphocytes induced by recombinant human granulocyte-macrophage colony-stimulating factor in patients with malignant lymphoma. Blood 1990; 75: 203–212. Hohaus S, Haas R, Ogniben E et al. Immunological characterization of blood stem cell autografts collected under administration of recombinant human granulocyte–macrophage colony-stimulating factor (rhGM-CSF). In: Freund M, Link H, Welte K (eds). Cytokines in Hematopoiesis, Oncology, and AIDS. Springer Verlag: Berlin, Heidelberg, 1991. Greenberg P, Advani R, Keating A et al. GM-CSF accelerates neutrophil recovery after autologous hematopoietic stem cell transplantation. Bone Marrow Transplant 1996; 18: 1057– 1064. Haas R, Mo¨hle R, Fru¨hauf S et al. Patient characteristics associated with successful mobilizing and autografting of peripheral blood progenitor cells in malignant lymphoma. Blood 1994; 83: 3787–3794. Menichella G, Pierelli L, Foddai ML et al. Autologous blood stem cell harvesting and transplantation in patients with advanced ovarian cancer. Br J Haematol 1991; 79: 444–450.

20 Bensinger W, Appelbaum F, Rowley S et al. Factors that influence collection and engraftment of autologous peripheralblood stem cells. J Clin Oncol 1995; 13: 2547–2555. 21 Schneider JG, Crown JP, Wasserheit C et al. Factors affecting the mobilization of primitive and committed hematopoietic progenitors into the peripheral blood of cancer patients. Bone Marrow Transplant 1994; 14: 877–884. 22 Dreger P, Kloss M, Petersen B et al. Autologous progenitor cell transplantation: prior exposure to stem cell-toxic drugs determines yield and engraftment of peripheral blood progenitor cell but not of bone marrow grafts. Blood 1995; 86: 3970–3978. 23 Demirer T, Buckner CD, Gooley T et al. Factors influencing collection of peripheral blood stem cells in patients with multiple myeloma. Bone Marrow Transplant 1996; 17: 937–941. 24 Bensinger WI, Longin K, Appelbaum F et al. Peripheral blood stem cells (PBSCs) collected after recombinant granulocyte colony stimulating factor (rhG-CSF): an analysis of factors correlating with the tempo of engraftment after transplantation. Br J Haematol 1994; 87: 825–831. 25 Weaver CH, Hazelton B, Birch R et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995; 86: 3961–3969. 26 Haas R, Hohaus S, Goldschmidt H et al. High-dose therapy and autografting with peripheral blood stem cells in malignant lymphoma: granulocyte–macrophage colony-stimulating factor for stem cell mobilization. Semin Oncol 1994; 21 (Suppl. 16): 19–24. 27 Ho AD, Young D, Maruyama M et al. Pluripotent and lineagecommitted CD34+ subsets in leukapheresis products mobilized by G-CSF, GM-CSF vs a combinant of both. Exp Hematol 1996; 24: 1460–1468. 28 Hohaus S, Pfo¨rsich M, Murea S et al. Immunomagnetic selection of CD34+ peripheral blood stem cells for autografting in patients with breast cancer. Br J Haematol 1997; 97: 881–888.