640
Effect of
peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy
haemopoietic growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) substantially shortens the period of severe neutropenia that follows high-dose chemotherapy and autologous bone-marrow infusion by stimulating granulopoiesis. Filgrastim also increases numbers of circulating progenitor cells. We have studied the ability of filgrastim to mobilise peripheralblood progenitor cells and assessed their efficacy when infused after chemotherapy on recovery of neutrophil and platelet counts. 17 patients with non-myeloid malignant disorders received filgrastim (12 µg/kg daily for 6 days) by The
follows marrow
high-dose chemotherapy and autologous boneinfusion can be substantially shortened by
with G-CSF .3GM-CSF is also beneficia1.5-7 However, neither growth factor affects the duration of severe thrombocytopenia. This feature is consistent with their lack of action on megakaryocyte colonies in vitro and on platelet production in vivo.1,2 In early studies, an unexpected finding was the high numbers of circulating progenitor cells in patients who received G-CSF or GM-CSF.8,9 We have examined the ability of G-CSF (filgrastim) to mobilise peripheral-blood progenitor cells; we have also assessed the feasibility of collection and efficacy of these cells when infused after treatment
high-dose chemotherapy.
continuous subcutaneous infusion. Numbers of
granulocyte-macrophage progenitors
in
blood increased a median of 58-fold over pretreatment values, and numbers of erythroid progenitors increased a median of 24-fold. Three leucapheresis procedures collected a mean total of 33 (SEM 5·7) × 104 granulocyte-macrophage progenitors per kg body weight. After high-dose chemotherapy in 14 of the patients (busulphan and cyclophosphamide), these cells were used to augment autologous bone-marrow rescue and posttransplant filgrastim treatment. Platelet recovery was in these patients than in controls who received the same treatment apart from the infusion of peripheral-blood progenitors; the platelet count reached 50 × 109/l a median of 15 days after infusion of haemopoietic cells in the study patients compared with 39 days in controls (p = 0·0006). The accelerated neutrophil recovery associated with filgrastim treatment after chemotherapy was maintained. This method may be widely applicable to aid both neutrophil and platelet recovery after high-dose chemotherapy; it will allow investigation of
significantly faster
peripheral-blood progenitor-cell allotransplantation. Introduction The
Patients and methods
peripheral
haemopoietic growth factors, granulocyte colonystimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were first identified by their ability to stimulate the clonal growth of haemopoietic progenitor cells in vitro.The purification and subsequent molecular cloning of these factors has allowed study of their effects in various clinical settings.2 The recombinant human G-CSF and GM-CSF produced in Escherichia coli now have the approved names filgrastim and ecogramostim. The period of severe neutropenia that
Eligible patients were those with non-myeloid malignant disorders of poor prognosis; some were in remission after initial chemotherapy but had poor prognostic features (acute lymphoblastic leukaemia and non-Hodkgin lymphoma) and others had had an inadequate response to, or relapse after, chemotherapy (acute lymphoblastic leukaemia, non-Hodgkin lymphoma, Hodgkin’s disease, and genn-cell tumour). The study took place under the ethical guidelines of the National Health and Medical Research Council of Australia and the US Food and Drug Administration. All patients gave informed consent. Recruitment started in December, 1989. All 17 study patients (table I) completed the leucapheresis phase of the study. After an initial leucapheresis, filgrastim (Amgen, Thousand Oaks, California, USA) was given as a continuous subcutaneous infusion (12 fig/kg daily) for 6 days, by way of a 23 gauge needle connected to a Conned infusion pump (Medina, New York, USA). Leucapheresis was repeated on days 5, 6, and 7 with a ’Fenwal CS-3000’ cell separator (Baxter, Deerfield, Illinois, USA); we used a modified mononuclear-cell collection program with the red-cell interface set at 020 units. Each leucapheresis continued until at least 7 litres of blood had been processed. The mononuclearcell product was further processed to reduce the volume and the final cell suspension was cryopreserved.10 The bone marrow of all patients was harvested and cryopreserved before they underwent chemotherapy; 3 patients did not receive high-dose chemotherapy (1 refused and 2 had progressive disease). High-dose chemotherapy consisted of oral busulphan (4 mgkg daily) on days - 7, - 6, - 5, and - 4 and intravenous ADDRESSES: Departments of Clinical Haematology and Medical Oncology (W P. Sheridan, FRACP, Prof R. M. Fox, PhD) and Diagnostic Haematology (C. G. Begley, PhD, D. Maher, FRACP, K. M. McGrath, FRCPA), Royal Melbourne Hospital, Victoria; Walter and Eliza Hall Institute of Medical Research, Melbourne (C G. Clinical Bone Marrow Begley); Haematology and Transplantation Unit, Royal Adelaide Hospital, South Australia (C A. Juttner, FRACP, L. Bik To, MD); Clinical Haematology and Bone Marrow Transplantation Unit, Alfred Hospital, Prahran, Victoria (J. Szer, FRACP); and Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Victoria, Australia
(G. Mostyn, PhD). Correspondence to Dr William P Sheridan, Department of Clinical Haematology and Medical Oncology, Royal Melbourne Hospital, c/o Post Office, Royal Melbourne Hospital, Victoria
3050, Australia
641
TABLE I-PATIENT CHARACTERISTICS
’Before high-dose chemotherapy. ALL=acute lymphoblastic leukaemia.
cyclophosphamide (60 mg/kg daily) on days -3 and - 2. Cryopreserved bone marrow and peripheral-blood mononuclearcell preparations obtained by leucapheresis were infused on day 0. Filgrastim was given after the cell infusions as previously described.3 Clinical status was assessed and complete blood count, including differential white-cell count, done daily during the leucapheresis phase and again after bone-marrow/peripheral-blood progenitorcell infusion until recovery. Bone-marrow samples were examined and scored as previously described.3 Standard criteria were used for starting and stopping parenteral antibiotics, administration of platelet and red-cell transfusions, use of parenteral nutrition, and discharge from hospital.’," Filgrastim administration was continued at outpatient visits if necessary. Haemopoietic progenitor cells were assayed in bone-marrow samples and in peripheral blood and leucapheresis product obtained before and after filgrastim treatment. Progenitor cells (colonyforming cells) were assayed by counting of colonies after 14 days’ culture.8,12 The number of progenitors per ml of sample was calculated as: frequency (per 105 mononuclear cells) x sample mononuclear-cell count. The results of the study patients were compared with those of two groups of control patients who met the same eligibility criteria and were treated in two previous studies. Control group I (patients treated between July, 1988, and February, 19903) received the same high-dose chemotherapy, autologous bone-marrow transplantation, and filgrastim after chemotherapy but no filgrastim-mobilised peripheral-blood progenitor cells. Control group II (patients treated between February, 1987, and June, 1988) received the same high-dose chemotherapy and autologous bonemarrow transplantation but no filgrastim. All clinical-care policies were maintained unchanged throughout the study and control
periods. Comparisons among groups were made with standard statistical tests by a computer statistics program (NCSS, Kaysville, Utah, USA). Student’s t test was used for continuous variables, Peto’s generalisation of Wilcoxon’s rank-sum test for time-dependent variables, and Fisher’s exact test for proportions. Results are given as mean and SEM or median (for time-dependent variables and fold
increases). Results As expected, during the 6 days of filgrastim treatment for collection of peripheral-blood progenitor cells, the total white-cell count rose from 5-3 (0-6) x 109/1 to 36-4 (46) x 109/1. These cells were predominantly mature and band neutrophils. Before filgrastim treatment, the number of
granulocyte-macrophage progenitor cells in the peripheral blood was low (0-04 [0’01] x 103/ml). However, after 5 days of treatment, the numbers had increased to 1-7 Student’s t test); the median (03)x103/ml (p 0-5 x 109/1) was 9 days in study patients (table II). Thus, with use of were
within
or
filgrastim-mobilised peripheral-blood progenitor-cells, neutrophil recovery was maintained but the severe thrombocytopenia was significantly
accelerated period of
Vv
Day after mfusion Fig 3-Probability of recovery of platelet count of at least 50x109/I after high-dose chemotherapy and infusion of haemopoietic cells. - =study group (n=14), ---=control group I (n=25); = control group(n=13) p
Effect of
peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy
haemopoietic growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) substantially shortens the period of severe neutropenia that follows high-dose chemotherapy and autologous bone-marrow infusion by stimulating granulopoiesis. Filgrastim also increases numbers of circulating progenitor cells. We have studied the ability of filgrastim to mobilise peripheralblood progenitor cells and assessed their efficacy when infused after chemotherapy on recovery of neutrophil and platelet counts. 17 patients with non-myeloid malignant disorders received filgrastim (12 µg/kg daily for 6 days) by The
follows marrow
high-dose chemotherapy and autologous boneinfusion can be substantially shortened by
with G-CSF .3GM-CSF is also beneficia1.5-7 However, neither growth factor affects the duration of severe thrombocytopenia. This feature is consistent with their lack of action on megakaryocyte colonies in vitro and on platelet production in vivo.1,2 In early studies, an unexpected finding was the high numbers of circulating progenitor cells in patients who received G-CSF or GM-CSF.8,9 We have examined the ability of G-CSF (filgrastim) to mobilise peripheral-blood progenitor cells; we have also assessed the feasibility of collection and efficacy of these cells when infused after treatment
high-dose chemotherapy.
continuous subcutaneous infusion. Numbers of
granulocyte-macrophage progenitors
in
blood increased a median of 58-fold over pretreatment values, and numbers of erythroid progenitors increased a median of 24-fold. Three leucapheresis procedures collected a mean total of 33 (SEM 5·7) × 104 granulocyte-macrophage progenitors per kg body weight. After high-dose chemotherapy in 14 of the patients (busulphan and cyclophosphamide), these cells were used to augment autologous bone-marrow rescue and posttransplant filgrastim treatment. Platelet recovery was in these patients than in controls who received the same treatment apart from the infusion of peripheral-blood progenitors; the platelet count reached 50 × 109/l a median of 15 days after infusion of haemopoietic cells in the study patients compared with 39 days in controls (p = 0·0006). The accelerated neutrophil recovery associated with filgrastim treatment after chemotherapy was maintained. This method may be widely applicable to aid both neutrophil and platelet recovery after high-dose chemotherapy; it will allow investigation of
significantly faster
peripheral-blood progenitor-cell allotransplantation. Introduction The
Patients and methods
peripheral
haemopoietic growth factors, granulocyte colonystimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were first identified by their ability to stimulate the clonal growth of haemopoietic progenitor cells in vitro.The purification and subsequent molecular cloning of these factors has allowed study of their effects in various clinical settings.2 The recombinant human G-CSF and GM-CSF produced in Escherichia coli now have the approved names filgrastim and ecogramostim. The period of severe neutropenia that
Eligible patients were those with non-myeloid malignant disorders of poor prognosis; some were in remission after initial chemotherapy but had poor prognostic features (acute lymphoblastic leukaemia and non-Hodkgin lymphoma) and others had had an inadequate response to, or relapse after, chemotherapy (acute lymphoblastic leukaemia, non-Hodgkin lymphoma, Hodgkin’s disease, and genn-cell tumour). The study took place under the ethical guidelines of the National Health and Medical Research Council of Australia and the US Food and Drug Administration. All patients gave informed consent. Recruitment started in December, 1989. All 17 study patients (table I) completed the leucapheresis phase of the study. After an initial leucapheresis, filgrastim (Amgen, Thousand Oaks, California, USA) was given as a continuous subcutaneous infusion (12 fig/kg daily) for 6 days, by way of a 23 gauge needle connected to a Conned infusion pump (Medina, New York, USA). Leucapheresis was repeated on days 5, 6, and 7 with a ’Fenwal CS-3000’ cell separator (Baxter, Deerfield, Illinois, USA); we used a modified mononuclear-cell collection program with the red-cell interface set at 020 units. Each leucapheresis continued until at least 7 litres of blood had been processed. The mononuclearcell product was further processed to reduce the volume and the final cell suspension was cryopreserved.10 The bone marrow of all patients was harvested and cryopreserved before they underwent chemotherapy; 3 patients did not receive high-dose chemotherapy (1 refused and 2 had progressive disease). High-dose chemotherapy consisted of oral busulphan (4 mgkg daily) on days - 7, - 6, - 5, and - 4 and intravenous ADDRESSES: Departments of Clinical Haematology and Medical Oncology (W P. Sheridan, FRACP, Prof R. M. Fox, PhD) and Diagnostic Haematology (C. G. Begley, PhD, D. Maher, FRACP, K. M. McGrath, FRCPA), Royal Melbourne Hospital, Victoria; Walter and Eliza Hall Institute of Medical Research, Melbourne (C G. Clinical Bone Marrow Begley); Haematology and Transplantation Unit, Royal Adelaide Hospital, South Australia (C A. Juttner, FRACP, L. Bik To, MD); Clinical Haematology and Bone Marrow Transplantation Unit, Alfred Hospital, Prahran, Victoria (J. Szer, FRACP); and Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Victoria, Australia
(G. Mostyn, PhD). Correspondence to Dr William P Sheridan, Department of Clinical Haematology and Medical Oncology, Royal Melbourne Hospital, c/o Post Office, Royal Melbourne Hospital, Victoria
3050, Australia
641
TABLE I-PATIENT CHARACTERISTICS
’Before high-dose chemotherapy. ALL=acute lymphoblastic leukaemia.
cyclophosphamide (60 mg/kg daily) on days -3 and - 2. Cryopreserved bone marrow and peripheral-blood mononuclearcell preparations obtained by leucapheresis were infused on day 0. Filgrastim was given after the cell infusions as previously described.3 Clinical status was assessed and complete blood count, including differential white-cell count, done daily during the leucapheresis phase and again after bone-marrow/peripheral-blood progenitorcell infusion until recovery. Bone-marrow samples were examined and scored as previously described.3 Standard criteria were used for starting and stopping parenteral antibiotics, administration of platelet and red-cell transfusions, use of parenteral nutrition, and discharge from hospital.’," Filgrastim administration was continued at outpatient visits if necessary. Haemopoietic progenitor cells were assayed in bone-marrow samples and in peripheral blood and leucapheresis product obtained before and after filgrastim treatment. Progenitor cells (colonyforming cells) were assayed by counting of colonies after 14 days’ culture.8,12 The number of progenitors per ml of sample was calculated as: frequency (per 105 mononuclear cells) x sample mononuclear-cell count. The results of the study patients were compared with those of two groups of control patients who met the same eligibility criteria and were treated in two previous studies. Control group I (patients treated between July, 1988, and February, 19903) received the same high-dose chemotherapy, autologous bone-marrow transplantation, and filgrastim after chemotherapy but no filgrastim-mobilised peripheral-blood progenitor cells. Control group II (patients treated between February, 1987, and June, 1988) received the same high-dose chemotherapy and autologous bonemarrow transplantation but no filgrastim. All clinical-care policies were maintained unchanged throughout the study and control
periods. Comparisons among groups were made with standard statistical tests by a computer statistics program (NCSS, Kaysville, Utah, USA). Student’s t test was used for continuous variables, Peto’s generalisation of Wilcoxon’s rank-sum test for time-dependent variables, and Fisher’s exact test for proportions. Results are given as mean and SEM or median (for time-dependent variables and fold
increases). Results As expected, during the 6 days of filgrastim treatment for collection of peripheral-blood progenitor cells, the total white-cell count rose from 5-3 (0-6) x 109/1 to 36-4 (46) x 109/1. These cells were predominantly mature and band neutrophils. Before filgrastim treatment, the number of
granulocyte-macrophage progenitor cells in the peripheral blood was low (0-04 [0’01] x 103/ml). However, after 5 days of treatment, the numbers had increased to 1-7 Student’s t test); the median (03)x103/ml (p 0-5 x 109/1) was 9 days in study patients (table II). Thus, with use of were
within
or
filgrastim-mobilised peripheral-blood progenitor-cells, neutrophil recovery was maintained but the severe thrombocytopenia was significantly
accelerated period of
Vv
Day after mfusion Fig 3-Probability of recovery of platelet count of at least 50x109/I after high-dose chemotherapy and infusion of haemopoietic cells. - =study group (n=14), ---=control group I (n=25); = control group(n=13) p
