Progenitor cell mobilisation Stem cell mobilisation with 16 g ... - Nature

Bone Marrow Transplantation (2002) 29, 727–730  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt

Progenitor cell mobilisation Stem cell mobilisation with 16 ␮g/kg vs 10 ␮g/kg of G-CSF for allogeneic transplantation in healthy donors N Kro¨ger1, H Renges1, S Sonnenberg1, W Kru¨ger1, K Gutensohn2, T Dielschneider2, L Cortes-Dericks1 and AR Zander1 1

Department of Bone Marrow Transplantation, University of Hamburg, Germany; and 2Department of Transfusion Medicine, University of Hamburg, Germany

Summary: We compared two doses of recombinant human granulocyte-stimulating factor (G-CSF) for stem cell mobilisation in 90 healthy donors for allogeneic stem cell transplantation in a retrospective analysis. Group I (n = 46) received 10 ␮g/kg G-CSF (filgrastim) given as 5 ␮g/kg twice daily, and group II (n = 44) received 16 ␮g/kg, given as 8 ␮g/kg twice daily with a 12-h interval. The groups were well-balanced for age and bodyweight. G-CSF application was performed on an outpatient basis, and leukapheresis was started in all donors on day 5. The most frequent side-effects of GCSF were grade I/II, bone pain, headache and fatigue in both groups, whereas grade III of bone pain, headache and fatigue occurred in the 2 ⴛ 8 ␮g/kg group only. One serious non-fatal event with non-traumatic spleen rupture occurred in the 2 ⴛ 5 ␮g/kg group. The CD34+ cell count in the first apheresis of all donors was 5.1 ⴛ 106/kg donor weight (range, 1.5–19.3). The CD34+ cell harvest was higher in the 2 ⴛ 8 ␮g/kg group than in the 2 ⴛ 5 ␮g/kg group (7.1 ⴛ 106/kg vs 4.9 ⴛ 106/kg; P = 0.09). The target of collecting ⬎5.0 ⴛ 106 CD34+ cells/kg donor weight with one apheresis procedure was achieved in 45% of group I and in 61% of group II, respectively. Administering G-CSF at a dosage of 8 ␮g/kg twice daily leads to a higher CD34+ cell yield than a dosage of 2 ⴛ 5 ␮g/kg, but is associated with increased toxicity and higher cost. Bone Marrow Transplantation (2002) 29, 727–730. DOI: 10.1038/sj/bmt/1703509 Keywords: stem cell mobilisation; granulocyte colonystimulating factor; healthy volunteer; allogeneic transplantation

Despite the fact that mobilised PBSC contain 10 times more T cells than bone marrow, the incidence of acute GVHD in allogeneic PBSC is similar to the incidence of aGVHD in allogeneic bone marrow transplantation.1,3 However, some investigators reported a higher incidence and severity of chronic GVHD after allogeneic PBSC transplantation.4 Mobilisation of progenitor cells in healthy donors is achieved with cytokines such as granulocyte colony-stimulating factor (G-CSF) or granulocyte–macrophage colony-stimulating factor (GM-CSF). G-CSF is preferred because of fewer side-effects and higher mobilisation efficacy.5 The possible advantages for the donor of PBSC include the avoidance of general anaesthesia, the less invasive procedure and the lack of hospitalisation.6 Although the mobilisation kinetics of CD34+ progenitor cells after G-CSF have been studied,5,7,8 neither the optimal dosage nor the optimal schedule have been established so far. The amount of cell harvest is of importance since recent reports suggest that the infusion of a high number of progenitor cells could improve outcome after allogeneic stem cell transplantation.9,10 Recent data in healthy donors as well as in cancer patients show a dose response effect to G CSF-dosages between 3 ␮g/kg and 30 ␮g/kg body-weight (BW).2,11–13 The adverse effect of G-CSF appears to be dose-dependent.14 A dose of 20 ␮g/kg and more resulted in a higher cell yield than the standard dose of 10 ␮g/kg, but all investigators compared a twice daily high-dose schedule with a once daily standard schedule (1 ⫻ 10 ␮g/kg). The split dose of G-CSF resulted in a significantly higher CD34+ cell yield as compared to giving the same dose once daily.15 A dose between 10 ␮g/kg to 16 ␮g/kg G-CSF is accepted as standard use by most centres world-wide. Herein we report our experience with 90 healthy donors who were divided into two groups: group I included 46 healthy donors who received 2 ⫻ 5 ␮g/kg G-CSF daily, and group II received 2 ⫻ 8 ␮g/kg G-CSF daily.

Peripheral blood progenitor cells (PBSC) instead of bone marrow can be used for allogeneic transplantation.1,2

Patients and methods

Correspondence: PD Dr N Kro¨ger, Bone Marrow Transplantation, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany Received 12 November 2001; accepted 14 February 2002

We report the results for PBSC mobilisation and collection in healthy donors for related or unrelated allogeneic stem cell transplantation at our institution between January 1998 and June 2001. Ninety healthy donors were enrolled in this

PBSC mobilisation in healthy donors N Kro¨ ger et al

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retrospective comparison. Two groups of donors were formed: group I: n = 46, 2 ⫻ 5 ␮g/kg G-CSF (filgrastim, Neupogen; Amgen, Munich, Germany) and group II: n = 44, 2 ⫻ 8 ␮g/kg G-CSF. Of group I, 25 patients participated in a comparative study, and the data have been recently published in a separate report.12 G-CSF was applied twice daily with an interval of 12 h. G-CSF dosage was rounded to standard size ampoules of 300 ␮g or 480 ␮g. G-CSF application was performed as an out-patient procedure. Leukapheresis was started in all donors on day 5. G-CSF application was continued until completion of leukapheresis, depending on the required cell dose of the recipient. For better comparison of the CD34+ cell harvest only the first apheresis was calculated for donor bodyweight rather than for recipient body-weight. The two groups were well-balanced for age and body-weight (Table 1). All donors were required to have a WHO performance index ⬍2 and adequate cardiac, renal, hepatic and haematological functions. Toxicity of filgrastim administration was performed according to the Common Toxicity Criteria (CTC). All donors gave written informed consent for administration of G-CSF and for collection of peripheral blood stem cells. The mobilisation protocol was approved by the local ethics commission. PBSC collection The collection of PBSC was performed identically after GCSF stimulation identically in all donors on day +5 with a Cobe Spectra using a 250 ml volume collection chamber. A total of 8–14 litres of blood per apheresis was processed at a flow rate of 50–70 ml/min; a mean volume of 250 ml was collected. Immunofluorescence staining and flow cytometry For quantification of CD34+ cells, 1 ⫻ 106 mononuclear cells separated by Ficoll–Hypaque density gradient centrifugation were incubated for 10 min at room temperature with phycoerythrin (PE)-conjugated monoclonal anti-CD34 antibodies (Clone 581, Beckman Coulter, Krefeld, Germany). The analysis was performed on a FACScan flow cytometer (EPICS XL-MCL, Beckman Coulter) with System II software Version 2.1 (Beckman Coulter) according to the method published by Sutherland et al.16 A minimum Table 1

Statistics Statistical analysis was performed using WinSTAT software (Kalmia, Cambridge, MA, USA). For comparison of the two different G-CSF groups, the independent t-test was applied. Correlation was performed with the Pearsontest. A P-value of ⬍0.05 was considered to be significant.

Results Tolerance of G-CSF Cytokine priming, as well as collection of peripheral blood stem cells (PBSC), resulted in more side-effects in group II than in group I: 48% and 42% mild fatigue (grade I/II CTC) was noted in group I and II, respectively. Grade III fatigue was noted only in 35% of group II. 88% and 16% of the patients experienced bone pain grade I/II in group I and II, respectively. Similarly grade III bone pain was observed only in 73% of group II. Headache grade I or II was noted by 72% and 73% of the donors in group I and II. In cases of bone pain or headache, treatment with oral paracetamol was started (Table 2). The side-effects disappeared after discontinuation of G-CSF. A discontinuation of G-CSF because of intolerable side-effects was not necessary in any case. One serious event was noted in the 2 ⫻ 5 ␮g/kg group. A young man with a body weight of 170 kg received 2 ⫻ 5 ␮g/kg G-CSF. He experienced bone pain grade III, and on day 5 he complained about severe pain in the upper abdomen. His leukocyte count was 41/nl, and haemoglobin was 11.2 g/dl. Ultrasonic test and CT scan showed an enlarged spleen with rupture. Leukapheresis was performed and the abdominal pain disappeared. No surgical intervention had to be performed, and the problem resolved completely. In heavily overweight donors the dose of GCSF should be calculated to an adjusted bodyweight. PBSC collection For all donors the median collected CD34+ cell count in first apheresis was 5.1 ⫻ 106/kg (range, 1.5–19.3) (Table

Donor characteristics

Variables

2 × 5 ␮g/kg

2 × 8 ␮g/kg

No. of donors Gender: male/female Median age (range)

46 30/16 45.5 (20–60) 80 (56–180) 6.2 (3.6–12.4) 11152 (6670–14 447)

44 18/26 37 (20–64) 74 (52–104) 6.6 (2.0–14.8) 10677 (6670–14 104)

Median (range) Median (range) Median volume

of 50 000 events was counted. To calculate the total of CD34+ cells, the number of MNC per leukapheresis product was multiplied by the percentage of CD34+ cells.

kg body weight WBC before G-CSF ml processed blood (range)

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Table 2

Toxicity of G-CSF administration (according to CTC)

Toxicity (CTC) Bone pain grade I or II Bone pain grade III Headache grade I or II Headache grade III Fatigue grade I or II Fatigue grade III

2 × 5 ␮g/kg

2 × 8 ␮/kg

88% — 72% — 60% —

16% 73% 73% 16% 42% 35%


3). The median leukocyte count before first apheresis was 51/nl (range, 21–91), and it correlated with the CD34+ cell yield (r, 0.35; P ⬍ 0.001). The median leukocyte count before apheresis was higher in the 2 ⫻ 8␮g/kg group compared to the 2 ⫻ 5 ␮g/kg group (58.5/nl vs 47/nl; P = 0.02). The CD34+ cell count in first apheresis was 4.9 ⫻ 106/kg (range, 2.4–13.3) in the 2 ⫻ 5 ␮g/kg group and 7.1 ⫻ 106/kg (range, 1.5–19.3) in the 2 ⫻ 8 ␮g/kg group (P = 0.09). The target of collecting ⬎3.0 ⫻ 106 CD34+ cells/kg with one apheresis procedure was achieved in 95% of the 2 ⫻ 5 ␮g/kg group and in 82% in the 2 ⫻ 8 ␮g/kg group. The target of collecting ⬎5.0 ⫻ 106 CD34+ cells/kg in first apheresis was achieved in 45% of the patients in group I and in 61% in group II, respectively. Discussion This retrospective study in healthy donors confirmed the reported results of a dose-related effect of G-CSF with respect to the CD34+ cell harvest after leukapheresis. Several investigators reported a dose–response effect to GCSF.5,11–13 and most studies have shown a dose–response effect up to 10 ␮g/kg G-CSF.14,17 Despite the fact that a dose of 10 to 16 ␮g/kg G-CSF is accepted as standard use by most centres world-wide, most of the high-dose G-CSF studies are performed with a dose of 20 to 24 ␮g/kg. In these trials a higher cell yield without obvious increase of toxicity was described for the high-dose G-CSF arm.9,11,13,18 Only a small trial by Majolini et al19 compared 16 ␮g/kg G-CSF to standard 10 ␮g/kg, but it failed to show a significant cell increase of the higher dose, probably due to the small number of only 11 donors. However, in all studies comparing low- to high-dose G-CSF a once daily schedule (1 ⫻ 10 ␮g/kg) was compared to a twice daily schedule (2 ⫻ 12 ␮g/kg, or 2 ⫻ 10 ␮g/kg), which might bias the results. A schedule dependency of G-CSF has been reported by several investigators when applying G-CSF as a single dose compared to a split schedule.15,20,21 The pharmacological profile of G-CSF in healthy donors and cancer patients shows a maximum serum concentration after subcutaneous application within 2–8 h,22 and due to the short elimination half-time of G-CSF of only about 3–4 h regardless of the route of administration the twice daily schedule might improve the CD34+ cell yield. Several retrospective studies of different G-CSF dosages in healthy donors and cancer patients suggested that splitting the G-CSF dosage into two injections with a 12-h interval might lead to a higher CD34+ cell harvest.22–24 Recently, our group showed in a prospec-

Table 3

CD34+ cell harvest

Variables WBC/nl prior to first LP (range) CD34+ × 106/kg in first LP (range) ⬎3 × 106/kg CD34+ in first LP ⬎5 × 106/kg CD34+ in first LP

2 × 5 ␮g/kg

2 × 8 ␮g/kg

47 (31–78) 4.9 (2.4–13.3) 95% 45%

58.5 (32–91) 7.1 (1.5–19.3) 82% 61%

tive trial in healthy donors that twice daily injections (2 ⫻ 5 ␮g/kg) resulted in a higher CD34+ cell harvest than a single application (1 ⫻ 10 ␮g/kg).15 Therefore, we investigated the effect of 10 and 16 ␮g/kg G-CSF both given twice daily as 2 ⫻ 5 ␮g/kg and 2 ⫻ 8 ␮g/kg in 90 healthy donors. The short-term side-effects of G-CSF were predominantly bone pain, headache and fatigue. Headache and bone pain, however, were more frequently observed in the 2 ⫻ 8 ␮g/kg group. A serious, but non-fatal event was observed in the 2 ⫻ 5 ␮g/kg group. A patient of 170 kg body weight experienced a non-traumatic spleen rupture, which could be treated without surgery. Spleen enlargement and spleen rupture should be kept in mind as possible serious events following G-CSF application.25 Apheresis was started on day 5, because several investigators reported a peak number of circulating CD34+ cells after filgrastim stimulation on day 5.5–7 To avoid prolonged exposure to G-CSF other investigators started collection on day 4, but comparative analysis showed a higher cell yield for the day 5 collection.26,27 A higher CD34+ cell yield can also be achieved by large volume apheresis, processing three to four times the total donor body weight per apheresis.28 In summary, our study confirmed a dose-dependency of G-CSF when applied for mobilisation of peripheral blood progenitor cells. In general, G-CSF application is safe, well-tolerated and effective in mobilising PBSC, however one serious event with spleen enlargement and non-traumatic spleen rupture was noted. Because of the increased number of side-effects of higher G-CSF dose (16 ␮g/kg), the split dose of 2 ⫻ 5 ␮g/kg is recommended. Longer follow-up of the donors is necessary to determine any late side-effects of G-CSF.

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