Lenograstim-mobilized peripheral blood progenitor cells in volunteer donors: an open label randomized split dose escalating study

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1 (2) 25, Macmillan Publishers Ltd All rights reserved / $15. Lenograstim-mobilized peripheral blood progenitor cells in volunteer donors: an open label randomized split dose escalating study N Basara, B Schmetzer, IW Blau, M Bischoff, S Günzelmann, D Kirsten and AA Fauser Clinic of and Haematology/Oncology and European Institute for Research and Development of Transplant Strategies, Idar-Oberstein, Germany Summary: Mobilization of peripheral blood cell progenitor cells was investigated in 36 healthy sibling donors using three different split doses of glycosylated rhg-csf (lenograstim). The donors were randomized into three groups: group 1 was given lenograstim at 8, group 2 at 11 and group 3 at 15 g/kg/day in two split doses, subcutaneously for 4 and 5 days, respectively. Leukapheresis was performed on day 4 or 5 depending on the WBC and CD34 cell count. We were able to demonstrate that there was a significant correlation between circulating CD34 cells on the day of harvest and CD34 cells in the apheresis products in all three groups. The number of CD34 cells pre-apheresis was inversely correlated with age in group 1 and group 2. However, in group 3, the number of CD34 cells preapheresis did not correlate with age. There was also a difference between the number of progenitor cells mobilized in the three dose groups regarding the time of harvest. Apheresis was performed in groups 1 and 2 on day 5 of mobilization in order to obtain a sufficient number of stem cells for allogeneic transplantation. In contrast, with the split dose of 15 g/kg/day, harvest could be routinely performed on day 4 of stimulation. We conclude that lenograstim given twice a day at doses of 8, 11 and 15 g/kg/day provided different CD34 cell yields in normal donors, in particular, with regard to the time of harvest. The number of CD34 cells preapheresis was not correlated with age in the group of donors mobilized with a split dose of 15 g/kg/day, indicating that this dosage might also be suitable for older donors. (2) 25, Keywords: lenograstim; mobilization; allogeneic stem cell transplant; volunteer donors Allogeneic peripheral blood stem cell transplantation (PBSCT) from sibling donors is increasingly used as an alternative source of haematopoietic stem cells rather than bone marrow-derived stem cells. 1 5 In 1996, more than Correspondence: Dr N Basara, Clinic of and Haematology/Oncology, Dr. Ottmar-Kohler Str. 2, Idar- Oberstein, Germany Received 13 May 1999; accepted 25 October PBSCT were reported to the European Group for Blood and Marrow Transplantation (EBMT) indicating a dramatic increase in the number of donors undergoing stem cell mobilization. 6 Collection of PBSC with a limited number of leukaphereses requires mobilization with haematopoietic growth factors. It has been shown that short-term administration of rhg-csf does allow the harvest of a large number of PBSC from healthy volunteer donors. 7,8 However, the optimal dosage for CD34 cell mobilization is not yet defined. 9 Based on earlier reported data 9 11 the most favourable time for stem cell collection might be day 4 to 5 after cytokine treatment. Continuation of rhg-csf administration beyond a 5-day course led to a rapid decline in the mobilization efficiency of CD34 cells. 11 It has been demonstrated that, at least for rhg-csf at doses between 5 and 1 g/kg/day, a dose-response relationship does exist with regard to the yield of CD34 cells. 12 Although rhg- CSF doses up to 24 g/kg/day have been reported, 13 experience with such high amounts remains limited. The purpose of this dose escalating study was to determine whether escalating split doses of lenograstim ranging from 8 to 15 g/kg do result in an increase in the number of CD34 cells harvested and whether higher doses of G-CSF might allow collection earlier. Donors and methods Donors Thirty-six healthy donors underwent stem cell mobilization between January 1998 and February 1999 at the Clinic of and Haematology. All donors were related to the recipients, 34 were siblings and one was a daughter, who donated at two different times. Evaluation included full medical history and physical examination. Investigations comprised complete blood counts, renal and liver profiles, ECG and chest X-ray. Donors were randomized to receive a dose of 8 g/kg, 11 g/kg or 15 g/kg/day. Lenograstim (Granocyte, Rhône- Poulenc Rorer, Frankfurt, Germany) was administered subcutaneously daily in two split doses for 5 days or for 4 days, respectively. With regard to donor characteristics there was no statistically significant difference between the three groups of donors concerning age, weight and sex distribution (Table 1). In addition, no difference was found in baseline WBC count, harvested blood volume and the duration of apheresis. The protocol used in this study was

2 372 Table 1 Donor characteristics Group 1 Group 2 Group 3 Dose ( g/kg/day) 8.9 ± ± ±.16 No. of donors (range) 34 (17 44) 39 (25 69) 47 (17 62) Weight 83 (48 15) 73 (56 1) 76 (53 1) Sex Female/Male 7/3 8/6 5/7 1. PBSCC on day WBC (1 9 /l) Baseline 4.8 ± ± ± 1.5 Pre-apheresis 46.7 ± ± ± 3.76 CD34 + (%) cells in PB Pre-apheresis.24 ±.2.2 ±.3.15 ±.2 CD34 + (1 6 /l) cells in PB Pre-apheresis 19 ± ± ± 15.9 is expressed as median (range). Values are expressed as mean ± s.e.m. approved by the IRB of the Clinic of BMT and Haematology/Oncology. The procedure, as well as the risks and benefits were explained in detail to the donors. Safety There was a total of 65 adverse events. Bone pain was the most frequently reported symptom (42 events). In addition, mild headache was reported in 23 donors. None of the donors had any serious adverse events. One donor experienced a transient WHO grade I increase in S-ALAT on day 5 of mobilization. Transient WHO grade I increase in LDH and AP was found in 29 donors. Harvesting of PBSC Following twice daily lenograstim administration, apheresis was scheduled and performed at day 4 or day 5 (Table 1) using a continuous flow centrifuge (Fenwal CS-3 Plus; Baxter, Unterschleißheim, Germany) for 3 to 4 h sessions processing 3 blood volume. The median processed blood volume was 15 l (range: 8 16 l). The study was designed to test if the exposure to higher doses of rhg-csf for a brief time could yield enough progenitor cells to ensure engraftment. A target number for collection was CD34 cells/kg b.w., and the apheresis was done at a blood CD34 cell count of /l. The results in this group of donors (group 3) were compared to the results in a group of donors with a standard and well described schema. Evaluation Peripheral blood counts, whole blood counts and platelet counts were performed before and after apheresis using a haematocytometer (Micro Diff II, Coulter, Krefeld, Germany). An aliquot of WBC was taken and used to calculate the CD34 cell count. Measurement of CD34 cells All PBSC products had a 1 ml sample removed and diluted 1:1 in autologous plasma. Routine analysis of these samples included a WBC, total nucleated cell count, progenitor cell determination and CD34 antigen analysis. The peripheral blood samples and leukapheresis products were analysed by double-colour flow cytometry as previously described. 14 Briefly, cells were stained simultaneously with the phycoerythrin (PE)-conjugated CD34 (HPCA-2)-antibody (Becton Dickinson, San Jose, CA, USA) and anti-cd45 (Becton Dickinson). Using the gating strategies according to the ISHAGE guidelines enumeration of the CD34 cells was performed on an EPICS XL (Coulter). Subsets of CD34 cells were determined by the expression of CD33, CD38 or HLA-DR antigens. The percentage of CD34 cells was converted to absolute numbers by multiplication with the leukocyte count in the sample obtained from the haematocytometer. Results were finally presented as the number of circulating CD34 cells per l peripheral blood or as the number of CD34 cells per leukapheresis product. T and NK cells were enumerated using the CD3-FITC, CD16,56-PE (Immunotech, Marseille, France) reagents as previously described. 14 CFU-GEMM, BFU-E and CFU-GM The assay for progenitor cells was carried out as already described. 15 Briefly, mononuclear cells were plated in 35 mm Petri dishes in 1 ml aliquots of IMDM, containing 3% FCS (HCC4434; Stem Cell Technologies, Vancouver, Canada). Progenitor cell growth was evaluated after 14 days of incubation in a humidified atmosphere. Two dishes were set up for each individual data point per experiment. CFU-GEMM, BFU-E and CFU-GM were scored from the same dish according to previously published criteria. 15

3 Statistical analysis When analysing the relationship between G-CSF dose and blood CD34 levels, only values obtained on the day of the first apheresis were used to avoid any influence of prior leukaphereses. The baseline numbers are shown as mean ± s.d. Multigroup comparison was performed by analysis of variance (ANOVA). 16 Differences between the mean values were evaluated by paired t-test. 16 To estimate correlation, Spearman s correlation coefficient (R) was calculated. All the statistical analyses were performed using the Excel program for windows 95. Results Since the optimal concentration for the mobilization of CD34 cells using rhg-csf and also the time frame for harvesting a sufficient number of CD34 cells remain to be determined in allogeneic transplantation, we conducted an open label randomized split dose escalating study in a total of 36 volunteer donors randomised in three groups receiving either 8, 11 or 15 g/kg/day in split doses over a period of 4 to 5 days. WBC count increased in all three groups of donors to a level of eight- to nine-fold (Table 1). When compared to the pre-treatment values, a slight reduction in the platelet count of 2% was noted in four patients. Apheresis was performed in groups 1 and 2 on day 5 of mobilization in order to obtain a sufficient number of stem cells for allogeneic transplantation. In contrast, harvest could be routinely performed on day 4 of stimulation with the split dose of 15 g/kg/day. The total number of WBC obtained by one apheresis was 4.83, 4.97 and in groups 1, 2 and 3, respectively. The total number of CD34 cells harvested was 4.29, 3.34 and in groups 1, 2 and 3, respectively. The differences in the total number of CD34 cells between the three groups were not significant (Table 2). The total number of CD34 cells was also calculated per kg body weight (b.w.) of recipient, per kg b.w. of donor per apheresis and as yield/blood volume processed. The differences between the three groups regarding CD34 cells per kg b.w. were also not significant. In addition to CD34 cells, the total number of CD3 and CD16,56 cells was determined. There was no significant difference in the total number of T cells between any of the three groups of donors. Correlation between CD34 cells in blood and the leukapheresis product The number of CD34 cells in the blood on the day of apheresis was significantly correlated with the number of CD34 cells in the apheresis product in groups 1, 2 and 3 (r =.68; P.1; r =.78; P.1; and r =.6; P.1, respectively) (Figure 1), as well as in the whole group of donors (r =.8; P.1). Correlation between donor characteristics and CD34 yield The number of CD34 cells pre-apheresis was inversely correlated with age in donor groups 1 and 2 (r =.46; r =.33; respectively) (Figure 2). In group 3, however, the number of CD34 cells pre-apheresis was not correlated with age. Circulating progenitor cells CFU-GM, BFU-E and CFU-GEMM were mobilized in peripheral blood at median numbers of 49.7, 59.2 and /kg b.w. of recipient, BFU-E 114, 61, /kg b.w. and CFU-GEMM at median numbers of 7.1, 3.7 and /kg b.w. of recipient. There was no significant difference between the number of progenitor cells mobilized in the three groups of donors (Table 2). 373 Table 2 Total No. of WBC, CD34 +, T, NK and CFC by one leukapheresis Dose of lenograstim (split dose) Group 1 Group 2 Group 3 (8 g/kg/day) (11 g/kg/day) (15 g/kg/day) No. of donors Total WBC ± ± ± 2.8 Total CD ± ± ± 2.95 CD /kg b.w. of recipient 6.19 ± ± ± 3.46 CD /kg b.w. of donor per apheresis 5.3 ± ± ± 2.86 CD cells/l of blood processed 29. ± ± ± 26.5 Total CD ± ± ±.62 CD3 1 8 /kg b.w. of recipient 2.34 ± ± ±.86 Total CD16 +, ± ± ± 1.4 CD16 +, /kg b.w. of recipient 26.4 ± ± ± 16.1 CFC 1 4 /kg b.w. of recipient CFU-GM 49.7 (18 25) 59.2 (15 15) 21.4 ( ) BFU-E 114 ( ) 61. ( ) 46.2 ( ) CFU-GEMM 7.7 ( ) 3.72 ( ) 1.95 ( ) Values are expressed as mean ± s.d. Number of CFC is expressed as median (range).

4 374 a b c 12 d Figure 1 Correlation between CD34 + cells in blood and leukapheresis product. The efficiency of mobilization and leukapheresis was determined by calculating the number of CD34 + in the final component per volume of processed blood. Expression in this way compensates for individual differences in blood volume, number of total blood volumes processed and duration of leukapheresis. (a) Group 1, Pearson r =.772 (P.8); (b) group 2, Pearsson r =.767 (P.47); (c) group 3, Pearson r =.9432 (P.1); (d) all three groups, Pearson r =.826 (P.1). a b c d Figure 2 Correlation between CD34 + in blood and age. (a) Group 1, Pearson r =.4587; (b) group 2, Pearson r =.339; (c) group 3, Pearson r =.1979; (d) all three groups, Pearson r =.4587 (P.5).

5 Notably BFU-E were mobilized to a larger extent than CFU-GM in all dose groups. Discussion The optimal dosage of rhg-csf for stem cell (CD34 cells) mobilization in normal donors is not well defined. Although rhg-csf doses up to 24 g/kg/day have been given, 5,11 experience with such a dose range remains limited. In this study, we performed a randomized open label dose-finding trial and analysed the quality of harvest product mobilized with three different doses of lenograstim aiming to evaluate if the dose of 15 g/kg/day given in a split regimen would result in the same or improved numbers of CD34 cells harvested earlier, ie at day 4 after stimulation. We used lenograstim, since it has been shown in a comparative study that lenograstim at 1 g/kg/day mobilized PBSC more efficiently than an identical dose of filgrastim. 12 We were able to demonstrate that there was a significant correlation between circulating CD34 cells on the day of harvest and the CD34 cells in the apheresis products in all three groups. In addition to this, the mobilization of colony-forming cells was similar in all three groups, although a trend for fewer progenitors in group 3 was observed. A possibility that earlier collection results in fewer useful progenitors could not be neglected. However, all of the patients receiving PBSCT from group 3 of donors had sustained engraftment, indicating that colony-forming cells are not the only cells responsible for successful engraftment. The most important finding of our study was that a similar number of CD34 cells in the apheresis product could be obtained with one apheresis by using a dose of 15 g/kg/day lenograstim for only 4 days. In addition, we showed that the number of CD34 cells pre-apheresis was not significantly inversely correlated with age, in the group of donors mobilized with 15 g/kg/day. However, the lack of this correlation could also be due to the small numbers of patients and variability in this group. In particular, the incidence of side events in this group of donors was not increased in comparison to the donors mobilized with lower doses of lenograstim. The dose of 15 g/kg/day in a split regimen might be considered for volunteer donors, who turn out to be poor mobilizers, such as elderly people. With regard to the dosage used to harvest volunteer donors after lenograstim administration, a consensus has not yet been achieved. There is evidence that doses of less than 1 g/kg/day are less effective in increasing circulating numbers of CD34 cells. 1 Factors influencing yields of progenitor cells for allogeneic transplantation have been recently discussed. 17 Luider et al 17 have suggested that a daily filgrastim dose of 7.5 to 1 g/kg (intermediate dose) is a convenient regimen giving adequate yields from a single collection on day 4 or 5 in most donors. However, according to these authors, a collection on day 4 may also be suitable for most donors mobilized with intermediateand high-dose ( 1 g/kg) filgrastim. Other groups have reported series of normal donors, who have been mobilized with rhg-csf using amounts ranging from 2.5 to 24 g/kg/day. 1,11,18 Waller et al 11 showed that a high-dose (24 g/kg/day) twice daily regimen of nonglycosylated rhg- CSF was well tolerated and resulted in high numbers of CD34 cells in apheresis products in healthy donors. Recently, Arbona et al 19 similarly demonstrated that mobilization with 6 or 8 g/kg/12 h of filgrastim was also associated with collection of significantly more CD34 cells in comparison with 1 g/kg/24 h. Weaver et al 2 recently evaluated doses of filgrastim up to 4 g/kg/day but in an autologous setting in patients with breast cancer. In patients with good marrow reserves, doses of filgrastim greater than 1 g/kg/day yielded a higher number of CD34 cells and may be useful when high numbers of CD34 cells are required. Lenograstim appears to be well tolerated during shortterm administration and there do not appear to be long-term adverse effects. With regard to safety issues, G-CSF has been administered after several years of follow-up 21 in allogeneic-related PBSCT. PBSCT from unrelated donors has been increasingly used in EBMT programs. 22 It should be noted that in our study a remarkable variability in the response to lenograstim was observed. Several investigators 18,23,24 have already reported this for lenograstim as well as for filgrastim. It would be important to identify donors with poor mobilization, in order to prime them by different strategies, usually including combinations of cytokines. 25 In conclusion, the mobilization of allogeneic PBPC is feasible using short-term administration of different doses of lenograstim. The collection of allogeneic PBPC is safe at day 4 if split doses of 15 g/kg/day are used for mobilization and could be effectively used also in older donors. References 1 Dreger P, Suttorp M, Haferlach T et al. Allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for treatment of engraftment failure after bone marrow transplantation. Blood 1993; 81: Russel NH, Hunter AE. Peripheral blood stem cells for allogeneic transplantation. Bone Marrow Transplant 1994; 13: Bensinger WI, Weaver CH, Appelbaum FR et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995; 85: Schmitz N, Bacigalupo A, Labopin M et al on behalf of the European Group for Blood and Marrow Transplantation (EBMT). Transplantation of peripheral blood progenitor cells from HLA-identical sibling donors. Br J Haematol 1996; 95: Bensinger WI, Clift R, Martin P et al. Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood 1996; 88: Schmitz N, Bacigalupo A, Hasenclever D et al. Allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 1998; 21: Russel NH, Hunter A, Roger S et al. 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