on bone marrow progenitor cells - Nature

Bone Marrow Transplantation, (1999) 23, 15–19  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Effects of short-term administration of G-CSF (filgrastim) on bone marrow progenitor cells: analysis of serial marrow samples from normal donors C Martıńez, A Urbano-Ispizua, M Rozman, M Rovira, P Marıń, N Montfort, E Carreras and E Montserrat Hematology Department, Postgraduate School of Hematology Farreras-Valentı´, Institut d’Investigacions Biome`diques August Pi i Sunyer, Hospital Clıńic, University of Barcelona, Spain

Summary: To determine the effect of G-CSF administration on both the total number of CD34ⴙ cells and the primitive CD34ⴙ subsets in bone marrow (BM), we have analyzed BM samples serially obtained from 10 normal donors in steady-state and during G-CSF treatment. Filgrastim was administered subcutaneously at a dosage of 10 ␮g/kg/day (n = 7) or 10 ␮g/kg/12 h (n = 3) for 4 consecutive days. Peripheral blood sampling and BM aspirates were performed on day 1 (just before G-CSF administration), day 3 (after 2 days of G-CSF), and day 5 (after 4 days of G-CSF). During G-CSF administration, a significant increase in the total number of BM nucleated cells was observed. The percentage (range) of CD34ⴙ cells decreased in BM from a median of 0.88 (0.47–1.44) on day 1 to 0.57 (0.32–1.87), and to 0.42 (0.16–0.87) on days 3 and 5, respectively. We observed a slight increase in the total number of BM CD34ⴙ cells on day 3 (0.66 × 109/l (0.13–0.77) ), and a decrease on day 5 (0.23 × 109/l (0.06–1.23) ) as compared with steady-state (0.40 × 109/l (0.06–1.68) ). The proportion of primitive BM hematopoietic progenitor cells (CD34ⴙCD38ⴚ, CD34ⴙHLA-DRⴚ, CD34ⴙCD117ⴚ) decreased during G-CSF administration. In parallel, a significant increase in the total number of CD34ⴙ cells in peripheral blood was observed, achieving the maximum value on day 5. These results suggest that in normal subjects the administration of G-CSF for 5 days may reduce the number of progenitor cells in BM, particularly the most primitive ones. Keywords: G-CSF; bone marrow CD34+ cells; normal donors

Peripheral blood stem cells (PBSC) are being increasingly used for both autologous and allogeneic transplantation. Numerous studies have shown that mobilized PBSC result in a more rapid hematopoietic reconstitution after myeloablative therapy as compared with bone marrow stem Correspondence: Dr C Martıńez, Hematology Department, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain Received 17 June 1998; accepted 10 August 1998

cells.1–4 A large amount of information about the effects of cytokines on the mobilization of progenitor cells in peripheral blood is available.5–10 Thus, it is well established that 4 to 5 days of G-CSF administration to patients or normal donors effectively increases the absolute number of circulating CD34+ cells, allowing the collection of sufficient quantities of progenitor cells to ensure sustained engraftment after transplantation.6–9 Data concerning the effects of G-CSF on bone marrow (BM), however, are scarce. In fact, it has not been elucidated whether the PBSC mobilization schedules currently in use result in the simultaneous enrichment of stem and progenitor cells in BM, or simply in a shift of these cells from BM to peripheral blood. Some clinical trials have recently been conducted to determine the progenitor cell content and engraftment capability of BM harvested after G-CSF administration, with controversial results.11–16 The main aim of this study was to analyze, in a population of normal donors, the effects of GCSF administration on the number of BM progenitor cells, with special emphasis on the immature cell subsets.

Materials and methods Donors and G-CSF administration Peripheral blood and BM samples were obtained from 10 healthy donors included in a program of allogeneic PBSC transplantation. Donors were thoroughly informed about the investigative nature of the procedure and gave their written consent. The protocol and consent forms were approved by the Hospital Clinic Ethics Committee and by the Spanish Ministry of Health. Median age of the donors was 48 years (range 20–57 years). Recombinant human GCSF (Filgrastim; Amgen, Thousand Oaks, CA, USA) was administered subcutaneously at a dosage of 10 ␮g/kg/day (n = 7) or 10 ␮g/kg/12 h (n = 3) for 4 consecutive days. Bone marrow aspirates from the sternum were performed under local anesthesia on day 1 (just before G-CSF administration), on day 3 (after 2 days of G-CSF), and on day 5 (after 4 days of G-CSF). The samples were collected with EDTA. In order to minimize peripheral blood cell contamination, no more than 1–1.5 ml of BM were obtained from each aspiration. Peripheral blood samples were collected by venipuncture from each donor on days 0, 3 and 5.

Short-term administration of filgrastim on BMPC C Martıńez et al

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Quantification and phenotyping of CD34+ cells Bone marrow samples were collected with EDTA and diluted 1:2 in phosphate-buffered saline (PBS). Cell counts were performed on the whole BM and peripheral blood using an automated cell counter (Sysmex F-800; Baxter Fenwal, Deerfield, IL, USA). CD34 quantification was performed on unfractioned peripheral blood and BM using FACS Lysing Solution (Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA, USA) and staining with the CD34-specific phycoerythrin (PE)-conjugated monoclonal antibody (MAb) 8G12 (HPCA-2-PE) (BDIS). The following MAbs were obtained from BDIS: FTIC-labeled and PE-labeled 8G12 against CD34, FTIC-labeled MAbs against CD3 (Leu-4), and CD19 (Leu-12), PE-labeled MAbs against CD56 (Leu-19), CD33 (anti-Leu-M9), CD38 (anti-Leu-17), and HLA-DR (anti-HLA-DR). The PE-labeled MAb against CD117 was obtained from Immunotech SA (Marseilles, France). The Simultest control (IgG1 FTIC + IgG2a PE) was purchased from BDIS. Flow cytometry was performed on a FACScan (BDIS) equipped with an air-cooled argon ion laser tuned to 488 nm. Lysis II software was used for data acquisition and analysis. For the purpose of CD34+ cell analyses, a gate for viable cells excluding erythrocytes and debris was set according to forward (FSC) and side light scatter (SCC). The CD34+ cells were analyzed in a fluorescence vs SCC plot. Thus, only cells with low SCC were counted as CD34+ cells. To obtain sufficient CD34+ cells for analysis of CD34+ subsets, an acquisition gate was set according to SSC and fluorescence intensity range comprising the cells with positive CD34 fluorescence signals. A minimum of 300 000 events were run through the cytometer per test, and 3000–5000 CD34+ cells within the acquisition gate were stored in listmode data files for further analysis.

median (range) of 1.7 (0.6–5.5) in steady-state to 1.9 (0.8– 7.2), and 7.5 (3.2–9.3) on day 3 and 5, respectively. Abnormal cytoplasmic vacuolization of promyelocytes was found on days 3 and 5 in seven cases. T lymphocytes and NK cells in bone marrow The relative numbers of CD3+ T lymphocytes and CD3+CD56+ NK cells decreased from a median (range) of 7.7% (4–21) and 1.65% (1.26–3.26) on day 1 to a median of 3.7% (2.4–6.5) and 0.9% (0.3–1.65) on day 5, respectively. The total number of T and NK cells did not change. Nucleated and CD34+ cells in bone marrow An increase in the total number of nucleated BM cells was observed during G-CSF administration, from a median × 109/l (range) of 41.5 (13.5–104) to 48 (35.5–120.8) (P = 0.06) on day 3, and 56 (36.4–176) (P = 0.02) on day 5. At the same points in time, the percentage (range) of CD34+ cells decreased from a median of 0.88 (0.47–1.44) to 0.57 (0.32–1.87) (P = 0.09) and to 0.42 (0.16–0.87) (P = 0.01) (Table 2). The total number × 109/l (range) of BM CD34+ cells increased slightly on day 3 (0.66 (0.13–0.77) ), and decreased on day 5 (0.23 (0.06–1.23) ) as compared with basal values (0.40 (0.06–1.68)) (Figure 1). These changes were not statistically significant, probably due to the small number of cases included in the study. Nucleated and CD34+ cells in peripheral blood

Median values and ranges for each parameter were determined. The Wilcoxon test (paired, nonparametric) was performed to compare the results of the different subpopulations.

The peripheral WBC count increased from a median × 109/l (range) of 5.2 (3.2–13.6) before G-CSF administration to 25.3 (23.8–37.8) (P = 0.02) and 39.4 (21.0–69.2) (P = 0.01) on days 3 and 5 of cytokine administration, respectively. The percentage of CD34+ cells rose from a median (range) of 0.002 (0.001–0.003) to 0.023 (0.007–0.030) (P = 0.06) on day 3, and 0.115 (0.028–1.62) (P = 0.01) on day 5 (Table 2). This resulted in a significant increment in the total number of CD34+ cells in the peripheral blood, the maximum value being achieved on day 5 (Figure 1).

Results

CD34+ cell subsets

Statistics

Overall bone marrow cellularity Bone marrow cellularity was highly enriched in myeloid cells during G-CSF administration. An increase in both early (myeloblasts and promyelocytes) and late myeloid precursors (myelocytes and metamyelocytes) was observed (Table 1). The myeloid:erythroid ratio changed from a Table 1

Day 1 Day 3 Day 5

Effect of G-CSF administration on bone marrow cellularity Promyelocytes

Myelocytes + metamyelocytes

Neutrophils

6 (3–11) 21 (10–23) 26 (21–39)

20 (11–30) 16 (12–33) 35 (24–54)

17 (11–40) 17 (2–64) 15 (7–28)

Values are expressed as percentage of total cellularity.

We analyzed CD34+ cell subsets from the BM in steadystate and during G-CSF administration, and from the peripheral blood on day 5. Since CD34+ cells were nearly undetectable in peripheral blood in steady-state, no accurate analysis of the changes in CD34+ cell subsets in peripheral blood could be done. FACS analysis showed that more than 90% of CD34+ cells in both compartments coexpressed the CD38 and HLA-DR antigens at baseline and during cytokine treatment. The proportion of primitive BM hematopoietic progenitors identified as CD34+CD38⫺, CD34+HLADR⫺ and CD34+CD117⫺ decreased during G-CSF administration (Figure 2). No significant changes were observed in the proportion of BM CD34+CD33+ and CD34+CD19+ cells during cytokine administration. The mean fluorescence intensity of CD34+ cells, used as a parameter for antigen density, was higher in BM CD34+ cells in steady-state as compared with those from days 3 and 5 (Figure 3).


Total nucleated cells, percentage of CD34+ cells and total CD34+ cells in bone marrow and peripheral blood before and during G-CSF admin-

Table 2 istration

Total nucleated cells ×109/l

% CD34+

CD34+ cells ×109/l

Bone marrow day 1 day 3 day 5

41.4 (13.5–104) 48 (35.5–120.8) 56 (36.4–176)

0.88 (0.47–1.44) 0.57 (0.32–1.87) 0.42 (0.16–0.87)

0.40 (0.06–1.68) 0.66 (0.03–0.77) 0.23 (0.06–1.23)

Peripheral blood day 1 day 3 day 5

5.16 (3.2–13.6) 25.3 (23.8–37.8) 39.4 (21–69.2)

0.04 (0.01–0.07) 0.08 (0.03–0.09) 0.16 (0.07–1.62)

0.002 (0.001–0.003) 0.023 (0.007–0.030) 0.115 (0.028–1.620)

Values are expressed as median and range. 200

1.0 BM

a 150

0.6 100

0.4 0.2

50

100 60 3.0 2.5

%

BM CD34+ cells ×109/l

PB

PB CD34+ cells ×106/l

0.8

2.0 1.5

0.0

1.0 0.5

0

–0.2 1

2

3

4

0.0

5

day 1

day 3

day 5

Days of G-CSF administration

Figure 1 Effect of G-CSF administration on bone marrow (BM) and peripheral blood (PB) CD34+ cells. Results are expressed as median ⫾ s.e.m.

CD34+CD38– b 100 60

Discussion

%

4 3 2 1 0 day 1

day 3

day 5

CD34+HLADR– c 100 70 50 40

%

Recent studies suggest that G-CSF administration might increase the content of progenitor cells in BM, and change the biological characteristics of bone marrow stem cells, making them similar to PBSC.11,12,17 These observations are the background for some recent clinical trials aimed at determining the engraftment capability of G-CSF-stimulated BM. Thus, it has been reported that the kinetics of engraftment with G-CSF-stimulated BM could be as rapid as engraftment with PBSC, and superior to conventional autologous bone marrow transplants;11,13–15 other authors, however, have found no differences after primed BM when compared with historical controls of unprimed BM.12,16 A preliminary report of G-CSF-stimulated BM allogeneic transplantation suggests that engraftment is rapid and stable.18 A high risk of chronic graft-versus-host disease has been reported after allogeneic PBSC transplants,19,20 probably due to the large number of T lymphocytes contained in leukapheresis products. Therefore, the administration of G-CSF before BM harvesting has been suggested as an alternative to increase the quantity of progenitor cells without a corresponding increase in the number of lymphocytes. Nevertheless, the effects of G-CSF on the total number of both progenitor cells and immature CD34+ subsets in bone marrow have been poorly investigated. In a recent study in mice, a great increase in the number of primitive BM hematopoietic progenitor cells after 2 days of G-CSF was observed, declining afterwards, to reach

30 20 10 0

day 1

day 3

day 5

CD34+ckit– Figure 2 Proportion of bone marrow primitive CD34+ subsets (a, CD34+CD38⫺; b, CD34+HLADR⫺; c, CD34+ckit⫺) in steady-state (day 1) and during G-CSF administration (days 3 and 5). Data are presented as mean ⫾ s.e.m. values.

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18

Mean fluorescence intensity

300

ing the optimal timing of G-CSF-stimulated BM collection to guarantee a sustained engraftment. To that end, our study suggests that a brief course of G-CSF could be enough to improve the myeloid engrafting capacity of BM. This would reduce the cytokine exposure time of normal donors and would avoid the possibility of immature progenitor cells migrating to the peripheral blood on day 5. Further studies to establish the optimal timing of G-CSF-stimulated BM collection, and the quality of engraftment and longterm hematopoietic reconstitution in allogeneic primed BM recipients are required.

250 200 150 100 50 0 day 1

day 3

day 5

Figure 3 Changes in mean fluorescence intensity of CD34+ cells in steady-state (day 1) bone marrow (BM) and during G-CSF treatment (days 3 and 5). Data are presented as mean ⫾ s.e.m. values.

basal level on days 5–6. Most of the reported progenitor cell expansion in BM occurred prior to the appearance of these cells in peripheral blood.21 We observed a slight increase in the absolute number of BM CD34+ cells on day 3 of G-CSF administration, whereas CD34+ cells decreased below steady-state values on day 5. Although it has been reported that 5 days of G-CSF administration results in a higher number of BM CFU-GM and BFU-E, compared with historical controls,11,12 in most studies no significant increase in BM CD34+ cell content has been observed.15,22,23 Indeed, we found that the proportion of primitive BM hematopoietic progenitors (CD34+CD38⫺, CD34+HLADR⫺ and CD34+CD117⫺ cells) decreased during G-CSF administration, with the lowest value being reached on day 5. In this regard, several studies suggest that cytokines can shift early repopulating stem cells out of the marrow into the peripheral blood. Thus, Bodine et al24 found that after 5 days of SCF/G-CSF, mice had only 25% of the baseline long-term repopulating stem cells in the marrow, and three times the amount of baseline stem cells had shifted to peripheral blood. Mandalam et al25 in a preliminary report on normal donors, suggest that the proportion of CD34+ primitive subsets in BM is lower in donors having received G-CSF for 3 days, than in those treated for 2 days, and in those who received no treatment. During prolonged G-CSF administration to mice, de Haan et al26 also showed a severe depletion of BM stem cells, particularly the most primitive ones, that increased in the spleen. In the clinical setting, Mavroudis et al27 have recently reported, on a group of 12 patients, three cases of incomplete engraftment and four cases that developed pancytopenia 2 to 3 months following T cell-depleted GCSF-stimulated BM allogeneic transplant. This suggests that after 5 days of G-CSF administration, the BM provided a sufficient number of engrafting cells which were, nonetheless, deficient in self-renewing, long-term repopulating cells. In conclusion, our study shows that in normal subjects the administration of G-CSF for 5 days may reduce the number of progenitor cells in BM, particularly the most primitive ones, basically by increasing their mobilization into peripheral blood. This poses the question of determin-

Acknowledgements This work has been supported in part by grants SGR 1996/0068 from Comissionat per a Universitats i Recerca, Generalitat Catalunya, and FIS-98/995 from Fondo de Investigaciones Sanitarias de la Seguridad Social, Spanish Ministry of Health.

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