Mobilization of peripheral blood progenitor cells (PBPC) in ... - Nature

Bone Marrow Transplantation, (1999) 23, 1101–1107  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Mobilization of peripheral blood progenitor cells (PBPC) in patients undergoing chemotherapy followed by autologous peripheral blood stem cell transplant (SCT) for high risk breast cancer (HRBC) I Benet, F Prosper, I Marugan, A Lluch, C Arbona, I Castillo, C Solano and J Garcia-Conde Department of Hematology and Medical Oncology, University Hospital, University of Valencia, Spain

Summary: We have determined the effect of delayed addition of G-CSF after chemotherapy on PBPC mobilization in a group of 30 patients with high risk breast cancer (HRBC) undergoing standard chemotherapy followed by high-dose chemotherapy (HDCT) and autologous SCT. Patients received FAC chemotherapy every 21 days followed by G-CSF at doses of 5 ␮g/kg/day starting on day +15 (groups 1 and 2) or +8 (group 3) after chemotherapy. PBPC collections were performed daily starting after 4 doses of G-CSF and continued until more than 2.5 × 106 CD34ⴙ cells had been collected. In group 1, steady-state BM progenitors were also harvested and used for SCT. Groups 2 and 3 received PBPC only. The median number of collections was three in each group. Significantly more PB CD34ⴙ cells were collected in patients receiving G-CSF starting on day 8 vs day 15 (9.43 ⴛ 106/kg and 6.2 ⴛ 106/kg, respectively) (P ⬍ 0.05). After conditioning chemotherapy all harvested cells including BM and PBPC were reinfused. Neutrophil and platelet engraftment was significantly faster in patients transplanted with day 8 G-CSF-mobilized PBPC (P ⬍ 0.05) and was associated with lower transplant related morbidity as reflected by days of fever, antibiotics or hospitalization (P ⬍ 0.05). Both schedules of mobilization provided successful long-term engraftment with 1 year post-transplant counts above 80% of pretransplant values. In conclusion, we demonstrate that delayed addition of G-CSF results in successful mobilization and collection of PBPC with significant advantage of day 8 G-CSF vs day 15. PBPC collections can be scheduled on a fixed day instead of being guided by the PB counts which provides a practical advantage. Transplantation of such progenitors results in rapid short-term and long-term trilineage engraftment. Keywords: mobilization; breast cancer; transplant; chemotherapy; G-CSF

Correspondence: Dr I Benet, Department of Hematology and Medical Oncology, Avd Blasco Ibanez 17, Hospital Clinico Universitario, University of Valencia, Valencia 46010, Spain Received 23 July 1998; accepted 13 January 1999

During the last few years the use of mobilized peripheral blood progenitor cells (PBPC) has replaced bone marrow (BM) for hematopoietic stem cell transplantation.1–3 Several studies have demonstrated that the use of PBPC instead of BM progenitors is associated with faster engraftment, decreased hospitalization and reduced transplant-related complications.4–7 PBPC transplantation has also been associated with successful long-term engraftment suggesting that mobilized PB progenitors contain enough stem cells to reconstitute long-term hematopoiesis after myeloablative chemotherapy.8,9 Hematopoietic progenitors for transplantation can be mobilized into the peripheral blood by treatment with growth factors, chemotherapy or a combination of both.9 Although multiple cytokines and growth factors have demonstrated their capacity to induce PBPC mobilization only the use of G-CSF and GM-CSF has been approved for mobilization in humans. Mobilization with G-CSF has been extensively studied both in normal donors as well as in patients with cancer.5,10–12 After treatment with G-CSF, the number of progenitor cells in the PB peaks between the 4th and 6th day with a five- to 20-fold increase in the number of CD34+ cells/ml in the peripheral blood over baseline values.12 G-CSF-induced mobilization is also dose and schedule dependent.12–14 When chemotherapy and growth factors are used for mobilization, treatment with G-CSF is usually started 24– 48 h after chemotherapy and continued until the target CD34+ cell dose has been collected.5,15,16 In comparison with growth factors, chemotherapy and growth factorinduced mobilization is associated with potential disadvantages such as an increase in morbidity and even mortality due to neutropenia and thrombocytopenia as well as difficulties in predicting the day after treatment on which PBPC collections should be initiated.9,17 On the other hand, the use of chemotherapy plus growth factor may increase the yield of PBPC and allows for the concomitant treatment of the underlying disease.15 Recent studies have suggested that dose intensity (highdose chemotherapy with stem cell support) and dose density (multiple courses of chemotherapy with short intervals) may have an impact on disease-free survival in patients with high risk breast cancer.18–21 The use of growth factors accelerates recovery after chemotherapy and allows administration of courses of chemotherapy in short intervals. New trials for breast cancer patients have been designed in which courses of chemotherapy every 15 days are followed by

Chemotherapy with delayed G-CSF induced mobilization I Benet et al

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high-dose chemotherapy with stem cell support.20 One of the problems of this approach is, however, to define the best method and time for PBPC collection. In this study, high risk breast cancer patients included in a program of consecutive courses of chemotherapy followed by high-dose chemotherapy with stem cell support were mobilized with G-CSF for procurement of stem cells. Administration of G-CSF for mobilization was initiated either on day 8 or day 15 after chemotherapy and PBPC collections were performed starting between 18 and 24 h after the fourth daily dose of G-CSF, independent of cell counts. We have analyzed the characteristics of mobilization, yield of progenitors and long-term and short-term engraftment after high-dose chemotherapy with stem cell support according to the type of mobilization. Patients and methods Patients Patients with pathological stage II or III breast cancer and ⭓10 axillary lymph nodes involved were eligible for adjuvant chemotherapy with 5-FU (600 mg/m2 day +1), doxorubicin (50 mg/m2 day +1) and cytoxan (600 mg/m2 day +1) (FAC)22,23 every 21 days for a total of six courses followed by high-dose chemotherapy and autologous hematopoietic stem cell transplantation. Additional inclusion criteria were age between 18 and 60 years old, ECOG performance status 0 or 1 and evidence of normal renal, liver and cardiac function. All patients signed an informed consent and the protocol was approved by the hospital ethics committee. Patient characteristics are shown in Table 1. Mobilization and cryopreservation of hematopoietic stem cells Patients without evidence of disease progression after six courses of adjuvant chemotherapy were consecutively enrolled in two different mobilization groups. The last course of FAC was used for mobilization and collection of PBPC. In each course, day +1 was considered the first day of chemotherapy. In group 1 (n = 10), G-CSF (5 ␮g/kg/day) Table 1

Characteristics of patients according to source of stem cells BM and PB Mobilized day 15 PB day 15 (group 1) (group 2)

No. Age Pathological stage II III Histology Invasive ductal Papilar Undifferentiated

Mobilized PB day 8 (group 3)

10 9 11 41 (29–58) 47 (35–60) 41 (37–55) 5 5

3 6

5 6

8 1 1

8 0 1

9 0 2

Day 15 G-CSF (5 ␮g/kg/day) was started on day +15 post chemotherapy. Day 8 G-CSF (5 ␮g/kg/day) was started on day +8 post chemotherapy. BM = bone marrow; PB = peripheral blood.

was started on day +15 and PBPC collections were performed daily starting after four doses of G-CSF until a target cell dose of 2.5 × 106 CD34+ cells per kilogram had been collected. Patients in group 1 underwent BM harvest at least 30 days after the last PBPC apheresis. Between the last PBPC collection and BM harvest no G-CSF was administered. BM progenitors were harvested by multiple aspirations from the posterior iliac crest under general anesthesia. In group 2 (n = 9), mobilization was performed as in group 1 but no BM harvest was performed. In group 3 (n = 11), G-CSF (5 ␮g/kg/day) was initiated on day +8 and PBPC collections were performed daily starting after four doses of G-CSF until a target cell dose of 2.5 × 106 CD34+ cells per kilogram had been collected. PBPC collections were performed with a Fenwal CS 3000 (Baxter, Deerfield, IL, USA) as previously described.14 PB and BM harvested cells were cryopreserved in 10% DMSO and stored in liquid nitrogen. High-dose chemotherapy and hematopoietic stem cell transplant Pretransplant conditioning chemotherapy included etoposide 7 mg/kg/day on days −6 to −3, cyclophosphamide 50 mg/kg/day on days −5 and −4 and melphalan 140 mg/m2 on day −2. On day 0 cryopreserved PBPC with or without BM were thawed and infused through a central venous access. All previously harvested cells were reinfused. The infused cell dose corresponds to the total dose of MNC cells, CD34+ cells or CFU-GM collected. Patients did not receive G-CSF after PBPC infusion. Patients received prophylactic antibiotics including trimethoprin-sulfamethoxazole from day −6 to discharge and norfloxacin from admission until the first episode of fever greater than 38°C when broad-spectrum antibiotics were started. Platelet transfusions and RBC transfusions were administered for platelet counts lower than 20 × 109/l or hemoglobin lower than 8.0 g/dl, respectively. Transplant-related toxicity including nausea and vomiting, mucositis and diarrhea were graded according to the WHO scale.24 Patients were discharged from the hospital when the neutrophil count was above 1.5 × 19/l and did not have evidence of ongoing infection or fever. Patients were followed in the outpatient clinic after discharge on days +14, +21, +30, +60, +90, +180 and +360 post-PBPC infusion. Blood counts Blood count and differential from PB and apheresis product were performed with an automated cell counter (H-3 Technicon; Bayer Diagnostics). Flow cytometry The CD34+ cell count was performed as previously described.14 Briefly, an aliquot of PB or apheresis product containing 1 × 106 cells was incubated with 10 ␮l of antibody to CD34 (anti HPCA-2; Becton Dickinson, San Jose, CA, USA) coupled to fluorescein isothiocyanate (FITC) for 20 min at 4°C. After lysis of red cells with ammonium chloride, the cells were washed with phosphate-buffered


saline solution (PBS) and then resuspended in 0.5 ml of PBS. IgG1 coupled to FITC was used as negative control. For analysis, 40 000 cells were acquired in list-mode using a flow cytometer (FACScan; Becton Dickinson) and software (LYSIS II; Becton Dickinson). Hematopoietic cell culture A total of 2–5 × 105 nucleated cells were plated in methylcellulose containing Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO Laboratories, Grand Island, NY, USA), supplemented with 20% fetal calf serum (FCS) (Hyclone, Logan, UT, USA) and 100 ng/ml of G-CSF (Amgen, Thousand Oaks, CA, USA). Cultures were incubated in a humidified atmosphere at 37°C and 5% CO2. The cultures were assessed on days 14–18 for the presence of CFU-GM as previously described.14 Statistics Statistical analysis was performed using SPSS software. Variables are expressed as median and range unless otherwise stated. Differences between groups were determined by the Mann–Whitney U test and Kruskal–Wallis for multiple groups. Correlations were calculated using the Pearson’s correlation coefficient. Distribution between categorical variables was examined by ␹2 test. Results Mobilization and collection of PBPC The total number of WBC, neutrophils, CD34+ cells, hemoglobin and platelet counts immediately before the first dose of G-CSF is depicted in Table 2. As patients in groups 1 and 2 received the same mobilization schedule comparisons were established between patients receiving G-CSF starting on day +8 post chemotherapy and patients receiving G-CSF from day +15. There were no statistically significant differences in the percentage or total number of CD34+ cells, number of platelets or hemoglobin before starting G-CSF between groups 1, 2 and 3. However, pre G-CSF values for WBC and neutrophil counts were significantly higher for patients in group 3 which most likely represents the fact

that the nadir of WBC and neutrophils had not been reached by day 8 after chemotherapy. After 4 days of G-CSF there was a statistically significant increase in the total number of CD34+ cells, WBC and neutrophils in the PB in both groups (P ⬍ 0.01) (Tables 2 and 3). The percentage of CD34+ cells was augmented in the group of patients receiving G-CSF from day 8 but not in those receiving G-CSF from day 15. However, the total number of CD34+ cells per ml of PB increased significantly in both groups (P ⬍ 0.001) (Tables 2 and 3). Interestingly, G-CSF induced a greater increase in the number of WBC and neutrophils in the group of patients receiving G-CSF from day 15. These results suggest that administration of G-CSF from day 8 may be cause mobilization of a higher percentage of progenitors while G-CSF started on day 15 may have a greater effect on mature cells vs progenitor cells. Owing to the fact that the total number of CD34+ cells per ml of blood depends both on the percentage and the total number of nucleated cells we did not find any statistically significant differences in the number of CD34+ cells per ml of blood between patients mobilized with day 8 or day 15 G-CSF (Table 3). To better characterize differences between both groups we analyzed the number of phenotypically defined progenitors per ml of blood at the time of the first apheresis. Significantly more CD34+ CD33+ cells per ml of blood were found in the PB of patients mobilized with G-CSF starting on day 8 which might reflect an increase in the number of myeloid committed progenitors (Table 3). There were no significant differences between both groups in the total number of CD34+ cells that coexpressed other cell surface markers including CD38, HLA-DR, CD13, CD19, CD71 or CD10 (data not shown). The median number of aphereses was three in each group. Although there were no differences in the number of MNC collected between patients mobilized with G-CSF starting on day 8 after chemotherapy or on day 15, significantly more CD34+ cells were obtained in patients receiving G-CSF from day 8 after chemotherapy (Table 4). The median number of CFU-GM collected was higher in the group of patients receiving G-CSF on day 8. However, these differences were not statistically significant probably due to the wide range. PBPC transplantation and hematopoietic engraftment

Table 2 Pre G-CSF values. Comparison between patients mobilized with G-CSF starting on day 8 or day 15 G-CSF from day 15 G-CSF from day 8 P value groups 1 and 2 group 3 No. of patients WBC × 109/l Neutrophils Hemoglobin g/dl Platelet × 109/l CD34+ % CD34 cells/ml

1.7 0.8 11.2 205 0.24 2675

19 (1.01–3.9) (0.18–1.68) (9.2–13) (105–447) (0.06–0.52) (1984–6601)

Results represent median and range. NS = not statistically significant.

3.32 1.94 10.7 184 0.11 4913

11 (2.39–6.82) (1.42–5.67) (9.3–12.6) (125–318) (0.04–0.28) (1307–9600)

0.01 0.009 NS NS NS NS

Leukocyte, neutrophil and platelet engraftment for patients in each group are shown in Table 5. Patients in group 1 received both mobilized PBPC and BM progenitors and the median number of BM MNC infused (according to the number of harvested cells) was 4.48 × 108/kg with 0.16 × 104/kg CFU-GM. Patients in group 3 had a faster WBC, neutrophil and platelet engraftment than patients in group 2. There were no differences between engraftment kinetics in patients receiving day 15 G-CSF-mobilized PBPC with or without the addition of BM progenitors. Transplantation with day 8 G-CSF-mobilized PBPC was also associated with a statistically significant decrease in platelet transfusions, days of fever and antibiotics and with a shorter hospitalization in comparison with group 2 (Table 5). Long-term engraftment was assessed by compar-

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Table 3

Cell values in the PB on the first day of apheresis in patients mobilized with G-CSF starting on day 8 or day 15

No. of patients WBC × 109/l Neutrophils Hemoglobin g/dl Platelet × 109/l CD34+ % CD34+ cells/ml CD34/CD33+ % CD34/CD33+ cells/ml

G-CSF from day 15

G-CSF from day 8

P value

19 28.15 (16.02–50.43) 23.82 (12.5–44.6) 11.2 (8.2–12.7) 205 (138–409) 0.18 (0.01–0.49) 49 569 (1622–83 210) 56.5 (24.3–96.3) 1453 (167–4390)

11 6.12 (2.76–32.51) 3.94 (0.39–24) 10.5 (8.9–12.5) 125 (75–282) 0.71 (0.1–1.2) 42 032 (2740–206 880) 94 (61.3–99.3) 2819 (109–16 523)

0.004 0.001 NS 0.001 0.004 NS 0.001 0.05


Table 4

Number of mobilized PB progenitors collected in each group Day 15 G-CSF

No. of patients 19 No. of collections 3 (2–5) 6.82 (4.9–9.5) MNC × 108/kg 6.2 (0.4–12) CD34+ cells × 106/kg 4 18.55 (2.7–623.5) CFU-GM × 10 /kg

Day 8 G-CSF

P value

11 3 (2–4) 6.31 (3.8–13.3) 9.43 (5–25.9) 38.92 (4.4–92)

NS NS 0.008 NS


Table 5 Transplant-related morbidity and engraftment kinetics after mobilized PBPC transplantation Group 1 WBC ⬎1.0 × 109/l ⬎3.0 × 109/l Neutrophils ⬎0.5 × 109/l ⬎1.0 × 109/l Platelets ⬎20 × 109/l ⬎100 × 109/l Platelet transfusion Antibiotics Fever Hospitalization

Group 2

Group 3

P value

11 (9–46) 14 (12–22) 12 (10–15) 16 (10–90) 22 (16–29) 13 (10–14)

0.05 0.05

12 (9–48) ND

15 (12–23) 13 (8–17) 21 (13–24) 13 (10–19)

NS 0.005

10 (9–90) ND 2 (1–8) 18 (0–50) 5 (0–13) 20 (14–53)

13 22 4 14 4 22

0.002 NS 0.002 0.01 0.02 0.04

(10–24) 9 (8–11) (21–23) 14 (12–17) (2–12) 1 (1–4) (0–23) 11 (9–14) (0–11) 1 (0–4) (15–28) 17 (10–21)

NS = not statistically significant; ND = not determined; P = comparison between group 2 and group 3.

ing the number of neutrophils, hemoglobin and platelets 1 year after mobilized PBPC transplant. As shown in Figure 1, engraftment was above 75% of pretransplantation counts for WBC, hemoglobin and platelet counts with no differences between the three groups. We finally determined the kinetics of BM and PB progenitor repopulation by analyzing the number of CFU-GM per 105 MNC in the BM and PB at different time points after transplantation. Although recovery of CFU-GM in BM and PB followed a different pattern, there were no statistically significant differences between patients receiving G-CSF on day 8 or 15 nor between patients receiving mobilized PBPC with or without BM (Figure 2).

Discussion Although PBPC mobilization with either growth factor or chemotherapy immediately followed by growth factor is considered the standard method to obtain stem cells for transplantation,25–27 in this report we demonstrate that adding G-CSF at different times after chemotherapy (8 or 15 days after chemotherapy) results in high yields of PBPC. Furthermore, transplantation with day 8 or day 15 mobilized PBPC provides rapid and sustained engraftment in patients with breast cancer undergoing high-dose chemotherapy and autologous SCT. The rationale behind the late addition of growth factor is supported by a recently published study by Haynes et al. Forty-two patients received cyclophosphamide and G-CSF for mobilization of PBPC. G-CSF was started on day +5 after chemotherapy. Delayed addition of G-CSF resulted in successful PBPC collections and subsequent SCT with significant cost saving.28 Our patients did not receive standard chemotherapy for mobilization. However, cyclophosphamide and doxorubicin have been used for mobilization in multiple studies. Furthermore, the goal of our therapy was to prevent delays in administration of chemotherapy and at the same time to facilitate collection of PBPC for SCT. Treatment with G-CSF was delayed until day 8 or day 15 after chemotherapy without compromising the number of PBPC collected. This finding may be particularly relevant in the context of new strategies for treatment of breast cancer patients where sequential therapy followed by HDC and SCT may offer an advantage in DFS and OS by increasing dose density and intensity.20 Our schedule of mobilization allows for harvesting of PBPC without delaying chemotherapy and without requiring growth factor for prolonged periods of time. One of the main problems that faces the use of chemotherapy plus growth factor for mobilization is the difficulty in predicting the exact day on which apheresis should be initiated.17,29 Generally, collections are initiated when the number of WBC or ANC increases above a threshold that varies according to the study. Aphereses are usually performed daily until the target CD34+ cell dose has been collected.5,29 In our study we established the day for the first collection independently of the WBC or neutrophil count by taking into account the number of days on G-CSF. In every patient, we were able to harvest enough PBPC for


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200

a

a BM CFU-GM × 105 MNC

% Neutrophil engraftment

100 90 80 70 60 50 Day +30

Day +180

150

100

50

0

Day +360

+14

+21

+30

+60

+90

+180

+360

+180

+360

Days after transplantation b

60

b

110

PB CFU-GM × 105 MNC

% Hemoglobin engraftment

120

100

90

80 Day +30

Day +80

50 40 30 20 10

Day +360 0 +14

% Platelet engraftment

100

c

+21

+30

+60

+90

Days after transplantation

90

Day 15 PBPC + BMPC

Day 15 PBPC

Day 8 PBPC

80

Figure 2 Kinetics of peripheral blood and bone marrow CFU-GM after PBPC transplantation. Number of CFU-GM per 105 MNC after PBPC transplant in BM (a) or PB (b) at different time points. Results represent the median for all patients in each group. Day 15: patients receiving G-CSF starting on day 15 post chemotherapy; day 8: patients receiving G-CSF starting on day 8 post chemotherapy.

70 60 50 Day +30

Day +180

Day 15 PBPC + BMPC

Day +360 Day 15 PBPC

Day 8 PBPC

Figure 1 Long-term engraftment after mobilized PBPC transplantation in patients with breast cancer. Percentage of neutrophil (a), hemoglobin (b) and platelet (c) engraftment in comparison with pretransplant levels. Results represent the median percentage of engraftment for all patients in each group. Day 15: patients receiving G-CSF starting on day 15 post chemotherapy; day 8: patients receiving G-CSF starting on day 8 post chemotherapy. BM, Bone marrow; PB, peripheral blood.

transplantation and the median number of CD34+ cells were well within the values reported for patients in similar conditions in which collection was performed according to the WBC or neutrophil count.5,30 The percentage of CD34+ cells after G-CSF treatment in the group of patients receiving G-CSF from day 8 was significantly higher than in patients receiving G-CSF from day 15 indicating a more selective effect on progenitors vs mature cells by adding G-CSF a few days earlier. Despite the fact that the total number of CD34+ cells per ml of blood was not significantly different between groups the

total number of CD34+ cells collected was greater in patients starting G-CSF after day 8. With a median number of three collections in both groups, the number of CD34+ cells collected was well within the range described for patients mobilized with chemotherapy immediately followed by G-CSF.15,30 The analysis of engraftment kinetics and transplantrelated morbidity (Table 5) also suggested the advantage of adding G-CSF on day 8 instead of day 15. Engraftment parameters were all within the limits reported by others25,28,31 but neutrophil and platelet engraftment was significantly faster in the group of patients receiving G-CSF from day 8. Faster engraftment may be related to the higher dose of CD34+ cells infused in patients mobilized with day 8 G-CSF. Days of fever, antibiotics and hospitalization were significantly reduced in patients receiving day 8 mobilized PBPC. Although addition of BM progenitors (group 1) to day 15 mobilized PBPC decreased the period to neutrophil and platelet engraftment these differences were not statistically significant. The number of CD34+ CD33+ cells has been associated with speed of engraftment in some studies.32 In accordance with that report,32 both the percent-


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age and total number per ml of CD34+ CD33+ cells was significantly higher in the group of patients treated with GCSF from day 8. The higher number of CD34+ cells infused as well as the higher number of CD34+ CD33+ cells in patients mobilized with day 8 G-CSF may explain at least in part the faster engraftment in this group. Although long-term engraftment after mobilized PBPC transplant has been less well studied than short-term engraftment there is significant clinical evidence that mobilized PBPC provide successful long-term neutrophil, platelet and hemoglobin engraftment after myeloablative therapy.33–36 In our study, there were no cases of graft failure and recovery of platelet, neutrophil and hemoglobin counts a year post transplantation was above 80% in comparison with baseline levels for all three groups of patients suggesting that PBPC mobilized by either G-CSF from day 8 or day 15 provided successful long-term engraftment. The frequency of CFU-GM slowly decreased in the PB and increased in the BM. In steady-state hematopoiesis most progenitors reside in the BM cavity and only a small percentage may be found in the PB.37 These results suggest that there may be increased trafficking of progenitors between the blood and BM compartment during the early period after transplant that subsequently returns to normal levels. It has been suggested that after transplantation there is an increase in the proliferation rate of progenitors in order to restore normal hematopoiesis and that proliferation of progenitors is associated with trafficking of progenitors.38 Our results would be in accordance with these observations. In conclusion, we demonstrate that addition of G-CSF on day 8 after standard doses of chemotherapy in patients with breast cancer results in adequate PBPC mobilization and subsequent engraftment after high-dose chemotherapy with the following advantages: significant cost saving by reducing the number of days requiring treatment with GCSF, prevents delays in the chemotherapy schedule and allows for programming of PBPC collections independently of WBC and neutrophil counts. Acknowledgements This work was supported in part by a grant from the Spanish Government, Ministerio de Sanidad (FIS 94/0525).

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