Progenitor Cell Mobilisation

A comparative study of sequential priming and mobilisation of progenitor cells with rhG-CSF alone and high-dose cyclophosphamide plus rhG-CSF

Abstract

Stem cell mobilisation can be achieved either by administration of rhG-CSF alone or after high-dose cyclophosphamide (HDCy) plus rhG-CSF. We have compared both mobilisation procedures intra-individually in 43 patients with haematological malignancies. Furthermore, the toxicity data were registered. The CD34+ cell yield was higher after mobilisation with HDCy plus rhG-CSF than after rhG-CSF alone in 21 out of 22 patients who were actually harvested after both procedures. If a patient mobilised insufficiently after rhG-CSF alone, the yield of CD34+ cells after the following HDCy priming was lower compared to patients who mobilised sufficiently after rhG-CSF priming alone. In 12 patients with B cell malignancies a reduced number of B cells such as CD10+, CD19+, CD20+ cells in bone marrow as well as in leukapheresis products was observed after HDCy plus rhG-CSF compared to rhG-CSF alone. Toxicity data revealed HDCy as a relatively toxic priming regimen with all patients hospitalised and 74% experiencing neutropenic fever and administration of intravenous antibiotics. In two patients, seizure-like episodes were observed during cyclophosphamide bolus infusion. In conclusion, HDCy increased the yield of CD34+cell and reduced B cells in leukapheresis products indicating reduced tumour cell load compared with rhG-CSF priming alone. The efficacy of HDCy priming is limited by its profound toxicity and morbidity. Studies evaluating efficacy and safety of lower doses of cyclophosphamide are needed. Bone Marrow Transplantation (2000) 26, 717–722.

Main

The use of peripheral blood stem cells (PBSC) to support high-dose chemotherapy in patients with malignancies is well-established and now preferred to bone marrow stem cells. Peripheral blood stem cells seem to produce more rapid engraftment than steady-state bone marrow stem cells123 and may at least from a theoretical point of view be less contaminated with tumour cells, although conflicting results have been reported.45 Mobilisation of a sufficient number of stem cells into the peripheral blood to ensure a rapid and sustained engraftment can be achieved by administration of growth factors such as rhG-CSF alone67 or by administration of myelosuppressive chemotherapy such as high-dose cyclophosphamide (4 g/m2).89 The combination of myelosuppressive chemotherapy and growth factors has been shown to be synergistic.10 It is, however, still uncertain which regimen is optimal regarding the maximum yield of progenitor cells and tumour cell contamination. Whether or not patients who fail to mobilise stem cells with growth factors alone will benefit from high-dose cyclophosphamide plus growth factor is also unclear. We have previously shown in a small group of patients that a higher yield of CD34+ cells was achieved after mobilisation with HDCy plus rhG-CSF than after rhG-CSF alone.11

In this study we have extended our previous investigation and compared progenitor cell mobilisation after rhG-CSF alone with HDCy mobilisation applied sequentially to the same cohort of patients with B cell malignancies. We have evaluated the yield of CD34+ cells and the proportion of different cell populations in bone marrow and leukapheresis products after both priming procedures. Finally the toxicity of the HDCy regimen has been evaluated.

Materials and methods

Patients

Between November 1993 and February 1997, 43 patients (25 male, 18 female, median age: 50 years, range 16–65) referred to the Department of Haematology, Herlev Hospital, University of Copenhagen for stem cell collections were included in a sequential study comparing rhG-CSF alone with high-dose cyclophosphamide (HDCy) plus rhG-CSF. All 43 patients had haematological malignancies (non-Hodgkin lymphoma: n = 17, multiple myeloma: n = 17, Hodgkin’s disease: n = 3, acute lymphoblastic leukaemia: n = 4, acute myeloid leukaemia: n = 1, chronic lymphoblastic leukaemia: n = 1). In the first sequence the patients were primed with rhG-CSF alone and a median of 9 weeks after (range 2–90) subsequently primed with HDCy plus rhG-CSF. Six patients were untreated at the time of rhG-CSF priming. These six and another six patients received chemotherapy in the interval between the two priming procedures, whereas the remaining 31 patients had no treatment in the interval. All but one had both procedures performed within 24 weeks, and the difference in time interval occurred mainly because of practical reasons. Bone marrow function assessed by blood leukocyte and platelet counts as previously described11 was normal at the time of rhG- CSF priming in 52% and in 53% at the time of HDCy plus rhG-CSF priming. A subgroup of 12 patients (multiple myeloma: n = 5, non-Hodgkin lymphoma: n = 7) were subjects for further flow cytometric analysis on bone marrow samples and leukapheresis products collected during both priming procedures. The study was approved by the ethics committee in Copenhagen County.

rhG-CSF priming

The patients were primed with daily subcutaneous injections of recombinant granulocyte colony-stimulating factor (rhG-CSF, Neupogen, Roche, Denmark) 10 μg/kg/day and continued until last day of leukapheresis.

Cyclophosphamide priming

After rhG-CSF priming stem cells were mobilised with high-dose (4 g/m2) cyclophosphamide (HDCy) supported by Mesna and hydration as described by To et al.8 At the end of the neutropenic period, when a rising white blood cell count exceeded 1.0 × 109/l and/or a rising platelet count exceeded 20 × 109/l (untransfused), rhG-CSF (10 μg/kg/ day) was added and continued until the last day of leukapheresis.

Timing of leukapheresis

In both priming procedures blood levels of CD34+ cells were monitored daily during priming and mobilisation. Leukapheresis was performed when the level of CD34+ cells exceeded 20 × 103/ml blood.11 Patients who never reached a CD34+ blood value of 20 × 103/ml had no leukapheresis performed and were characterised as insufficient mobilisers.

Bone marrow sampling

Bone marrow (BM) aspirates and biopsy specimens from the posterior iliac crest taken on the first day of leukapheresis were evaluated in 12 patients after both priming procedures. The first 2 ml aspirated were used for morphological studies. The subsequent 38 ml of marrow were sampled during systematic withdrawal of the aspiration needle by aspiration of 0.5–1.0 ml per mm retraction over a distance of 15–40 mm. This 38 ml sample was used to determine the number, type and functional status of BM cells in individual patients. The marrow was anticoagulated with heparin 1000 IU/ml.

Leukapheresis and cryopreservation

Leukaphereses were performed with a Fenwal CS 3000 Plus blood cell separator (Fenwal Laboratories, Deerfield, IL, USA). A total blood volume of 10 l per apheresis was processed at a flow rate of 50–70 ml/min. Leukapheresis was performed on 3 consecutive days or shorter if a yield of at least 2 × 106 CD34+ cells/kg body weight were obtained. Leukapheresis and bone marrow products were cryopreserved in DMSO using a controlled rate liquid nitrogen freezer (Planer Biomed, Sunbury-on-Thames, UK) and stored in liquid nitrogen.

Flow cytometry

The numbers of CD34+ cells in fresh bone marrow samples and leukapheresis products were estimated by flow cytometry as described previously.12

Frozen bone marrow samples and leukapheresis products after both priming procedures from 12 patients were analysed with the following moAbs: anti-CD10 FITC (Calla; BDIS, Broendby, Denmark), anti-CD19 FITC (Leu 12 FITC; BDIS), anti-CD19; FITC (B4 FITC; Coulter Clone, Ramcon, Birkeroed, Denmark), anti-CD20 FITC (Leu 16 FITC; BDIS), anti-CD21 FITC (Leu 21 FITC; Immunotech, Ramcon, Birkeroed, Denmark).

Subset analysis of CD34+ cells was performed on cells acquired in a CD34 SSC/fluorescence gate. Cells were incubated with anti-CD34 PE-conjugated moAbs and one of the following conjugated moAbs: anti-CD19 FITC (Leu 12 FITC, BDIS), anti-CD19 FITC (B4 FITC; Coulter Clone).

In order to detect plasma cells a double staining analysis was performed with anti-CD38 PE (Leu 17 PE, BDIS) and anti-CD45RA FITC (Immunotech). Ten thousand events were collected in list mode with debris eliminated. In quadrant statistics (FL-1 vs FL-2) the plasma cells (CD38++/CD45RA) were identified in the upper left quadrant.

Toxicity registration

Toxicity data after both priming procedures were registered. In addition, the medical records of all patients (n = 99) undergoing HDCy priming but not included in the study during the same period were reviewed and toxicity data registered.

Statistical analysis

The yield of CD34+ cells as well as the different cellular subsets in bone marrow and leukapheresis products from the two priming procedures were compared using the non-parametric test for paired design (Wilcoxon test). The yield of CD34+ cells harvested after HDCy priming depending on previous insufficient or sufficient mobilisation by rhG-CSF alone was made using the non-parametric test for unpaired design (Mann–Whitney test). In both tests a P value less than 0.05 was considered significant.

Results

Stem cell mobilisation after rhG-CSF vs HDCy plus rhG-CSF

Of the 43 patients only 23 (53%) mobilised sufficiently after priming with rhG-CSF alone, ie reached a CD34+ blood level of 20 × 103/ml and had leukapheresis performed. Of the 20 patients who mobilised insufficiently after rhG-CSF alone, 13 mobilised sufficiently after the following HDCy priming regimen. Twenty-two patients were actually harvested after both priming procedures. In 21 of the 22 patients a higher yield of CD34+ cells was achieved after priming with HDCy plus rhG-CSF than after rhG-CSF alone (Figure 1). The difference was statistically significant (P < 0.0001). There was only one patient who mobilised sufficiently after rhG-CSF alone but did not mobilise after the subsequent HDCy priming.

Figure 1
figure1

Mean yield of CD34+ cells (total yield of CD34+ cells/number of leukapheresis days) harvested in 22 patients after mobilisation with rhG- CSF alone and after high-dose cyclophophamide plus rhG-CSF.

If the mobilisation was insufficient after rhG-CSF priming alone the yield of CD34+ cells/kg harvested after the following HDCy priming was significantly lower (median 1.69 × 106/kg, range 0.50–11.12) compared to the yield after HDCy priming in patients who mobilised sufficiently by rhG-CSF alone (median 7.05 × 106/kg, range 1.78–25.13) (P = 0.0007) (Figure 2).

Figure 2
figure2

Median yield and range of CD34+ cells harvested after priming with high-dose cyclophosphamide plus rhG-CSF in 15 patients who previously mobilised insufficiently and 16 patients who previously mobilised sufficiently with rhG-CSF alone.

Flow cytometry analysis

All the 12 patients included in the flow cytometry analysis had B cell malignancies (multiple myeloma: n = 5, NHL: n = 7). The purpose of this substudy was to estimate the proportion of B cells in both bone marrow and leukapheresis products after both priming procedures by a panel of different B cell markers. B cell subsets of CD34+ cells, which have been reported to be the cell pool containing clonally related premalignant cells,1314 were also evaluated.

The percentage of CD34+ cells was increased after HDCy priming compared to rhG-CSF priming alone. However, there was no difference in bone marrow CD34+ cell percent after the two priming procedures, indicating a selective mobilisation of CD34+ cells after HDCy priming (Table 1).

Table 1  Percentage of different antigens in bone marrow and leukapheresis products after priming with rhG-CSF alone and high-dose cyclophosphamide plus rhG-CSF in 12 patients

In bone marrow samples the frequencies of CD19+ and the CD20+ cells were reduced after HDCy priming compared to rhG-CSF priming (Table 1). In leukapheresis products all B cell populations (CD19+, CD20+, CD21+) were reduced after HDCy priming (Table 1).

The small population of pro-B cells belonging to the CD34+ cell pool (CD34+CD19+) was, however, not reduced after HDCy priming (Table 1). The content of plasma cells (CD38++CD45RA) was in all cases very low (less than 1%) and was not evaluable in a quantitative setting in this analysis.

Toxicity

The 43 patients were given a median dose of 7200 mg cyclophosphamide (range 3000–8400). Successful mobilisation was achieved in 34 (79%) patients and the median number of days from cyclophosphamide infusion to start of leukapheresis was 13 days (range 11–22). All patients were admitted to hospital for a median of 19 days (range 7–27).

Almost all patients experienced leukopenia (93%). Fever and infections were the most frequent complications resulting in treatment with i.v. antibiotics in the majority (70%) of the patients (Table 2). Bacteria were most often isolated from blood (Staphylococcus aureus: n = 1, Streptococcus haemolytica: n = 1, Klebsiella pneumoniae: n = 1, Escherischia coli: n = 2, Pneumococcus: n = 1) or the central venous catheter (Staphylococcus aureus: n = 3, Streptococcus haemolytica: n = 1, Klebsiella pneumoniae: n = 1, Escherischia coli: n = 1).

Table 2  Toxicity in 43 high-dose cyclophosphamide priming procedures

Bleeding episodes were relatively infrequent and seen in only six patients (14%). The needs for transfusion of blood products are given in Table 2.

The most serious complication beside infectious complications was seizure-like attacks seen in two patients during infusion of cyclophosphamide. In both cases the infusion was disrupted and the patients recovered quickly. There was no difference in toxicity between patients who mobilised sufficiently and patients who did not mobilise stem cells after HDCy priming. The same pattern and frequence of toxicity was observed in the 99 patients who underwent HDCy plus rhG-CSF priming at our institution during the same period.

After priming with rhG-CSF alone no toxicity was observed apart from mild bone pain in a few cases. This side-effect was effectively controlled by administration of paracetamol.

Discussion

We have shown that HDCy plus rhG-CSF mobilises more progenitor cells into the blood than rhG-CSF alone when administered sequentially in a cohort of patients with B cell malignancies. Many studies have suggested that mobilisation with a combination of chemotherapy and cytokines stimulates the circulation of significantly more progenitor cells than either chemotherapy or cytokines alone.81015 In most previous studies the different priming regimens have been given to different patient groups. The difference found in these studies may be due to differences in the two patient populations. In our study, when the two priming procedures were applied sequentially to the same patient in 43 patients, we found a higher yield of CD34+ cells after high-dose cyclophosphamide plus rhG-CSF than after rhG-CSF alone in 21 out of 22 cases. To our knowledge there have only been two studies with the same design. In a study by Möhle et al,16 a higher yield of CD34+ cells after priming with chemotherapy plus growth factors than after growth factor priming alone was also found. However, the patient population was different from ours as it consisted of breast cancer patients with normal bone marrow function, and furthermore, the chemotherapy used was not cyclophosphamide but ifosfamide and epirubicin. Our patients had haematological malignancies and not all had normal bone marrow function. Cremer et al17 also found higher yield of CD34+ cells after high-dose cyclophosphamide plus rhG-CSF in a small study of eight myeloma patients. Although HDCy plus rhG-CSF in all cases resulted in a higher yield of stem cells, patients who mobilised poorly with rhG-CSF alone had a lower yield after HDCy priming than patients who mobilised sufficiently after rhG-CSF alone. This finding indicates a general impaired ability to mobilise stem cells in the former group, which may be due to marrow damage from previous cytotoxic treatment.18

We have previously shown that prior chemotherapy and bone marrow function are important factors affecting the yield of CD34+ cells by leukapheresis.11 In the present study, these factors seemed not to be more important than the priming regimen. There was no difference in the proportion of patients with normal and abnormal bone marrow function in the two priming procedures. Furthermore, HDCy plus rhG-CSF priming yielded more CD34+ cells despite the fact the some of the patients had received more chemotherapy at that time than after priming with rhG-CSF alone.

In parallel with the improved harvest of CD34+ cells priming with high-dose cyclophosphamide may also be beneficial as it reduces the tumour load before transplantation. The different B cell populations were reduced in both bone marrow and leukapheresis products after priming with HDCy. We have not evaluated the tumour cell contamination in bone marrow and leukapheresis products by more specific methods in this study, but it has been shown that clonal cells in both B cell lymphoma and myeloma patients reside within the B cell compartment.192021 A reduction in the number of B cells may thus predict a reduction in tumour load as well. In a small study of eight multiple myeloma patients the percentage of malignant cells detected by a quantitative PCR assay involving CDR3-specific primers was lower in leukapheresis products harvested after HDCy plus rhG-CSF than after rhG-CSF alone.17 Mobilisation of tumour cells to the blood in multiple myeloma after priming with rhG-CSF alone has also been reported.22

We have found that the proportion of CD34+CD19+ cells was unaltered after HDCy priming. It has previously been postulated that this cell population contains clonally related cells in both NHL14 and multiple myeloma,13 but these findings have not been confirmed, and in mutiple myeloma several studies have shown that the clonally related cells do not express the CD34 antigen.232425

In contrast to priming with rhG-CSF alone, the HDCy regimen is rather intensive and causes some toxicity and morbidity. All our patients were hospitalised during the neutropenic period, and fever which resulted in treatment with intravenously antibiotics was the most frequently observed event being seen in 74% of the patients. This incidence is somewhat higher than reported in other studies in which the same dose of cyclophosphamide was given. In these studies the incidence of fever ranges from 2 to 53% resulting in treatment with intravenous antibiotics in 2 to 50%.8262728 In the reported studies, rhG-CSF was given immediately after HDCy and continued until after leukapheresis. In our HDCy regimen rhG-CSF administration was delayed with start of rhG-CSF at the end of the neutropenic period. This delay of rhG-CSF treatment may in part explain the difference in the incidences of fever and antibiotic treatment. In the study by Kotasek et al,28 patients who had been treated with a higher dose of cyclophosphamide (7 g/m2) all experienced fever and needed treatment with intravenous antibiotics. The same trend of higher side-effects with higher doses of cyclophosphamide was found by Rowlings et al29 comparing 4 g/m2 with 7 g/m2 of cyclophosphamide. Toxicity and morbidity are related to the dose of cyclophosphamide2829 and in many new mobilisation protocols the cyclophosphamide dose is reduced to 2 g/m2. It remains, however, to be evaluated whether this lower dose results in the same outcome regarding CD34+ cell yield, reduced tumour cell in the leukapheresis product alongside with lower toxicity and morbidity of the priming regimen.

In conclusion, we have found a better yield of CD34+ cells, and in addition, a reduced B cell compartment indicating reduced tumour cell load after priming with high-dose cyclophosphamide plus rhG-CSF compared with rhG-CSF alone. The benefit of high-dose cyclophosphamide is somewhat compromised by the relatively high haematological toxicity and morbidity of the regimen. A new strategy with a lower dose of cyclophosphamide or combinations of growth factors has been initiated in many centres and the results of this with respect to harvest outcome and side-effects are awaited.

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Acknowledgements

This study was supported by grants from the Danish Cancer Society, Danish Medical Association (Højmosegård Foundation), Ebba Celinders Foundation and the Foundation of 17-12-1981.

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Knudsen, L., Jensen, L., Gaarsdal, E. et al. A comparative study of sequential priming and mobilisation of progenitor cells with rhG-CSF alone and high-dose cyclophosphamide plus rhG-CSF. Bone Marrow Transplant 26, 717–722 (2000). https://doi.org/10.1038/sj.bmt.1702609

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Keywords

  • high-dose cyclophosphamide
  • G-CSF
  • progenitor cells
  • CD34+ cells
  • stem cell mobilisation

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