Stem Cells, Vol. 15, No. 2, 112-118,
March 1997
© 1997 AlphaMed Press
In Vitro Production of Megakaryocytes from PIXY321 versus GM-CSF-Mobilized Peripheral Blood Progenitor Cells
P. Lefebvrea,
J.N. Winterb,
A.W. Rademakerc,
C. Goolsbyd,
I. Cohena
a Department of Cell and Molecular Biology,
b Department of Medicine,
c Department of Preventive Medicine,
d Department of Pathology and the Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois, USA
Key Words. Megakaryocytes • PIXY321 • GM-CSF • Thrombopoietin • Cyclophosphamide • Peripheral blood progenitor cells
Dr. Isaac Cohen, Northwestern University, RIC 1407, 345 East Superior Street, Chicago, IL 60611, USA.
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Abstract
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The generation of megakaryocytes (MK) from cultured peripheral blood progenitor cells (PBSC), harvested via apheresis, from 18 female breast cancer patients treated with either PIXY321 or GM-CSF was compared. Nonadherent mononuclear cells (MNC) were cultured in liquid suspension with 50 U/ml thrombopoietin (TPO) and 2.5% autologous heparinized plasma for 12 days. Flow cytometric analysis was used to measure the percentage of CD34+ on day 1 and CD41+ cells on day 12. The frequency of CD34+ cells was greater in GM-CSF-mobilized samples than in PIXY321-mobilized samples, and MK/MNC yields correlated directly with the number of CD34+ cells seeded. PIXY321-mobilized samples produced more MKs per CD34+ cell than GM-CSF-mobilized samples. Overall, there was no significant difference in the MK/MNC yield between PIXY321- and GM-CSF-mobilized samples. Cyclophosphamide (CY) increased the frequency of CD34+ cells and the corresponding MK/MNC yield for both cytokines, but had no effect on the MK/CD34+ yield. Compared to GM-CSF, PIXY321 mobilization resulted in increased CD34+ cell commitment to the MK lineage.
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Introduction
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Treatment of metastatic breast cancer with high-dose chemotherapy is limited by myelosuppression and its complications. Reinfusion of cytokine-mobilized peripheral blood hematopoietic progenitors significantly shortens the period of neutropenia and thrombocytopenia when compared to conventional bone marrow autografts, although platelet engraftment may be delayed in comparison to neutrophil recovery [1-5].
PIXY321 is a recombinant chimeric polypeptide consisting of the binding and active domains of the cytokines GM-CSF and interleukin 3 (IL-3) [6], that has been shown to have enhanced stimulatory activity on megakaryocytopoiesis [7]. PIXY321 promotes more rapid platelet recovery in rhesus monkeys than GM-CSF and IL-3 combined [5], and has been shown to effectively mobilize peripheral blood progenitor cells (PBPC) in lymphoma patients [8]. Its capacity for ameliorating hematopoietic suppression has been the subject of recent clinical studies [9]. In phase I/II trials, PIXY321 administration has resulted in more rapid platelet recovery in patients receiving conventional-dose chemotherapy [10] and in those undergoing autologous bone marrow transplantation [11] when compared to historical controls. A recently reported prospective randomized trial, however, showed no advantage for PIXY321 over GM-CSF with regard to platelet nadirs or duration of thrombocytopenia in breast cancer patients receiving standard-dose chemotherapy [12].
Our goal is to develop megakaryocyte (MK) ex vivo expansion methods for clinical use. In view of the purported effects of PIXY321 on megakaryocytopoiesis, we hypothesized that PIXY321-mobilized PBPCs would have a greater capacity for generating MKs than GM-CSF-mobilized PBPCs. To test this hypothesis, nonadherent mononuclear cells (MNCs) from apheresis specimens of patients enrolled in a phase III randomized trial comparing PBPC mobilization by PIXY321 and GM-CSF [13] were cultured in the presence of thrombopoietin (TPO), the putative growth factor responsible for the proliferation and maturation of MKs [14-18]. The megakaryocytopoietic activity of the two populations was then measured and compared.
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Materials and Methods
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Clinical Protocol
Women age 60 years or less, with advanced-stage breast cancer (either stage IV or IIIB, if inflammatory), were eligible for entry onto this multi-institutional clinical study. A total of 26 patients were randomized on this study, 18 at Northwestern Memorial Hospital, to receive either PIXY321 or GM-CSF to enhance the collection of PBPCs in the steady state and again following myelosuppressive chemotherapy (Fig. 1
). This protocol was approved by the Northwestern University Institutional Review Board, and informed consent was obtained in all cases. Only those patients who were previously untreated for metastatic disease or who had received three or fewer cycles of chemotherapy for advanced disease were admitted to this trial. Women who had previously received adjuvant chemotherapy were eligible for study only if relapse had occurred six or more months after the completion of adjuvant treatment. Prior exposure to mitomycin C or carboplatin disqualified patients from entry. A minimum interval of three weeks from prior chemotherapy, irradiation, or surgery was required. Minimum hematologic parameters included an absolute neutrophil count of 1,500/µl, platelets 
108/ml, and hemoglobin 8 gm/dl. Induction therapy consisted of cyclophosphamide (CY) (2,400 mg/m2) and adriamycin (60 mg/m2). Patients who had not previously been treated for metastatic disease received three cycles, whereas those who had received one to three cycles of chemotherapy for advanced disease at the time of entry on study received only two. The last two patients from whom samples were obtained for this study of megakaryocytopoiesis received CY at 1,200 mg/m2, because of concerns that the relatively poor mobilization observed on both arms was attributable to the high dose intensity. Taxol was substituted in two cases randomized to PIXY321 because of prior anthracycline exposure. Patients were then restaged, and only those who demonstrated a response to therapy went on to mobilization and stem cell transplant. Randomization was stratified according to the presence or absence of neoplastic cells in the bone marrow. After placement of an apheresis catheter, patients received either GM-CSF (Leukine®, Immunex Corp.; Seattle, WA), 375 µg/m2 given once daily as a s.c. injection on days 1-7, or PIXY321 (Immunex), 375 µg/m2 twice daily as a s.c. injection. Leukapheresis was performed on days 5, 6, and 7, using the CS-3000 Blood Cell Separator (Fenwal Division, Baxter Healthcare Corporation; Deerfield, IL). Aliquots of peripheral blood progenitor cells were stored in ampules containing 2 x 108 cells/ml, cryopreserved in a final concentration of 10% DMSO using a controlled rate freezer, and stored in vapor phase of liquid nitrogen. One week later, patients were treated with myelosuppressive doses of CY (4 gm/m2) followed by either PIXY321 or GM-CSF, as per the earlier randomization and at the same dose. PBPC collections began when the absolute neutrophil count exceeded 1,000/µl. Three sequential ten-liter collections were performed, and aliquots of the PBPCs were stored in ampules and cryopreserved as above for future study. Patients subsequently underwent high-dose chemotherapy with CY, thiotepa, and carboplatin, followed by stem cell reinfusion. Reinfusion was followed by either GM-CSF or PIXY321, according to a second randomization.
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Figure 1. Schema of clinical protocol. Patients previously treated for metastatic disease who had received up to three cycles of treatment for advanced disease at the time of study entry received two additional cycles of cyclophosphamide and adriamycin, whereas those who were untreated at study entry received three cycles. The peripheral blood progenitor cell harvest samples used for this study are highlighted in bold.
*The last two patients from whom samples were obtained for this study received cyclophosphamide at 1,200 mg/m2 because of concerns that the higher dose was sufficiently myelosuppressive to prevent mobilization; one was randomized to PIXY321 and one to GM-CSF.
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Elimination of Adherent Cells
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Frozen samples from all 18 Northwestern Memorial Hospital patients who underwent mobilization of PBPCs with either PIXY321 or GM-CSF, in the steady state and during recovery from myelosuppressive chemotherapy, were available for study. Ampules were thawed rapidly in a 37°C waterbath and transferred to a thawing medium of Iscove's modified Dulbecco's medium (IMDM; GIBCO; Gaithersburg, MD) containing 20% fetal bovine serum (GIBCO), 12.5 µg/ml DNase I (Boehringer Mannheim; Indianapolis, IN), 12.5 U/ml heparin (GIBCO), 100 µM Dulbecco's modified Eagle's nonessential amino acids (NEA; GIBCO), 100 U/ml penicillin/100 µg/ml streptomycin (PS; Boehringer Mannheim) at 4°C and kept on ice for greater than 20 min. Erythrocytes were lysed by mixing the cell suspension 1:3 with 100 µM EDTA, 1 mM KHCO3, 0.17 M NH4Cl, pH 7.3, for 5 min [19]. This was underlaid with 5 ml 10% human serum albumin (HSA, Baxter Hyland; Deerfield, IL) in Dulbecco's phosphate-buffered solution (D-PBS; GIBCO), and centrifuged 6 min at 380 x g. The pellet was washed in 40 ml thawing medium and centrifuged 10 min at 260 x g. The cells were resuspended at 106 cells/ml in 50% thawing medium/IMDM and incubated 2 hours or overnight at 37°C in 5% CO2 in a Cellstar 2720 incubator (Queue Systems; Asheville, NC).
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Cell Culture
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Nonadherent MNCs were collected and washed in 1% HSA/IMDM and resuspended at 106 cells/ml in a culture medium of IMDM, PS, NEA, 1% HSA, 2.5% autologous plasma, 2 mM glutamine (Sigma; St. Louis, MO), and 50 U/ml TPO, where 10 U = quantity that stimulates one-half maximal proliferation of BaF3/mpl cells (TPO; Zymogenetics; Seattle WA). The autologous plasma was made by collecting blood on 3.3 U/ml preservative-free heparin (GIBCO) in siliconized tubes and centrifuging blood three times at 1,000 x g.
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Phenotypic Analysis
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Viable cells were counted via the trypan blue exclusion method (samples were generally >85% viable) and up to 2.5 x 105 cells were washed in special phosphate-buffered albumin containing 13.6 mM sodium citrate•2H2O, 11 mM dextrose, 1 mM theophylline, 2.2 µM prostaglandin E1, 10% bovine serum albumin (Sigma) in D-PBS, pH 7.4. The cells were stained for 15 min on ice in the dark with either 1 µg phycoerythrin (PE)-conjugated-anti-CD34+ (HPCA-2, Becton Dickinson; San Jose, CA) (day 1) or fluorescein isothiocyanate (FITC)-conjugated anti-CD41+ (Immunotech/Amac; Westbrook, ME) (day 12). Corresponding negative controls were PE-anti-mouse IgG1 (Becton Dickinson) and FITC-anti-mouse IgG1 (Immunotech/Amac) used at equivalent IgG1 concentrations. Cells were washed again in special phosphate-buffered albumin and fixed with 1% paraformaldehyde containing 0.067 M sodium cacodylate in saline. Flow cytometric analysis was performed using a Coulter Cytometry XL (Coulter Co.; Hialeah, FL) dual laser flow cytometer. Fluorescence attributable to FITC- and PE-labeled antibodies was determined using excitation by an argon laser operating at 488 nm. Emission from FITC and PE was measured using band pass filters of 525 nm and 585 nm, respectively; 28,500 ± 11,900 total events were counted per sample. The integration region for the CD34+ cells was set around the uppermost positive population of cells. The integration region for the CD41+ cells was set at 1% positive in the negative population. The percent positive cells was calculated by subtracting the percent positive of the isotype control within the same integration region.
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Statistical Analysis
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The mean and standard error of the mean were calculated for each parameter. The differences, after cytokine treatment and after cytokine-stimulated recovery from CY, in the percentage of CD34+ cells in the MNC population and yields of MK per seeded MNC or CD34+ cells within each cytokine, were compared using the two-tailed Wilcoxon signed rank test. Differences between the PIXY321 and GM-CSF populations across the same study conditions were analyzed using the two-tailed Mann-Whitney U rank sum test. These nonparametric tests were used due to the non-normal distribution of the data. Results of the MK/CD34+ calculations after cytokine treatment and after cytokine-stimulated recovery from CY were pooled for each cytokine before analysis. The correlation of MK/MNC with the percentage of CD34+ for each cytokine alone and when pooled was measured using the Spearman's rank correlation coefficient. Analysis was also done with outliers removed to assure that they did not significantly affect results. (Data not shown, as outliers did not affect statistical significance.)
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Results
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CD34+ Cell Mobilization
Table 1 shows the clinical features of patients whose PBPCs were mobilized with either GM-CSF or PIXY321. There were no significant differences between the two arms with regard to sites of disease, prior treatment for advanced disease, and mean interval from chemotherapy to first dose of PIXY321 or GM-CSF (35 days versus 32 days). More patients on the GM-CSF arm had received adjuvant chemotherapy. While more adjuvant chemotherapy may have resulted in more cumulative myelosuppression, and thus poorer mobilization, this turned out not to be the case. When PBPCs were mobilized with cytokine alone, GM-CSF patients had a significantly higher percentage of CD34+ cells in the nonadherent MNC population compared to PIXY321 patients (GM-CSF, 0.35% ± 0.09 versus PIXY321, 0.10% ± 0.014, p = 0.006). In apheresis products collected during cytokine-stimulated recovery from CY treatment, the mean percentage of CD34+ cells in GM-CSF-mobilized MNC was three times higher than the mean for PIXY321-mobilized MNC, but did not reach statistical significance due to high variance (CY followed by GM-CSF, 1.61% ± 0.56 versus CY followed by PIXY321, 0.56% ± 0.15, p = 0.08). Mobilization with myelosuppressive chemotherapy (CY) followed by cytokine produced a significantly higher percentage of CD34+ cells in the nonadherent MNC population in both patient groups (PIXY321 versus CY followed by PIXY321, p = 0.006; GM-CSF versus CY followed by GM-CSF, p = 0.008).
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Megakaryocytes per Mononuclear Cell Seeded
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There were no significant differences between the MK/MNC yields of GM-CSF and PIXY321-mobilized patient samples, either when mobilized with cytokine alone (GM-CSF, 0.025 ± 0.007 versus PIXY321, 0.017 ± 0.003, p = 0.56) or CY followed by cytokine (CY followed by GM-CSF, 0.197 ± 0.149 versus CY followed by PIXY321, 0.064 ± 0.018, p = 0.61). MNCs collected during cytokine-stimulated recovery from CY produced significantly more MK/MNC than cytokine mobilization alone in every patient tested (PIXY321 versus CY followed by PIXY321, p = 0.002; GM-CSF versus CY followed by GM-CSF, p = 0.016).
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Correlation of MK/MNC with the Percentage of CD34+ Cells Seeded
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The number of MK/MNC recovered at day 12 correlated directly with the percentage of CD34+ in the MNC at day 1 for both PIXY321- and GM-CSF-treated patients, both when mobilized with cytokine alone or CY followed by cytokine (Fig. 2). This was true whether the two groups were analyzed separately (PIXY321, r = 0.69, p < 0.001; GM-CSF, r = 0.91, p < 0.001), or combined (Fig. 2).
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Megakaryocytes per CD34+ Cell Seeded
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The yield of MK per CD34+ cell seeded was calculated. There was no statistical difference in the yield of MK/CD34+ between PBPCs mobilized with cytokine alone and CY followed by cytokine (PIXY321, 23.0 ± 7.8 versus CY followed by PIXY321, 20.6 ± 8.3, p = 0.32; GM-CSF, 6.3 ± 2.8 versus CY followed by GM-CSF, 7.8 ± 2.7, p = 0.99). When the results for mobilization with cytokine alone and CY followed by cytokine were combined, PIXY321-mobilized PBPCs had a higher mean MK/CD34+ cell seeded than GM-CSF-mobilized PBPCs (PIXY321 ± CY, 21.8 ± 5.53 versus GM-CSF ± CY, 8.37 ± 1.92, p = 0.037).
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Discussion
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PIXY321 is being tested in clinical trials for its capacity to mobilize PBPCs and to alleviate post-transplant thrombocytopenia, based on the positive effects seen on megakaryocytopoiesis in vitro and platelet production in vivo. To determine if PIXY321-mobilized PBPCs produce more MKs ex vivo than standard treatment GM-CSF-mobilized PBPCs, we studied the production of MKs in liquid culture from apheresis samples obtained from patients receiving either PIXY321 or GM-CSF for the mobilization of PBPCs. The results of this study show that PIXY321-mobilized CD34+ cells produced more MKs than GM-CSF-mobilized CD34+ cells, while GM-CSF mobilized more CD34+ cells per MNC collected than PIXY321. Together, this led to no net difference in the number of MKs produced per MNC cultured. One explanation for this finding may be that PIXY321 mobilizes a different subpopulation of CD34+ cells than GM-CSF, with a greater commitment to the megakaryocytic lineage, but is a less effective mobilizer of hematopoietic progenitors overall.
In view of the small number of positive cells in certain samples, we ascertained the statistical validity of our results in three different ways. First, the CD34 frequencies were confirmed by another laboratory that analyzed the same patient samples for another study. While their sample preparation protocol was different from ours (in particular, they did not fractionate out adherent cells), their results and ours correlated to a significance of p < 10–6. Second, the statistical method used was a nonparametric rank sum test, better suited to the non-normal distribution of the data. Samples were analyzed based on their relative rank to each other, not on their absolute values. By this method, the relative ranks of the samples would not be expected to change very much even if the absolute values were to change. Third, the statistical significance of the findings did not change when the absolute values were varied by their inherent uncertainty. Thus, we feel confident the results obtained are real.
Bruno et al. [7] first reported that human bone marrow cells cultured in vitro with PIXY321 sustained greater long-term megakaryocytopoiesis than GM-CSF alone. Our results indicate that PIXY321 exerts a similar effect ex vivo. The MK-promoting activity of PIXY321 may derive from the fact that it is a GM-CSF/IL-3 fusion protein. GM-CSF promotes the maturation of myeloid progenitors, making it effective in promoting neutrophil recovery [20, 21], whereas IL-3 promotes proliferation and differentiation of early progenitors, including those of the megakaryocytic lineage [22-24]. Clinically, IL-3 has been shown to induce a dose-dependent increase in thrombopoiesis in patients with normal bone marrow function and in patients with secondary bone marrow failure [25]. Taken together, these laboratory and clinical findings suggest that the IL-3 portion of PIXY321 could promote the mobilization of more MK progenitors compared to GM-CSF alone, and that culturing these progenitors in the presence of TPO would produce greater numbers of MKs [26]. Our results support this hypothesis.
There was no significant difference between PIXY321 and GM-CSF in the number of MKs generated per mononuclear cell. This was true whether PBPCs were mobilized by cytokine administration alone or by cytokine-stimulated recovery from myelosuppressive chemotherapy. If there is a relationship between PBPC mobilization and cytokine-stimulated progenitor recovery following chemotherapy, then these results may explain the recently reported observations of a phase III trial comparing PIXY321 and GM-CSF administration following conventional-dose chemotherapy for breast cancer [12]. No differences were found in platelet nadirs, duration of thrombocytopenia, or need for platelet transfusions. This may be explained by the fact that when compared to GM-CSF, PIXY321 is ineffective in mobilizing CD34+ cells [13]. If the effect of PIXY321 on the kinetics of mobilization and recovery are the same, then even if PIXY321 enhances the proliferation of CD34+ cells committed to the MK lineage compared to GM-CSF, the smaller pool of CD34+ cells generated by PIXY321 would account for the lack of clinical impact seen in the randomized trial.
Given that MK progenitors express the CD34+ marker, the relationship between the percentage of CD34+ cells seeded and the MK/MNC produced by each sample was analyzed. As expected, the number of MK/MNC produced at day 12 correlated with the percentage of CD34+ cells in the MNC culture on day 1, regardless of the cytokine used for mobilization. This suggests that, in general, the number of CD34+ cells will determine the yield of MKs in ex vivo expansion. The clinical correlate of this finding was shown in the studies of Takamatsu [4], which showed that platelet recovery after autologous PBPC transplantation could be predicted accurately by the number of colony-forming units-granulocyte/macrophage ([CFU-GM], which in most studies correlates with CD34+ cell content) infused, and that the number of CFU-GM in the autograft was equal to CFU-MK in predicting time-to-platelet recovery.
Myelosuppressive doses of CY followed by cytokine mobilized more CD34+ cells per MNC than the same cytokine alone. However, there was no significant difference seen in the generation of MKs per CD34+ cell. This shows that CD34+ cells mobilized by cytokine alone are similar to those mobilized during cytokine stimulated recovery from CY in their capacity to generate MKs. Myelosuppressive doses of CY have been shown to mobilize greater numbers of PBPCs [27, 28], and these are believed to be equivalent to bone marrow cells immunologically and in their in vitro growth characteristics [29]. Our results confirm these observations.
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Conclusion
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This study shows that there is a difference in the type of CD34+ cell mobilized by PIXY321 compared to GM-CSF: PIXY321 mobilizes more cells committed to the MK lineage. Successful ex vivo expansion of MKs will depend on both the total number of CD34+ cells available (in this case, enhanced by GM-CSF), and their capacity for generating MKs. Therefore, the particular cytokine or cytokines used for mobilization may have profound effects on the results of an ex vivo expansion protocol.
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Acknowledgments
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The authors would like to thank Dr. Don Foster of Zymogenetics (Seattle, WA) for supplying the TPO, Drs. Leslie Garrison and David Green for reviewing the manuscript, and Vicki Mossiman for assistance with flow cytometry.
This study was supported by United States Army Medical Research Acquisition Activity grant DAMD17-94-J-4465.
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accepted for publication November 21, 1996.
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Abstract
Introduction
) and GM-CSF (
) samples were combined. Graph was extended to show outliers. r = 0.70, p < 0.001.