Patients and methods

The present study was conducted in patients who underwent an autologous transplant at the Hematology Departments of the Institut Curie and the Institut Gustave Roussy. The study was approved by the local ethics committee of both Institutions and by the French Drug and Food Safety administration (AFSAPPS). The preclinical conditions for the generation of MK differentiation and expansion from CD34+ cells were established according to protocols previously defined in the laboratory,³ which was adapted to the clinical practice because of the original use of proteins from bovine origin and research degree lipids. The protocol for clinical grade cell expansion used X-Vivo 20 medium, containing human albumin (1.5%), clinical grade Medialipids (180 g/ml of culture), stem cell factor 25 ng/ml (kindly provided by Amgen) and PEG-rhMGDF 10 ng/ml (kindly provided by Kirin Bewery Company, Japan). The culture was performed in the Clinical Cell Therapy facility of the Gustave Roussy Institute at 37°C for 10 days, in Lifecell culture bags. At day 10, cultures were evaluated visually by the presence of cells with MK morphology and proplatelet-forming mature MKs, and by phenotypic characterization using of CD41, CD61, CD34, CD33, and CD15 markers. In these conditions, all preclinical tests have shown the ability to induce expansion of CD34+ cell cultures by 10-fold in terms of total nucleated cells in 10 days and concomitant differentiation of the cells towards MK-differentiation pathway, with approximately 50% of the generated cells expressing CD41.

Nine consecutive patients with relapsed non-Hodgkin's lymphoma (NHL) and one patient with relapsed Hodgkin's disease were included in the study. After obtaining written informed consent, all patients underwent peripheral blood stem cell (PBSC) mobilization with DHAP (dexamethasone, cytarabine and cisplatinum), IVA (doxorubicine, etoposide, ifosfamide), or high-dose cyclophosphamide regimens plus G-CSF (granulocyte colony-stimulating factor). One to three apheresis procedures were performed to obtain at least 4 10⁶ CD34+ cells/kg. Figure 1 summarizes the scheme of the trial design. Two fractions were cryopreserved: a first fraction of at least enough cells for 2 10⁶ CD34+ cells/kg as nonmanipulated PBPC and a second fraction that was obtained after CD34+ selection (Isolex 300i, Baxter) of 1–2 10⁶ CD34+ cells/kg. At 10 days before ASCT, this fraction was thawed and cultured in X-Vivo medium in the conditions described above. At day 0, the cultured cells were washed to eliminate the medium culture, debris and platelets generated from the proplatelet-producing cells. Before infusion, the cells were evaluated using viability counts, cytology, phenotyping and bacteriology. The patients received high-dose BEAM (BCNU, cyclophosphamide, cytarabine, and melphalan) chemotherapy and were reinfused on day 0 with ex vivo expanded progenitor cells through filters, which have been shown to allow the transfer of MK and standard PBSC. Owing to reinfusion of cells cultured in the presence of SCF and in order to prevent any allergic reactions, patients received cetirizine and ranitidine from days –1 to 1. No hematopoietic growth factor was administered after PBSC transplantation. Platelet transfusion was performed when platelet count reached 15 000/l or less or in case of hemorrhagic syndrome, red blood cell (RBC) transfusion was indicated when hemoglobin level was lower than 8 g/dl. Toxicity was evaluated according to World Health Organization (WHO) criteria.

Figure 1.

Scheme of the trial design.

Full figure and legend (49K)

Results

Among the included 10 patients, two were not infused with cultured cells, one because of insufficient apheresis product, and the other because of a cardiac contraindication. Therefore, eight patients were evaluated for the nadir and duration of thrombocytopenia after reinfusion of ex vivo expanded MK precursor cells and nonmanipulated PBSC. No toxicity was observed after reinfusion, particularly no modification of the breathing frequency. All data from cultured and nonmanipulated cells are presented in Table 1. At day 10 of culture, the mean fold expansion for the eight evaluated patients was 9.27 (range: 4.85–16.77) for total nucleated cells, 2 (range: 0.94–6.33) for CD34+ cells, and 676 (range: 142–1183) for CD41+ cells and 627-fold for CD61+ cells (range: 38–1519). The mean number of infused CD41+ and CD61+ cells was 10.6 10⁶ cells/kg (range: 2–16 10⁶/kg) and 12 10⁶cells/kg (range: 6.92–16.3 10⁶/kg), respectively. Figure 2 shows a representative flow cytometry profile of the CD34+ cells obtained (Figure 2a and b) in UPN 1 at the start of the culture (99% pure CD34+) and after 10 days of culture in the presence of SCF and rhMGDF. As can be seen in this figure, at day +10 of the culture, 84% of the cells were CD41+ and 77% of the cells were CD61+, whereas before expansion 1.5 and 0.5% of CD34+ cells expressed CD41 and CD61, respectively. May–Grunwald–Giemsa stain of the expanded cells before infusion showed evidence of polyploid MKs (Figure 2e).

Figure 2.

Flow cytometry and morphological analysis of CD34+ cells before (a and b) and after expansion (c–e) in the presence of SCF and rhMGDF for 10 days in patient UPN1. Cells were 99% CD34+ before expansion. At day +10, the expanded cell population contained 84% CD41+ cells (c) and 77% CD61+ cells (d) with the generation of MKs (e).

Full figure and legend (59K)

Table 1 - Characteristics of ex vivo expanded and reinfused cells.

Full table

The patients' platelet recoveries are represented in the Figure 3. The median date of platelet transfusion independence (>15 000/l) was day 8 (range: 7–12). Five patients received one platelet transfusion, two patients two platelet transfusions, and one patient received three platelet transfusions. Patients recovered absolute neutrophil counts >500/l on a median day 12 (range: 10–15). One patient did not receive RBC transfusion, and three and four patients received one and two RBC transfusions, respectively. Nonhematologic toxicity of grades 3 and 4 occurred in three patients, without any relationship between toxicities and reinfusion of CD34+ expanded cells: one patient presented diarrhea, anorexia, and Varicella zoster virus infection grade 3, one patient had grade 3 anorexia, and one patient died suddenly at home on day 35; no autopsy was performed, but no relationship could be established between this patient's death and the reinfused ex vivo cultured PBSC. This patient was heavily pretreated before autologous transplantation, with a calculated dose of 760 mg/m² of doxorubicin.

Figure 3.

Patients' platelet recovery after reinfusion of ex vivo expanded progenitor cells and standard PBSC (patients 1–4 and 5–9).

Full figure and legend (24K)

Discussion

Since 1993, it has been demonstrated that ex vivo expanded PBPC from cancer patients mobilized by chemotherapy plus G-CSF can be administered along with a nonmanipulated graft enabling reconstitution of hematopoiesis,^{4, 5} without alteration of immature hematopoietic progenitors⁶ and tumor cell amplification.^{7, 8, 9} However, the participation of ex vivo expanded cells in long-term reconstituion remains to be shown. In the CD34+ cell expansion trials in which the graft was performed only by the use of expanded cells, a rapid reconstitution of hematopoiesis was observed only in patients receiving no myeloablative conditioning.^{10, 11} To decrease the duration of high-dose chemotherapy-induced thrombocytopenia, numerous culture conditions have been tested in vitro,^{12, 13, 14, 15} but the clinical applicability of these techniques is not established because of the components used in the culture. A pioneering clinical trial was performed in 1997 by Bertolini et al¹⁶ with a combination of several growth factors including SCF, MGDF, IL-3, IL-11, l-Flt3, and MIP-1 alpha. In this trial, two patients among 10 receiving highest doses of CD61+ cells did not receive any platelet transfusions, but the conditioning regimen in these two patients treated for breast cancer included thiotepa and L-PAM, a much less myelotoxic regimen as compared to BEAM. In our study, we show that, with a combination of MGDF plus SCF for a total ex vivo culture of 10 days, it is possible to obtain a mean 676-fold expansion of CD41+ cells from CD34+ cells in clinically suitable conditions. Previous studies from the laboratory have shown that the combination of SCF and MGDF is optimal for the generation of MK in vitro from CD34+ cells in serum-free medium and the presence of well-defined lipids.¹² Considering these in vitro data and that observed in a baboon model of autologous transplantation in which a significant decrease of the median delay of platelet recovery was observed in a CD34+ expansion study using four cytokines (SCF, TPO, IL-3, and L-Flt3),³ we tested whether MGDF plus SCF is an optimal combination for ex vivo expansion of MK progenitor cells.

Our study demonstrates the safety of ex vivo expanded MK precursor cells reinfused after high-dose chemotherapy in patients with relapsed malignant lymphoma. This observation is in concordance with that previously reported in the baboon model of autologous transplantation.³ Among all reported studies, platelet transfusion support and duration of thrombocytopenia lower than 20 000/l can not be compared because of various high-dose chemotherapy regimens and eventual coreinfusion of standard PBPC. In the report of Bertolini and co-workers, eight patients among the 10 evaluated cases required platelet transfusions and, in two of the four patients receiving the highest doses of cultured MK progenitors, platelet transfusion support was not needed, suggesting that the total number of ex vivo expanded cells (CD34+ and/or MK progenitor cells) could influence the delay of platelet recovery after high-dose chemotherapy. However, the patient population in the study of Bertolini et al¹⁶ was different from ours, in particular, in terms of conditioning regimens used. Despite this fact, it is worth noting that these two patients received the highest doses of CD61+ cells. This observation is in concordance with others showing that there is a strong inverse correlation between the expanded cell dose and the time to neutrophil or platelet recovery.^{17, 18, 19} In our study, this correlation could explain the fact that, despite a short median date of platelet transfusion independence, all patients received at least one platelet transfusion. It could therefore be proposed to perform a future ex vivo expansion trial of MK progenitors by a combination of MGDF+SCF after starting the culture with a much higher number of CD34+ cells (>5 10⁶/kg) than that used in our study (1–2 10⁶ CD34+/kg). However, it should be pointed out that the mean numbers of CD41+ and CD61+ cells/kg of body weight transplanted to our patients was very significant (10 and 12 10⁶ cells/kg, respectively) and the absence of clinical efficacy could suggest an abnormal homing and maturation of the ex vivo generated MK precursors. In a recent clinical trial using ex vivo expanded MKs after culture with SCF, MGDF, and hIL3, generated grafts contained up to 3–25% CD41+ cells in patients receiving nonmyeloablative intensification regimens.²⁰ However, this study also showed the absence of clinical efficacy in terms of shortening thrombocytopenia, suggesting that clinically useful ex vivo expansion of functional and proplatelet-forming MKs will be a difficult task in clinically acceptable conditions. The culture conditions could in parallel be modified depending on the future availability of well-defined lipids and/or other cytokines to improve the quality of the MKs generated ex vivo, despite the presence of proplatelet-forming MKs that we observed in preclinical conditions, the optimal maturation might not have been achieved in large-scale cultures.

In conclusion, ex vivo expansion of MK progenitor cells in clinically acceptable conditions is feasible and could be used to treat patients receiving an autograft as a salvage therapy for relapsed lymphoma. This procedure was safe but failed to prevent thrombocytopenia <15 000/l. This result could be attributed to a lower PBPC count collected for ex vivo cultures that should be optimized.

References

1. Westermann AM, Holtkamp MM, Linthorst GA et al. At home management of aplastic phase following high-dose chemotherapy with stem-cell rescue for haematological and non- haematological malignancies. Ann Oncol 1999; 10: 511–517. | Article | PubMed | ISI | ChemPort |
2. Archimbaud E, Ottmann OG, Yin JA et al. A randomised, double-blind, placebo-controlled study with pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) as an adjunct to chemotherapy for adults with de novo acute myeloid leukaemia. Blood 1999; 94: 3694–3701. | PubMed | ISI | ChemPort |
3. Norol F, Drouet M, Mathieu J et al. Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate hematologic recovery in a baboon model of autologous transplantation. Br J Haematol 2000; 109: 162–172. | Article | PubMed | ISI |
4. Brugger W, Mocklin W, Heimfeld S et al. Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin. Blood 1993; 81: 2579–2584. | PubMed | ISI | ChemPort |
5. Henschler R, Brugger W, Luft T et al. Maintenance of transplantation potential in ex vivo expanded CD34(+)-selected human peripheral blood progenitor cells. Blood 1994; 84: 2898–2903. | PubMed | ISI |
6. David S, Boiron JM, Dupouy M et al. Expansion of blood CD34+ cells: committed precursor expansion does not affect immature hematopoietic progenitors. J Hematother 1997; 6: 151–158. | PubMed | ISI |
7. Widmer L, Pichert G, Jost LM, Stahel RA. Fate of contaminating t(14; 18)+ lymphoma cells during ex vivo expansion of CD34-selected hematopoietic progenitor cells. Blood 1996; 88: 3166–3175. | PubMed | ISI | ChemPort |
8. Stiff P, Chen B, Franklin W et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood 2000; 95: 2169–2174. | PubMed | ISI | ChemPort |
9. Yao M, Fouillard L, Lemoine FM et al. Ex vivo expansion of CD34-positive peripheral blood progenitor cells from patients with non-Hodgkin's lymphoma: no evidence of concomitant expansion of contaminating bcl2/JH-positive lymphoma cells. Bone Marrow Transplant 2000; 26: 497–503. | Article | PubMed | ISI | ChemPort |
10. Holyoake TL, Alcorn MJ, Richmond L et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant 1997; 19: 1095–1101. | Article | PubMed | ISI | ChemPort |
11. McNiece I, Jones R, Cagnoni P et al. Ex-vivo expansion of hematopoietic progenitor cells: preliminary results in breast cancer. Hematol Cell Ther 1999; 41: 82–86. | Article | PubMed | ISI | ChemPort |
12. Norol F, Vitrat N, Cramer E et al. Effects of cytokines on platelet production from blood and marrow CD34+ cells. Blood 1998; 91: 830–843. | PubMed | ISI |
13. van den Oudenrijn S, de Haas M, Calafat J et al. A combination of megakaryocyte growth and development factor and interleukin-1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes. Br J Hematol 1999; 106: 553–563. | Article |
14. Drayer AL, Sibinga CT, Blom NR et al. The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors. Br J Haematol 2000; 109: 776–784. | Article | PubMed | ISI |
15. Blair A, Baker CL, Pamphilon DH, Judson PA. Ex vivo expansion of megakaryocyte progenitor cells from normal bone marrow and peripheral blood and from patients with haematological malignancies. Br J Haematol 2002; 116: 912–919. | Article | PubMed | ISI |
16. Bertolini F, Battaglia P, Pedrazzoli GA et al. Megakaryocyte progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients. Blood 1997; 89: 2679–2688. | PubMed | ISI | ChemPort |
17. Williams SF, Lee WJ, Bender JG et al. Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer. Blood 1996; 87: 1687–1691. | PubMed | ISI | ChemPort |
18. Engelhardt M, Douville J, Behringer D et al. Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy. Bone Marrow Transplant 2001; 27: 249–259. | Article | PubMed | ISI |
19. Paquette RL, Dergham ST, Karpf E et al. Culture conditions affect the ability of ex vivo expanded peripheral blood progenitor cells to accelerate hematopoietic recovery. Exp Hematol 2002; 30: 374–380. | Article | PubMed | ISI | ChemPort |
20. Scheding S, Bergmann M, Rathke G et al. Additional transplantation of ex-vivo generated Megakaryocytic cells after high-dose chemotherapy: results of a pilot study. Blood 2002; 100: 3291 (abstract).