A pediatric patient with very early meningeal relapse of his CD34+ CD133− pre-B-ALL was transplanted with 2.5 × 106/kg CD133 selected autologous progenitor cells. Enrichment of CD133+ cells resulted in a purity of 92.3 ± 3.5% CD133+. Hematopoietic engraftment with >1.0 × 109/l neutrophils and >50 × 109/l platelets was reached within 13 and 24 days, respectively. At a follow-up of 11½ months after autologous transplantation, the patient is in complete remission. To our knowledge, the successful transplantation with a CD133 selected graft is the first one to be reported worldwide. CD133 selected cells may serve as an alternative in the case of CD34+ malignancy.
Pediatric patients with high-risk of relapse of ALL are frequently treated with transplantation of allogeneic bone marrow (BM) or peripheral blood stem cells (PBSC). However, despite allogeneic transplantation, prognosis for patients with a very early meningeal relapse of leukemia remains poor. Few case reports suggest a better outcome in these patients after autologous transplantation.1 It has been shown that clonogenic tumor cells within the graft can cause relapse of the disease.2 To avoid relapse from reinfusion of grafts, CD34 selection of stem cells is a well-established strategy to remove contaminating tumor cells from autografts in CD34-negative solid tumors of pediatric patients.3 To reach high purity of the stem cell transplant, refined selection strategies are needed. In order to eliminate potentially CD34-positive tumor cells from autologous grafts, we established a clinical scale CD133 selection procedure for pediatric patients with neuroblastoma.4 CD133 is a novel stem cell glycoprotein antigen selectively expressed on CD34bright progenitor cells.5 Here, we report on the application of this new selection strategy for purification of an autologous graft and transplantation of a boy with relapsed CD34-positive CD133-negative ALL.
Patients and methods
An 11-year-old boy with a pre-B-ALL suffered very early isolated meningeal relapse. Initially, he had been treated with polychemotherapy according to ALL-BFM 95 (German/Austrian multicenter study) from July 1999 to February 2000. At that time, flow cytometric immunophenotyping of the leukemic blasts showed positivity for antigenes CD19, CD20, CD24, CD22, cyCD22, CD10, CD34, HLA-DR but negative for CD133. Moreover, the blasts were negative for the fusion transcripts TEL-AML1, BCR-ABL and MLL–1 as determined by PCR. Fourteen months later, isolated meningeal relapse was confirmed by magnetic resonance imaging and lumbar and bone marrow puncture. From October 2000, the boy was treated according to ALL-BFM-Rez 96 including intrathecal application of methotrexate, cytosine arabinoside and prednisolone and intravenous application of dexamethasone, methotrexate, vincristine and PEG-asparaginase. For the poor prognosis of both conventional chemotherapy and allogeneic transplantation in this case, we decided on high-dose chemotherapy and autologous transplantation.
Autologous stem cell harvesting
Autologous peripheral blood stem cells (PBSC) were harvested after five courses of chemotherapy. Course 5 of the ALL-BFM-Rez 96 protocol consisted of oral application of dexamethasone (20 mg/m2/day) and mercaptopurine (100 mg/m2/day) from day 1 to day 5; vincristine (i.v. 1.5 mg/m2/day) on days 1 and 6, methotrexate (1 g/m2; 36 h infusion) on day 1, cytosine arabinoside (2 g/m2/twice a day, 3 h infusion) on day 5, PEG-asaparaginase (500 U/m2; 1 h infusion) on day 6, and intrathecal application of methotrexate (12 mg) and cytosine arabinoside (30 mg) and prednisolone (10 mg) on day 1. From 2 days after the end of chemotherapy to the last day of harvest, stem cells were mobilized with s.c. 5 μg/kg rhG-CSF (Neupogen; Amgen, Munich, Germany). At the time of apheresis the patient weighed 40 kg. PBSC I, II, III was collected on days 9, 10 and 11 of mobilization using a Cobe Spectra (Cobe Laboratories, Lakewood, CO, USA).
CD133+ cells were immunomagnetically selected from aphereses using the automated CliniMacs system (Miltenyi/Biotec, Bergisch-Gladbach, Germany). Cells were processed as described for CD34 selection4,6 under GMP conditions. Briefly, both the pooled PBSC I+II and PBSC III in a separated run were washed twice with CliniMacs PBS/EDTA buffer (Miltenyi/Biotec) supplemented with 0.4% human serum albumine (BSD Hessia, Frankfurt, Germany) for platelet reduction and cells were resuspended to a total volume of 80 ml. To avoid nonspecific binding, cells were incubated with 5 ml intraglobine (50 μg/ml; Biotest, Frankfurt, Germany) for 5 min. Cells were then labeled with anti-CD133 antibodies (CD133+ microbeads, kindly provided by Miltenyi/Biotec) for 30 min at 4°C. After incubation, cells were washed twice with CliniMacs PBS/EDTA buffer by centrifugation at 1100 g for 15 min. Finally, CD133 positive selection was performed using the software programme ‘enrichment 2.1’ on the CliniMacs system.
Conditioning regimen and ASCR
The patient received TBI on days −7, to −4 (12 Gy total body, 18 Gy cranial) and etoposide on day −3 (60 mg/kg). Seventy-two hours after the completion of the radio-chemotherapy, cryopreserved stem cells were thawed rapidly and a total of 2.5 × 106 CD133+/CD34+ cells/kg were reinfused.
Flow cytometric analysis
Three- and four-color flow cytometric analyses were performed on a Coulter Epics XL (Coulter, Krefeld, Germany) to determine the percentage of leukocytes expressing CD133+ and CD34+ in PBSC and CD133+ selected preparations. Moreover, we searched for residual leukemic blasts. After transplantation, we monitored the reconstitution of immunological subtypes in the peripheral blood. Briefly, 2 × 105–1 × 106 cells were labeled for 20 min in separate tubes with CD34-FITC/CD133-PE/CD45-PC5 for determination of progenitor cells. Also, CD45FITC/CD34PE/7-AAD including stem-count beads (Stem-Kit/Coulter Immunotech and 7-AAD) was used to evaluate the absolute count of CD34+ cells according to a modified ISHAGE protocol. For detection of residual leukemic cells prior to and after CD133 selection, cells were labeled with CD19-FITC/CD10-PE/CD45-ECD/CD34-PC5, CD19-FITC/CD133-PE/CD45-ECD/CD34-PC5 as well as CD19-FITC/CD34-PE/PI. Immune reconstitution was monitored in T cell subsets, NK cells and B cells in weekly peripheral blood samples during the first 100 days post transplant, then monthly samples or according to the current patient status. For this, cells were FITC/PE/ECD/PC5-labeled with CD45/CD4/CD8/CD3, CD45/CD56/CD19/CD3, HLA-DR/CD38/CD3/CD8, HLA-DR/CD38/CD3/CD4, CD45RA/CD45RO/CD3/CD4, CD45RA/CD45RO/CD3/CD8 and CD4/CD69/CD3/CD8. CD133–1-PE and CD133–2-PE were obtained from Miltenyi/Biotec; all other antibodies were from Coulter Immunotech (Marseille, France). Dead cells were detected and excluded from analysis by 7-AAD and propidium- iodide (PI) staining. Additionally, IgG1-FITC/IgG1-PE/IgG1-ECD/IgG1-PC5 and IgG1-FITC/IgG1-PE/CD45-ECD/IgG1-PC5 staining served as controls.
Collection and positive selection of CD133+ cells
Three leukaphereses were performed. The number of leukocytes collected was 4.66 × 1010 CD45+ cells with a total number of 299 × 106 CD34+ cells (7.5 × 106 CD34+/kg) and 226 × 106 CD133+ cells (5.7 × 106 CD133+/kg). After cryopreservation of 80 × 106 CD34+ cells as an unselected back up product, CD133 selection in two separate runs led to a total number of 105 × 106 CD34+ cells (2.6 × 106 CD34+/kg) and 106 × 106 CD133+ cells (2.6 × 106 CD133+/kg). The percentages of CD34+ and CD133+ cells in the different PBSCs prior to and after CD133 selection are shown in Table 1.
Enrichment of progenitor cells by CD133 selection from the pooled PBSC I+II and from PBSC III resulted in a purity of 92.3 ± 3.5% CD133+ and 91.3 ± 4.6% CD34+ cells. Examplarily, the percentages of progenitor cells in PBSC I and after CD133 selection in the pooled PBSC I and II are shown in Figure 1.
Prior to selection, 71% of the CD34+ cells were also positive for CD133+. After CD133 enrichment >99% of the CD34+ cells and >97% of the CD133+ cells were CD34+CD133+ cells. Moreover, purification led to an increased frequency of CD34−CD133+ cells (0.9% and 2.6%).
Viability of the cells was >97% prior to and after purification. Recovery of CD133+ hematopoietic progenitor cells resulted in 63.9 ± 5.5% while the recovery of CD34+ cells was 48.1 ± 4.2%, respectively (Table 1).
Detection of residual leukemic cells
Due to nonspecific staining of membrane particles, which cannot be excluded from analysis by 7-AAD or propidiumiodide, flow cytometric analysis did not allow for definitive exclusion of residual leukemic blasts (CD45+CD34+CD19+CD10+) in apheresis products I, II, III and one selected graft (in both cases, <0.02%). The second purified graft stained negatively. Molecular genetic screening for blasts was not possible, since the leukemic blasts were negative for the fusion transcripts TEL-AML1, BCR-ABL and MLL–1 determined by PCR.
Engraftment was seen on days 13, 13 and 24 for leukocytes (>1.0 × 109/l), neutrophils (>1.0 × 109/l), and platelets (>50.0 × 109/l), respectively. This is comparable with the engraftment after reinfusion of CD34+ selected cells as shown for the absolute number of neutrophils count (ANC) of some of our pediatric patients (four neuroblastoma, one Ewing's sarcoma, one osteosarcoma, one rhabdomyosarcoma; Figure 2). These patients were transplanted with a similar dose of autologous CD34+ stem cells (1.8–4.0 × 106/kg). Generally, to accelerate recovery, all our patients were treated with rhG-CSF for 14–15 days after autologous transplantation.
Monitoring of different immunological subtypes is illustrated in Figure 3. CD19+ B cell recovery started as early as 1.5 months after transplantation. CD56+CD16+CD3− natural killer cells (NK) increased early and reached normal levels within 1 month. No marked decrease in the absolute number of CD4+ T helper cells was observed and CD8+ cytotoxic cells reconstituted fairly rapidly resulting in a slightly inversed CD4/CD8 ratio, only. CD45 isoforms confirmed rapid recovery of memory cytotoxic T cells (CD45RO+CD8+CD3+) and naive cytotoxic T cells (CD45RA+CD8+CD3+). By contrast, the naive helper T cells subset (CD45RA+CD4+CD3+) was somewhat reduced (not shown in Figure 3). No striking difference to other patients was observed in the activation markers on the various T cell subsets.
Clinical status after transplantation
Infusion of CD133 selected progenitor cells was well tolerated. The patient did not show any allergical reactions or side-effects due to the remaining CD133 microbeads on the surface of the progenitor cells. No viral or fungal infection or other kind of complication during and after hematopoietic recovery was seen. No additional late toxicities have been observed. With a follow-up of 11½ months after transplantation, the boy is alive with a good performance status in continuous complete remission.
Very early leukemic relapse in the CNS continues to be a difficult therapeutic challenge. Intensification of chemotherapy prior to allogeneic transplantation did not improve prognosis. Because a few cases are reported with similar or better outcome after autologous transplantation, high-dose chemotherapy with reinfusion of autografts seems to be an alternative worth exploring for these patients.1 For autologous grafts, a refined selection technique is indispensable to yield a high purity of progenitor cells and to avoid a relapse due to residual leukemic contamination. For pediatric patients with neuroblastoma, Brenner et al2 showed direct evidence that neuroblastoma cells present in patients’ bone marrow or peripheral blood stem cell grafts can contribute to relapse. The commonly used CD34 selection is an excellent technique to achieve a high purity of CD34 cells.7 In various trials, clinical scale CD34 selection led to a median purity of 96–97% CD34+ cells.4,6,7 However, the use of a CD34 enrichment technique is not to be recommended in cases of CD34 positive malignancies. Rather, enrichment with the new progenitor antigen CD133 may present a preferable purification strategy in selected cases. Several studies demonstrated that the majority of ALLs are double positive for CD133 and CD34.8,9 Therefore, purification with CD133 can be used for a minority of ALLs, only.
In our previous work, we compared large scale CD34 and CD133 purification of autologous grafts of pediatric patients with neuroblastoma.4 Both selection strategies led to similar high median purities of 98.0% and 97.3% CD34+ cells. No contaminating neuroblastoma cells remained as determined by RT-PCR for tyrosine hydroxylase. These patients were transplanted with CD34 selected progenitor cells, only. Experimentally data demonstrate a higher proliferating and self-replicating capacity of CD133+CD34+ cells in comparison to CD133−CD34+ cells. The latter contain committed progenitors, which do not have full reconstituting capacity.5,10,11,12 An extremely rare population of cells lacking CD34 and lineage commitment markers (CD34−lin−CD38−) is positive for CD133 and shows a very high reconstituting ability.12 This gives rise to the thought that CD133 selected cells might be superior for stem cell transplantation to CD34 selected cells. We saw that CD133 selection led to enrichment of this rare CD133+CD34− progenitor cell population together with the CD133+CD34+ double positive cells. Therefore, fewer progenitor cells in the graft might be sufficient to reach full reconstitution. However, hematopoietic recovery of our patient reported here did not differ significantly from that of our patients receiving the same number of conventionally selected stem cells. Additionally, reconstitution of the immunological subsets did not show remarkable differences to patients treated with CD34-enriched grafts. To date, the literature lacks any data about transplantation with CD133 enriched progenitors. Therefore, to our knowledge, the successful transplantation with a CD133 selected graft of our patient with a CD34+CD133− ALL is the first one to be reported worldwide.
In summary, large scale CD133 positive selection is feasible and achieves equivalent high purities of progenitor cells equivalent to that previously reported for CD34 selection. Tumor and leukemic cell depletion is effective with this method. CD133 selected grafts can lead to full hematopoietic reconstitution. Therefore, CD133 purification presents a favorable alternative to CD34 selection of autologous grafts in patients with CD34-positive but CD133-negative malignant cells.
Messina C, Valsecchi MG, Arico M et al. Autologous bone marrow transplantation for treatment of isolated central nervous system relapse of childhood acute lymphoblastic leukemia Bone Marrow Transplant 1998 21: 9 14
Brenner MK, Rill DR, Moen RC et al. Gene-marking to trace origin of relapse after autologous bone marrow transplantation Lancet 1993 341: 85 86
Handgretinger R, Lang P, Schumm P et al. Isolation and transplantation of autologous peripheral CD34+ progenitor cells highly purified by magnetic-activated cell sorting Bone Marrow Transplant 1998 21: 987 993
Koehl U, Esser R, Zimmermann S et al. Large scale purification of progenitor cells by AC133+ selection Bone Marrow Transplant 2001 27: 296 (Abstr. P736)
Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells Blood 1997 12: 5002 5012
Koehl U, Gunkel M, Gruettner HP et al. Positive selection of hematopoietic progenitor cells for autologous and allogeneic transplantation in pediatric patients with solid tumors and leukemia Transplant Hematol Oncol 1999 1: 159 168
Schumm M, Lang P, Taylor G et al. Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device J Hematother 1999 6: 5 11
Barsch G, Baumann M, Ritter J et al. Expression of AC133 and CD117 on candidate normal stem cell populations in childhood B-cell precursor acute lymphoblastic leukemia Br J Haematol 1999 107: 572 580
Bühring HJ, Seiffert M, Marxer A et al. AC133 antigen is not restricted to acute myeloid leukemic blasts but is also found on acute lymphoid leukemia blasts and on subset of CD34+ B-cell precursors Blood 1999 2: 832 833
Kobari L, Giarratana MC, Pflumic F et al. CD133 cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells J Hematother Stem Cell Res 2001 10: 273 281
Matsumoto K, Kazuta Y, Yamashita N et al. In vitro proliferation potential of AC133 positive cells in peripheral blood Stem Cells 2000 18: 196 203
Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7 Blood 2000 9: 2813 2820
This project was supported by ‘Hilfe für Krebskranke Kinder Frankfurt eV’, by ‘Frankfurter Stiftung für Krebskranke Kinder’ and by ‘Paul und Ursula Klein-Stiftung’. We acknowledge the excellent technical support of Andrea Brinkmann, Ilse Bühler and Stephanie Grohal.
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Cite this article
Koehl, U., Zimmermann, S., Esser, R. et al. Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia. Bone Marrow Transplant 29, 927–930 (2002). https://doi.org/10.1038/sj.bmt.1703558
- progenitor cells
- CD133+ positive selection
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