Positive Selection and Transplantation of Peripheral CD34+ Progenitor Cells: Feasibility and Purging Efficacy in Pediatric Patients with Neuroblastoma

Summary:Many poor-risk neuroblastomas and tumours of the Ewing's sarcoma family (ET) recur despite autologous transplants. Recurrence may be due to tumor cells contained in the BM harvests or PBSC harvests. The objectives of this prospective study were to: (1) determine the incidence and degree of tumor cell contamination in paired BM and PBSC harvests; and (2) determine the efficacy of tumor cell purging by immunomagnetic CD34 ؉ cell selection. 198 samples from 11 consecutive patients with neuroblastoma or Ewing's sarcoma were analyzed. We assayed tumor contamination by RT-PCR assay for PGP 9.5, plus immunohistochemistry for neuroblastoma-specific antigens (the latter in neuroblastoma only). None of these patients had tumor cells detected in their BM by clinical histology immediately before BM or PBSC harvests. However, 82% of PBSC and 89% of backup BM harvests were contaminated with tumor by RT-PCR and/or immunocytochemistry assays. Unselected PBSC and BM harvests contained similar quantities of tumor cells (median, ෂ200 000 cells). Cyclophosphamide plus G-CSF mobilization did not affect the incidence or level of contamination in PBSC harvests, as compared to blood obtained before mobilization. Immunomagnetic CD34 ؉ cell selection depleted tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for BM harvests. Keywords: neuroblastoma; Ewing's sarcoma; autologous transplantation Most childhood solid cancers are sensitive to chemotherapy and radiotherapy, but the prognosis for patients with recurrent, metastatic or refractory pediatric solid tumors remains dismal. 1,2 Recent studies suggest that high-dose myeloablative radio-chemotherapy with autologous bone marrow or peripheral blood stem cell rescue provides the possibility of cure for some patients with advanced neuroblastoma, rhabdomyosarcoma, Wilms' tumor, germ cell tumors, Ewing's sarcoma and peripheral primitive neuroectodermal tumors. [3][4][5][6][7] Nevertheless, only a minority of patients with poor-risk solid cancers treated by high-dose myeloablative radio-chemotherapy are long-term survivors. Relapse posttransplantation can arise from tumor cells in the patient that survive high-dose myeloablative radio-chemotherapy. Gene marking studies have shown that tumor cells which contaminate the hematopoietic graft can also contribute to relapse. 8,9 Previously, we developed a combination highdose myeloablative radio-chemotherapy regimen for a broad range of childhood cancers. 10 We then developed an immunomagnetic CD34 + cell selection technique to 'reverse purge' autologous bone marrow harvests from pediatric patients. 11 With a prototype Isolex device, the estimated median tumor cell depletion was 2.6 logs for bone marrow harvests. However, the tumor cell content was modelled, not actually measured, in this prior study. The efficacy of tumor cell purging by immunomagnetic CD34 + cell selection of mobilized PBSC harvests from pediatric patients has not been reported.PBSC harvests have been reported to contain fewer tumor cells and to be less frequently...

Section: Discussionmentioning

confidence: 99%

Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing’s sarcoma

Leung¹,

Chen²,

Klann

et al. 1998

“…[10][11][12] Preclinical studies have suggested several clinical applications of highly purified CD34 ϩ cells, including tumour cell purging. Enrichment of CD34 ϩ cells has been proven to deplete tumour cells from autografts, [13][14][15][16][17][18] and is necessary for ex vivo expansion of progenitor cells. Furthermore, highly purified CD34 ϩ cells are essential for optimal cytokine-mediated expansion and differentiation in short-term culture.…”

mentioning

confidence: 99%

Selective loss of progenitor subsets following clinical CD34+ cell enrichment by magnetic field, magnetic beads or chromatography separation

Johnsen

Hutchings

Taaning

et al. 1999

Summary:In this preclinical evaluation we have compared the efficacy of three clinical CD34 ؉ enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34 ؉ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88-98%), 86% (62-97%), and 49% (18-85%), and median yield of 65% (54-100%), 40% (21-74%), and 30% (8-55%), respectively. Subset analysis recognised a selective loss of CD34 ؉ /61 ؉ after enrichment, most likely due to class I-II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2-4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34 bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin-avidin and insufficient physical manipulation for CD34 ؉ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (Ͻ10 ؊5 ) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a twostep procedure including CD34 selection and subsequent depletion of the tumour cells in question.

“…However, we have noted first a persistent decrease in CD4 + lymphocyte levels and a high incidence of serious and life-threatening late complications (two patients had EBV-associated lymphoproliferation, six children had severe VZV infection and six severe septic shock events occurred after completion of hematopoietic reconstitution); second, a delayed platelet recovery which is longer than in historical series. 78,79 Other published experience of PB CD34 + cell selection in children 43,69 confirms the feasibility of this technique and a successful short-term engraftment of PB CD34 + cells. However, in children as well as in adults' questions concerning the use of ex vivo modified PBPCs remain open: to define categories of patients for whom selection is beneficial and to balance the advantages and disadvantages of grafting selected cells.…”

Section: Collection In the Smallest Of Childrenmentioning

confidence: 65%

“…[43][44][45][46][47][48] Few teams use peripheral venous access together with a central catheter installed at diagnosis. 12,49 Only when it is deemed impossible to ensure adequate blood flow with peripheral veins is a CVC installed under local anesthesia and sedation.…”

Section: Venous Access and Related Complicationsmentioning

confidence: 99%

“…61,62 However, the published clinical trials of CD34 + cell transplantation have mainly been performed in adults [63][64][65][66] and there are only a few reports of significant studies performed in children. 43,[67][68][69] The majority of these pediatric patients are children with advanced neuroblastoma. One of the potential benefits of CD34 + enrichment of PBPC or bone marrow autografts is the concomitant depletion of tumor cell levels.…”

Section: Collection In the Smallest Of Childrenmentioning

confidence: 99%

See 1 more Smart Citation

Using peripheral blood progenitor cells (PBPC) for transplantation in pediatric patients: a state-of-the-art review

Díaz

Kanold

Vicent

et al. 2000

Summary:This paper presents a state-of-the-art review of using mobilized-peripheral blood progenitor cells (PBPC) for transplantation in children. Our own data and those from Medline searches and meeting reports, are analyzed and presented for the different sections that involve transplantation. Recommendations concerning the choice of mobilization regimens, venous access, priming of separator extracorporeal line, anticoagulation, and number of CD34 + cells to infuse for rapid engraftment are proposed. In the allogeneic setting, we analyze ethical and safety aspects of pediatric donor mobilization and collection. Data from the literature suggest that the use of cytokine-mobilized PBPC for allogeneic transplantation appears to be safe both for pediatric donors and patients leading a rapid hematopoietic engraftment with a similar incidence of acute graftversus-host disease (GVHD). The high incidence of chronic GVHD and its management emerge as the most concerning aspect in allogeneic PBPC transplantation. Bone Marrow Transplantation (2000) 26, 1291-1298.