Stem Cells, Vol. 17, No. 6, 373-376,
November 1999
© 1999 AlphaMed Press
Clinical Utility in Maximizing CD34+ Cell Count in Stem Cell Grafts
Richard K. Burt
Allogeneic Transplantation, Northwestern University, Chicago, Illinois, USA
Dr. Richard K. Burt, Allogeneic Transplantation, Northwestern University, 250 East Superior, Wesley Pavilion, Room 1456, Chicago, Illinois 60612, USA.
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Introduction
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Manipulation of hematopoietic stem cell grafts has focused on purging autologous tumor cells to decrease relapse rate or depleting allogeneic lymphocytes to decrease graft-versus-host disease (GVHD). In practice, these approaches have limited application. Most relapses occur from failure of the conditioning regimen to eradicate residual disease, not from infusion of tumor cells with the graft. Depletion of lymphocytes from an allograft diminishes GVHD but is complicated by an increased relapse rate, graft failure, and opportunistic infections from delayed immune reconstitution. Rather than emphasizing removal of a non-stem-cell component, we proposed engineering a hematopoietic stem cell graft to optimize the "stem cell" content. In this paper, we will review the advantage of maximizing the CD34+ stem cell content in both autologous and allogeneic grafts.
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Cell Markers
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A marker unique to the hematopoietic multilineage stem cell has not been identified. However, progenitor cells expressing the surface antigen CD34+ are capable of long-term engraftment. Within this CD34+ population resides a subset of multipotent (lineage-nonspecific) stem cells capable of myeloid, lymphoid, erythroid, or megakaryocyte commitment (Fig. 1
). CD34 is also found on progenitor cells that have already committed toward lineage specificity. Characterization of CD34+ cells by flow cytometry allows identification of these different subsets. The earliest multilineage stem cell is CD34+, MDR-1+, c-kit+, and CD45RO+, but CD38–, HLA-DR–, and lineage-differentiation marker negative. In contrast, lineage-specific progenitor cells coexpress CD34 antigens associated with myeloid (CD33 and CD13), megakaryocytic (CD41 and CD61), erythroid (CD71), B-lymphoid (CD19), or T-lymphoid (CD7) development.
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Figure 1. Stem cell subsets. Multipotent cells are able to give rise to myeloid, lymphoid, erythroid, and megakaryocytic cells, and, possibly, veto cells.
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In autologous transplantations, maximizing the CD34+ content of a graft results in more rapid engraftment kinetics and improved late hematopoietic reconstitution [1-3]. In allogeneic transplantations, CD34+ dose correlates with early and late hematopoiesis and hospital charges. It is possible but unproven that CD34+ dose may impact on survival, and possibly GVHD [4, 5].
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Autologous Transplantation
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In 1995, Weaver et al. reported that CD34+ cell dose correlated with time to engraftment after autologous peripheral blood transplantation [1]. This multicenter analysis involved 692 patients with a variety of malignancies (breast cancer, lymphoma, ovarian cancer, multiple myeloma, and sarcoma), preparative regimens (BEAC, BuCY, CTCb, and ICE), and post-infusion growth factors (recombinant human [rHu]G-CSF, rHuGM-CSF, none). The most important factors affecting neutrophil recovery were CD34+ dose and the administration of post-infusion growth factor. A dose-response relationship existed between the number of CD34+ cells infused and both neutrophil and platelet recovery. The median time to an absolute neutrophil count (ANC) >0.5 x 109/l (for two consecutive days) decreased with increasing CD34+ stem cell dose: 11 days for <2.5 x 106 CD34+ cells/kg; 10 days for 2.5 to 7.5 x 106 CD34+ cells/kg; nine days for 7.5 to 12.5 x 106 CD34+ cells/kg; and eight days for >12.5 x 106 CD34+ cells/kg.
Kiss et al. reported that after autologous peripheral blood stem cell transplantation, CD34+ cell dose correlated with both early engraftment kinetics and late peripheral blood counts [2]. A threshold effect between rapid and slow engraftment occurred at 5 x 106 CD34+ cells/kg. In addition, hemoglobin and platelets were significantly higher at 180, 360, and 540 days after transplantation for those patients who received >5 x 106 CD34+ cells/kg. Therefore, for autologous transplantations, a higher CD34+ cell dose appears to correlate with improved long-term hematopoiesis.
Dercksen et al. also reported a similar correlation between CD34+ cell dose and engraftment kinetics [3]. Beyond the larger number of CD34+ cells infused, they reported that the relative proportions of CD34+ lineage-specific subsets may be important. For example, platelet recovery correlated with the number of CD34+/CD41+ cells infused. Patients receiving <0.5 x 108 CD34+/CD41+ cells/kg had a median platelet recovery of 19 days versus 11 days for patients who received >0.5 x 106 cells/kg. This suggests that subsets of stem cells, or, in other words, stem cell component therapy, may be important for graft engineering.
From the published data, it appears that maximizing the number of infused autologous CD34+ progenitor cells results in fewer complications, fewer blood product requirements, more rapid hospital discharge, and better long-term peripheral blood counts. The optimal number of CD34+ cells infused should be >5 x 106/kg. Mobilizing large numbers of CD34+ cells may be difficult in some patients. Improved progenitor cell collection may occur by increasing the dose of rHuG-CSF (16 or 32 µg/kg) or combining rHuG-CSF and stem cell factor (rHuSCF).
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Allogeneic Transplantation
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Compared with the technical limitations of a marrow harvest, peripheral blood stem cell mobilization permits repetitive collections to achieve high numbers of stem cells. This facilitated the comparison of autologous stem cell dose and transplantation outcome. In contrast, allogeneic peripheral blood transplantations are complicated by an increased risk of chronic GVHD. Therefore, the short-term favorable engraftment kinetics of allogeneic peripheral blood stem cells appear to be offset by an increase in morbidity and mortality from chronic GVHD [6-18].
A blood graft, relative to a marrow transplantation, may increase chronic GVHD because of increased numbers of T cells in the peripheral blood relative to the marrow, or because growth-factor mobilization skews the T-cell phenotype toward a Th2 cytokine profile. One approach for decreasing the risk of chronic GVHD is T-cell depletion of the peripheral blood graft. T-cell depletion of a marrow allograft reduces GVHD but is associated with increased rates of graft failure and leukemic relapse. It is reasonable to anticipate that the same pitfalls would occur with lymphocyte depletion of a peripheral blood allograft. Mavroudis et al. reported that T-cell-depleted blood allografts are associated with delayed cytopenias and graft failure [19]. To avoid graft failure or an increase in leukemia relapse while minimizing the risk of GVHD, a partial lymphocyte depletion of the blood allograft may be performed. However, the number of T cells necessary to preserve engraftment and prevent relapse while not affecting the risk of GVHD is unknown. It is even unclear if the allogeneic graft-versus-leukemia (GVL) effect is separable from GVHD. Titration to an ideal number of T cells may be impossible or difficult to predict due to variability in T-cell subsets, patient age, type of disease, disease stage, or minor antigen disparities between recipient and host.
To circumvent these problems while maintaining the beneficial effect on CD34+ stem cell dose, we initiated a protocol of infusing allografts composed of unmanipulated marrow supplemented with lymphocyte-depleted (i.e., CD34+-enriched) peripheral blood (Fig. 2) [4]. This approach would contribute to the rapid engraftment kinetics of peripheral blood stem cells, allowing for an increase in stem cell dose without an increased risk of chronic GVHD. It would also preserve the beneficial effect of unmanipulated marrow in terms of engraftment and GVL (Fig. 2). The combination of unmanipulated marrow with CD34+-enriched peripheral blood results in a graft of >5 x 106 CD34+ cells/kg from which the peripheral blood contributes most of the stem cells but only 1% of the T cells (Fig. 3) [4].
We recently reported that using this approach to maximize the allogeneic stem cell dose without changing the number of lymphocytes present in a marrow allograft results in rapid engraftment (ANC >0.5 x 109/l on day 10; platelet count >20 x 109/l on day 12), and no apparent increase in either acute or chronic GVHD. This engraftment rate has resulted in a marked decrease in both hospital charges and complications. At our institution, the average hospital discharge occurs 14 days after infusing a combined marrow and CD34+-enriched blood allograft. This compares with a day-28 discharge after an unmanipulated marrow graft.
Increasing the CD34+ stem cell dose (without altering the number of infused T cells in a normal marrow graft) is designed to achieve rapid engraftment and better long-term hematopoiesis without affecting the incidence of GVHD. Although unproven, this approach may decrease GVHD. Aversa et al. reported that HLA-mismatched transplantation using megadose CD34+ cells (>107/kg) may decrease GVHD by a "veto effect" [5]. It has been speculated that a subset of CD34+ progenitor cells may present antigens without costimulatory molecules predisposing to tolerance [20-22]. Although controversial, this theory highlights not only the importance of high doses of CD34+ cells in hematopoietic transplantation but also possible future directions for graft engineering to obtain optimal subsets of CD34+ progenitor cells.
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Summary
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Many different stem cell subsets have been identified. Some of these subsets are multipotent, able to develop into any one of several cell types, while other stem cell subsets are committed to differentiated myeloid, lymphoid, erythroid, or megakaryocytic lineages.
Autologous transplantation studies have suggested that the dose of CD34 cells infused correlates with rapid engraftment. Allogeneic transplantation studies suggest that CD34 cell number correlates not only with rapid engraftment but also with early hospital discharge and, although as yet unproven, possibly long-term outcomes including late hematopoiesis, GVHD, and survival.
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Footnotes
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Reprinted from The Oncologist 1999;4:265-268.
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References
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Weaver CH, Hazelton B, Birch R et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995;86:3961-3969.[Abstract/Free Full Text]
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Kiss JE, Rybka WB, Winkelsein A et al. Relationship of CD34 cell dose to early and late hematopoiesis following autologous peripheral-blood stem-cell transplantation. Bone Marrow Transplant 1997;19:303-310.[Medline]
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Dercksen MW, Rodenhuis S, Dirkson MK et al. Subsets of CD34+ cells and rapid hematopoietic recovery after peripheral blood stem cell transplantation. J Clin Oncol 1995;13:1922-1932.[Abstract/Free Full Text]
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Burt RK, Kuzel TM, Fishman M et al. Stem cell component therapy: supplementation of unmanipulated marrow with CD34 enriched peripheral blood stem cells. Bone Marrow Transplant 1999;23:381-386.[Medline]
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Aversa F, Tabilio A, Velardi A et al. Treatment of high-risk leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998;339:1186-1193.[Abstract/Free Full Text]
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Mavroudis DA, Read EJ, Molldrem J et al. T cell-depleted granulocyte colony-stimulating factor (G-CSF) modified allogeneic bone marrow transplantation for hematological malignancy improves graft CD34+ cell content but is associated with delayed pancytopenia. Bone Marrow Transplant 1998;21:431-440.[Medline]
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Bachar-Lustig E, Rachmim N, Li HW et al. Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice. Nat Med 1995;1:1268-1273.[Medline]
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Rachamim N, Gan J, Segall H et al. Potential role of CD34 stem cells in tolerance induction. Transplant Proc 1997;29:1935-1936.[Medline]
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Introduction
Cell Markers
