Stem cell in vitro purging

As stated at the outset of this chapter, due to the nature of harvesting stem cells in a marrow disorder such as leukemia, one can expect tumor contamination of grafts. A number of approaches to reduce tumor burden among harvested grafts have been undertaken to reduce the likelihood of relapse following ASCT. Such relapses can be expected to arise from one of the two sources: harvested cells in the autograft, or unharvested, drug-resistant clones that survive the preparative regimen. A combination of the two etiologies is likely. Studies of relapses after syngeneic transplantation demonstrate relapse rates of more than 50%, demonstrating that the myeloablative regimen itself may be a greater source of therapeutic failure than inability to achieve a tumor-free graft at the time of reinfusion.13 The corollary to the syngeneic data, however, shows that residual leukemia is frequently present at the time of harvesting. It is therefore not surprising that grafts should have a high risk of tumor contamination, and that—even with improvements in myeloablative regimens—this may contribute to relapse.

Contamination of autografts at the time of harvesting occurs frequently and is readily confirmed by immunophenotype, clonogenic assay, or PCR. Experiments in which harvested marrow was trans-fected with a retroviral construct containing the neomycin-resistance gene were found to show incorporation of the marker gene in a subset of leukemia cells at the time of relapse. This suggests that a component of the relapse was indeed caused by leukemia cells from the reinfused harvest product.14 Purging of the graft therefore makes intuitive sense to minimize the risk of reinfusion of clonogenic leukemia cells.

Purging of marrow is accomplished by chemical, immunologic, or physical means. Chemotherapeutic agents such as the prodrugs 4-hydroperoxycyclophos-phamide (4-HC) or mafosfamide are particularly toxic to committed progenitors and leukemic blasts, but spare the earliest hematopoietic elements. These stem cells express relatively high levels of the detoxifying enzyme aldehyde dehydroxygenase, decreasing intra-cellular generation of active metabolites such as phos-phoramine mustard and limiting stem cell injury from either agent.15 Regardless of this fact, purging with cyto-toxic agents lowers stem cell dose. In combination with the slow self-renewal rates of early progenitors, it is not surprising that engraftment is typically delayed following purging with cytotoxic agents, and this may be associated with increased bleeding or risk of infection.

Numerous studies have demonstrated that purging can decrease the degree of leukemic burden in the autograft, and that this is associated with a lower relapse rate.1617 There are no prospective randomized trials to examine whether purging is associated with improved survival as compared to unmanipulated autografts. The largest study investigating the benefits of purging in AML is a retrospective analysis of nearly 300 autologous transplants from the Autologous Blood and Marrow Registry.18 In all of these cases, transplantation occurred within 6 months of complete remission, minimizing the bias of delay prior to transplantation that confounds numerous studies of purging. A multivariate analysis of this data showed purging with 4-HC was associated with an improved leukemia-free survival (56% vs 31% in first CR; 29% vs 10% in second CR, respectively) and overall survival (62% vs 40% in first CR; 46% vs 17% in second CR, respectively), with comparable transplant-related morbidity and mortality. Significant differences were noted between the two groups in terms of time to engraftment, with a median time to ANC >500 of 40 days in the purged group and 30 days in those receiving unpurged transplants. Differences in regimen-related toxicities, as well as some disparities in baseline characteristics of the patients receiving each type of transplant may have contributed to the study's findings. However, the overall survival of 46% among purged transplants beyond the first remission deserves note, as this survival is more typical of the survival plateau among historical studies of unpurged transplants in first remission. Indeed, there are few, if any large series of unpurged transplants that significantly exceed this level, regardless of disease status.

A number of monoclonal antibodies have been employed to purge contaminating early hematopoietic cells, such as anti-CD33 and CD14, along with immunomagnetic beads or complement fixation.19-22 Other methods that have been used include density-gradient centrifugation, elutriation, hyperthermia, and cytokines, such as interleukin-2.23 Like the effect of chemotherapeutic purging, molecular and immunologic purging of autografts, even when associated with reduction in leukemic burden, lowers stem cell count. Numerous retrospective and phase II studies have shown that graft manipulation delays engraft-ment. Attempts to use myeloid growth factors, such as granulocyte or granulocyte-monocyte stimulating factors have not been pursued to determine if these agents can minimize some of this toxicity. Alternatively, amifostine has been examined as a cyto-protective agent, to allow for the sparing of normal hematopoietic stem cells in the graft.24

Despite a sound rationale and both laboratory and clinical evidence for the feasibility and efficacy of autograft purging, the benefit of purging remains speculative. The labor-intensive procedure adds substantial cost to transplant, as well as the potential for increased transplant-related morbidity from delayed engraft-ment. This has kept the practice from widespread acceptance. From available literature, patients that seem to benefit the most from the practice are those who are beyond the first remission at the time of transplant. However, this is a heterogeneous group and this complicates analysis of data from various centers and purging agents. It is also unclear if there would be a benefit of purging marrow obtained in second remission above that of using stem cells harvested and stored early in the course of disease (i.e., as soon as feasible once in first remission). In the absence of randomized data, the practice of autograft purging for leukemia remains investigational at present.

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