Several factors influence the engraftment of allogeneic hematopoietic stem cells including host immune status, histocompatibility, hematopoietic """"""""space"""""""", stem cell dose, and subsets and doses of T cells within the allograft. Host immune status has not been emphasized, relative to allogeneic stem cell engraftment, when myeloablative conditioning regimens are utilized for allogeneic HSCT, as they markedly attenuate the HVG response that mediates graft rejection. However, when non-myeloablative and reduced-intensity conditioning regimens are utilized prior to allogeneic HSCT, it has been both observed in murine models and inferred from clinical trials data that engraftment is inversely correlated with host immune status. As there can be significant variability in the immune status of patients being considered for alloHSCT, we surmised that individual patients required different levels of host immune depletion in order to achieve the rapid and complete engraftment of allogeneic hematopoietic stem cells, especially if they were from someone other than an HLA-matched sibling. To address the problem of host immune status variability, we developed a novel treatment approach, referred to as targeted lymphocyte-depletion(TLD). The foundation for the TLD approach is based upon a pre-clinical murine engraftment model developed in the Fowler laboratory (ETIB). In an F1 into parent model, mice received repetitive cycles of fludarabine and cyclophosphamide at sub-clinical doses. This resulted in a significant depletion of circulating T cells with minimal myelosuppression. This level of T cell depletion, equivalent to that seen with lethal doses of myeloablative conditioning, was sufficient to permit the engraftment of fully MHC-mismatched allografts. Based on the these data, we hypothesized there exists a level of host T cells in humans which can be quantified and obtained without myeloablative conditioning that is sufficient to consistently permit the engraftment of allogeneic hematopoietic stem cells. TLD utilizes repetitive cycles of disease-specific immunosuppressive chemotherapy at conventional doses that is administered until a pre-determined level of host immune depletion, as determined by circulating T cell numbers, has been achieved prior to proceeding to reduced-intensity allogeneic stem cell transplantation. The number of TLD cycles is based on reaching a target lymphocyte number; patients with higher lymphocyte counts count required more cycles. As such, TLD provides a personalized approach to the highly variable pre-transplant host immune status in order to abrogate graft rejection and facilitate rapid and full donor chimerism following RIST. The TLD approach has been implemented through a systematic, step-wise progression of studies in clinical settings in which one of the barriers to engraftment (e.g. T cell depletion, HLA-disparity) was increased, while other engraftment variables were kept constant. In our first trial, 99-C-0143, we demonstrated that TLD could result in rapid and sustained complete donor engraftment in patients receiving T-cell replete (TCR) allografts from an HLA matched sibling. However, a significant proportion of patients treated on this study experienced relatively high rates of acute GVHD associated with cyclosporine monotherapy for GVHD prevention. We therefore designed a study, 03-C-0077, of TCR allografts from HLA-matched related donors using TLD and a standard dual-agent GVHD regimen, cyclosporine plus methotrexate.
An aim of this study was to evaluate the relationships between the homeostatic cytokines, interleukin-7 (IL-7) and interleukin 15 (IL-15), with immune depletion, engraftment kinetics, GVHD, and immune reconstitution. Consistent with their homeostatic effects, we observed that IL-7 and IL-15 levels rose from baseline as transplant conditioning induced lymphopenia, and then decreased with lymphocyte recovery. These inverse correlations were strongest within 1 month post-alloHSCT. IL-7 and IL-15 levels varied inversely according to lymphocyte counts after transplant. The IL-7 levels at day +7 (P = 0.01) and day+14 (P = 0.00003) post-transplant were associated with the subsequent development of acute GVHD. Among patients who developed acute GVHD, median IL-7 levels were 25.7 and 20.9 pg/ml at days +7 and +14, respectively. Patients who did not develop acute GVHD had median IL-7 levels of 11.8 and 8.22 pg/ml at days +7 and +14, respectively. Higher IL-7 levels at day+14 were strongly associated with more severe grades of acute GVHD (P<0.0001) Logistic regression analysis confirmed that the IL-7 level at day+14 was the strongest single parameter associated with acute GVHD. Using a cutoff value of 13 pg/ml, the IL-7 level at day+14 predicted the subsequent development of acute GVHD with a sensitivity of 85.7% and specificity of 88.2%. The positive and negative predictive values for this single-variable model were 85.7% and 88.2%, respectively. Based on the above results, we initiated a clinical trial, 07-C-0195 utilizing TCR allografts from HLA-matched volunteer unrelated donors (URD) and the TLD approach to study its effect upon engraftment. The use of stem cells from URD increases the availability of allogeneic cellular therapy to an additional 30-50% of patients;however, reduced-intensity alloHSCT from HLA-matched URD are associated with higher degrees of graft rejection and mixed chimerism due to differences in minor histocompatibility antigens between donor and recipient. Patients on this study are randomized to receive one of two GVHD prophylaxis regimens, which have been reported to have relatively good results in regard to acute GVHD in the URD setting. The first regimen contains alemtuzumab with cyclosporine, and the second regimen constains tacrolimus, methotrexate, and sirolimus. The two regimens work through biologically distinct mechanisms, and their effects upon immune reconstitution, which have not previously been well studied with either regimen, are predicted to be markedly different, regardless of the presence or absence of acute GVHD. This study will provide preliminary data relative to multiple aspects of immune reconstitution, which is important and essential to the understanding of this biology and development of strategies to improve outcomes related to GVHD and immune dysregulation. These studies are a natural extension of our prior work and take advantage of the unique expertise and resources within the laboratories of the ETIB, intramural programs of the NCI, and expertise on CDR3 spectratyping at Hackensack University with whom we have a formal collaboration. Preliminary results from this trial have demonstrated that the TLD approach has results in rapid and complete donor chimerism. based upon these preliminary results we have expanded the eligibility criteria to accept URD with HLA-mismatches to further investigate the TLD in this setting. The preliminary results of 07-C-0195 has also led to the development of a protocol, 09-C-0210, which will investigate the TLD in the setting of umbilical cord blood transplantation. This is a natural progression of this project's overall goal of increasing the general applicability of allogeneic HSCT. The URD and UCB transplant initiatives are an inter-institutional (NCI, NHLBI, and NIAID), interagency (NIH and DOD) and extramural (National Marrow Donor Program) collaboration.
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