Mixed Donor-recipient Chimerism Research Articles

In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation

AbstractStable, mixed-donor–recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient–red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

The range of haploidentical transplant protocols in sickle cell disease: all haplos are not created equally.

The ideal curative therapy for sickle cell disease (SCD) must be applicable across all ages and include individuals with strokes and preexisting heart, lung, and kidney disease. Myeloablative, matched sibling donor hematopoietic stem cell transplant (HCT) for children with SCD has shown excellent outcomes over the past 3 decades but has been restricted due to the limited availability of a human leukocyte antigen-matched sibling donor (10%-15%) and increased treatment-related death in adults with myeloablative conditioning. To overcome these 2 significant barriers to curative therapy in SCD, related haploidentical HCT has become an active area of research. The use of related haploidentical donors (first- and second-degree relatives) increases the donor pool to at least 90% of those eligible across the life span. Importantly, most adults, even with strokes or significant comorbidities, can tolerate the nonmyeloablative conditioning regimen without treatment-related death. Since 2013, at least 3 related haploidentical HCT strategies have emerged as potential curative therapies for SCD: (1) a nonmyeloablative, T-cell replete, bone marrow transplant with thiotepa and posttransplant cyclophosphamide with a goal of complete donor chimerism; (2) a nonmyeloablative, in vivo T-cell depletion, using peripheral blood stem cells (PBSCs) with a goal of stable mixed donor-recipient chimerism; and (3) a myeloablative, ex vivo T-cell depletion using PBSCs and advanced-technology graft manipulation, with a goal of complete donor chimerism. We review the similarities, differences, outcomes, and gaps in knowledge with these 3 haploidentical HCT approaches for SCD.

BEAM-Campath Allogeneic Stem Cell Transplant for Patients with Relapsed/Refractory Lymphoma: High Incidence of Long-Term Mixed Donor-Recipient Chimerism and the Response to Donor Lymphocyte Infusions

BiCNU (carmustine), etoposide, Ara-C, melphalan (BEAM) and Campath conditioning was developed to reduce the high transplant-related mortality in patients with lymphoma while delivering intensive antilymphoma immunotherapy, as well as to some extent a platform for allogeneic stem cell engraftment. Significant numbers of patients appeared to have persistent recipient-derived hematopoiesis, and therefore we retrospectively analyzed patients with lymphoma undergoing BEAM-Campath conditioned allogeneic stem cell transplantation at our center (2003 to 2017) to characterize the patterns of chimerism and patient outcomes. Chimerism was analyzed with short tandem repeat PCR. Mixed donor-recipient chimerism (MDRC) was defined as 5% to 94.9% donor. Fifty-two patients (n=30 male), with a median age of 45 years, were identified with histologic diagnoses of Hodgkin lymphoma (n=13), diffuse large B cell lymphoma (n=7), low-grade non-Hodgkin lymphoma (n=16), mantle cell lymphoma (n=10), and T cell lymphoma (n=6). Pretransplant, 93% achieved complete response (52%) or partial response (41%) with a median of 3 prior therapies (n=3 prior autologous stem cell transplantation). Donors were Matched sibling donors (MSD) (n=21), matched unrelated donors (MUD) (n=24), miss-matched unrelated donors (MMUD) (n=6), and syngeneic (n=1). Acute graft-versus host disease (GVHD) developed in 52% (81% grade I to II) and chronic GVHD (83% extensive) in 12%. MDRC of T cells (MDRCt) developed in 62% (n=32), and 29% (n=15) developed MDRC of myeloid cells (MDRCm) at a median onset of 100 days. Donor lymphocyte infusion (DLI) was given to 17 patients, with a median starting dose of 1×106/kg. The first DLI was given at a median of 225 days post-transplant (range, 99 days to 5.3 years). Of these, 9 developed acute post-DLI GVHD and 2 limited chronic GVHD. Conversion to full donor occurred in 47% MDRCt and 50% MDRCm. Multivariate analysis identified sibling donor type as associated with increased MDRCt (P=.035; hazard ratio [HR], 0.17) and reduced total nucleated cell dose with increased MDRCm (P=.021; HR, 0.76). The median follow-up was 6 years, and 2-year NRM cumulative incidence was 16% (95% confidence interval [CI], 7% to 27%). Ten-year progression and extensive GVHD-free survival was 45% (95% CI, 28% to 61%), and overall survival was 66% (95% CI, 50% to 78%). One-year landmark analysis identified no increased GVHD or relapse risk with MDRCt or MDRCm but reduced nonrelapse mortality (NRM) risk with MDRCt (P=.001). BEAM-Campath allografts for high-risk lymphoma achieve long-term disease-free survival with low rates of GVHD and transplant-related mortality. The frequent development of myeloid MDRC demonstrates that BEAM-Campath is a nonmyeloablative conditioning regimen in almost a third of patients. MDRCt is associated with reduced NRM, but neither MDRCt or MDRCm is associated with increased GVHD or relapse.

Efficacy and Toxicity of Donor Lymphocyte Infusions in the Management of Mixed Donor-Recipient Chimerism Following Allogeneic Stem Cell Transplantation for Lymphoid Malignancies:- a Study of the LWP-EBMT

Efficacy and Toxicity of Donor Lymphocyte Infusions in the Management of Mixed Donor-Recipient Chimerism Following Allogeneic Stem Cell Transplantation for Lymphoid Malignancies:- a Study of the LWP-EBMT

Allogeneic Stem Cell Transplantation after Fanconi Anemia Conditioning in Children with Ataxia-Telangiectasia Results in Stable T Cell Engraftment and Lack of Infections despite Mixed Chimerism

Ataxia-telangiectasia (A-T) syndrome is an autosomal recessive chromosomal breakage syndrome caused by mutation of the ataxia-telangiectasia mutated gene manifested by progressive neurodegeneration, telangiectasias of sclera and skin, immune deficiency with sinopulmonary infections, and increased incidence of lymphoid malignancies and solid tumors. Three children with A-T underwent allogeneic stem cell transplantation (SCT) using protocols for Fanconi anemia. All 3 patients were engrafted with a mixed donor–recipient chimerism, but the full donor engraftment was observed in the T lymphocyte compartment. Immunologic recovery resulted in T cell production and lack of symptomatic infections. Regular intravenous immunoglobulin supplementation was needed until IgG production recovered, which depended on pretransplant serotherapy. During the observation period patients did not require hospital admission, and none of the transplanted patients developed sinopulmonary infections. Neurologic functions in reported patients were impaired and slowly deteriorated after transplantation, but no immediate toxicities were observed. The following hallmark features of A-T were present after SCT: neurologic symptoms, growth failure, telangiectasia formation, or increased serum alpha fetoprotein. SCT can help control immune deficiency constituting 1 of the features of A-T, and elimination of autologous hematopoiesis reduces the risk of lymphoid malignancies. Resolving crucial problems with qualification for SCT depends on balancing the risk and benefits of transplant therapy.

Relationship between Mixed Donor–Recipient Chimerism and Disease Recurrence after Hematopoietic Cell Transplantation for Sickle Cell Disease

Mixed donor chimerism after hematopoietic cell transplantation for sickle cell disease (SCD) can result in resolution of disease symptoms, but symptoms recur when donor chimerism is critically low. The relationship between chimerism, hemoglobin S (HbS) level, and symptomatic disease was correlated retrospectively in 95 patients who had chimerism reports available at day 100 and at 1 and 2 years after transplantation. Recurrent disease was defined as recurrence of vaso-occlusive crises, acute chest syndrome, stroke, and/or HbS levels > 50%. Thirty-five patients maintained full donor chimerism (myeloid or whole blood) through 2 years. Donor chimerism was less than 10% (defined as graft failure) in 13 patients during this period. Mixed chimerism was reported in the remaining 47 patients (range, 10% to 94%). The lowest documented donor chimerism without symptomatic disease was 26%. Of 12 surviving patients with recurrent disease, 2 had recurrence of symptoms before documented graft failure (donor chimerism of 11% and 17%, respectively). Three patients underwent second transplantation for graft failure. None received donor leukocyte infusion to maintain mixed chimerism or prevent graft failure. We conclude stable donor chimerism greater than 25% is associated with resolution of SCD-related symptoms, and HbS levels in transplant recipients should be interpreted in context of the sickle trait status of the donors.

Successful Allogeneic Hematopoietic Stem Cell Transplantation in XIAP Deficiency Using Reduced‐Intensity Conditioning

Hematopoietic stem cell transplantation (HSCT) is currently the only available curative therapy for X-linked inhibitor of apoptosis (XIAP) deficiency. Myeloablative conditioning regimens are associated with high mortality rates. Reduced-intensity conditioning (RIC) is recommended in order to decrease treatment-related toxicities, but RIC regimens increase the risk for mixed donor-recipient chimerism that may progress to graft loss. We report our experience with a patient with XIAP deficiency who was successfully treated with allogeneic HSCT using a RIC protocol. Post-transplant chimerism was vigilantly monitored and maintained with donor lymphocyte infusions and a stem cell boost to a level that prevented hemophagocytic lymphohistiocytosis recurrence.

Mixed chimerism and graft loss in pediatric recipients of an alemtuzumab-based reduced-intensity conditioning regimen for non-malignant disease.

Reduced-intensity conditioning (RIC) regimens can mitigate the toxicity of hematopoietic cell transplantation (HCT) in children with non-malignant diseases, but are associated with increased risk for post-transplant mixed donor/recipient chimerism (MC) and/or graft loss (GL). Intervention with donor lymphocytes or stem cell boosts (DLI/boost) may be necessary, but there is limited information about timing and results of intervention. We retrospectively evaluated 31 consecutive pediatric recipients of an alemtuzumab-based RIC HCT at the Children's Hospital of Philadelphia from May 2007 to December 2012 to determine the incidence of MC, GL, and use of DLI/boost. All patients received alemtuzumab with either fludarabine (150 mg/m(2) )/melphalan (140 mg/m(2) ) (n = 30) or fludarabine/busulfan (n = 1), and unmanipulated marrow grafts from related (48%) or matched unrelated (52%) donors. Of surviving patients, 67% and 44% displayed MC and MC with ≤80% donor contribution (MC ≤ 80%), respectively. Rates of MC, MC ≤ 80%, DLI/boost, and GL were significantly higher in recipients of proximal/intermediate (100%, 73%, 46%, and 46%, respectively) compared to distal alemtuzumab (44%, 25%, 6%, and 6%, respectively). Event-free and overall survival was significantly lower in HLH compared with non-HLH patients. Twenty percent of patients required DLI/boost, and DLI/boost did not affect the incidence of GL. RIC with proximal/intermediate alemtuzumab is associated with high rates of MC, need for DLI/boost, and GL.

Orak Hücre Anemisinde Hematopoetik Kök Hücre Transplantasyonu

Sickle cell anemia is one of the most common hemoglobinopathies in the worldwide. Sickle cell anemia characterized by crises and organ failure develops over time. Myeloablative stem cell transplantation is curative but it has been performed in children younger than 16 years of age. Modest modifications in the conditioning regimen and supportive care have improved outcome such that the majority of children with a suitable HLA-matched sibling donor can expect a cure from this approach. But nonmyeloablative protocols are crucial for the future of Hematopoietic Stem-Cell Transplantation for older sickle cell anemia patients with organ failure. A protocol for nonmyeloablative allogeneic hematopoietic stem-cell transplantation that includes total-body irradiation and treatment with alemtuzumab and sirolimus can achieve stable, mixed donor–recipient chimerism. Stem cell transplantation is recommended in the presence of HLA-matched siblings in patients at risk.

Mixed Chimerism and Graft Loss in Pediatric Recipients of an Alemtuzumab-Based Reduced-Intensity Conditioning Regimen for Nonmalignant Disease

Reduced-intensity conditioning (RIC) regimens are increasingly used to reduce transplant-related mortality (TRM) for pediatric patients (pts) undergoing hematopoietic cell transplantation (HCT) for nonmalignant diseases. However, these pts are at increased risk for post-transplant mixed donor/recipient chimerism (MC) and/or primary and secondary graft failure (GF). Intervention with donor lymphocytes (DLI) or second transplants may be necessary, but there is limited information about timing and results of intervention. We retrospectively evaluated 31 consecutive pediatric recipients of an alemtuzumab-based RIC HCT at the Children’s Hospital of Philadelphia from May 2007 to December 2012 to determine the incidence of MC and GF, and timing of interventions. All pts received alemtuzumab (Campath) with either fludarabine (150mg/m2)/melphalan (140 mg/m2) (n=30) or fludarabine/busulfan (n=1). The timing of Campath differed according to disease. Pts with hematologic disorders and IPEX received Campath beginning day -21 or -22. Pts with hemophagocytic lymphohistiocytosis (HLH) received proximal (N=8) or intermediate (n=2 ) Campath starting at day -9 or -13, respectively. Diagnoses included HLH in 10 pts, hemoglobinopathy in 8 pts, primary immunodeficiency in 8 pts, and bone marrow failure in 5 pts. All grafts were unmanipulated bone marrow from matched sibling donors (45%), matched unrelated donors (52%) or mismatched related donor (3%). Donor chimerism was evaluated using polymerase chain reaction of microsatellite markers/short tandem repeats at regular intervals post-transplant. Four pts died of TRM (n=3) or early disease progression (n=1) and were not included in the analysis. Of the 27 surviving pts, 18 (66.7%) displayed MC (donor chimerism <100%) on at least one occasion, with 12 (44.4%) reaching a nadir MC level <80%. Two pts with MC≥80% experienced recurrent disease, one with HLH and one with auto-immune syndrome. Of the 12 pts with nadir MC<80%, 6 received DLI with 2 subsequent GFs. Of the remaining 6 pts who did not receive DLI, there were also 2 GFs. The overall incidence of GF/recurrence was 22.2% (6/27). Within the HLH group 7/7 surviving pts developed MC, with 5 (71.4%) reaching a nadir <80%. Despite DLI/boost in all 5 of these pts, 2 progressed to GF. The incidence of GF/recurrence in the surviving HLH pts was 42.9% (3/7) By contrast, in the non-HLH pts, only 11 of 20 (55.0%) surviving pts developed MC with 7 (35.0%) reaching a nadir<80%. Of these 7 with MC<80%, 2 progressed to GF for an overall incidence of GF/recurrence of disease of 15% (3/20). In summary, we observed a high incidence of MC and GF in recipients of Campath-based RIC, particularly in HLH pts. The role for DLI/boost in preventing GF in pts receiving RIC HCT for non-malignant disease who experience low/falling MC requires further study.

Increased Relapse Associated with Mixed Donor-Donor Chimerism Following Double Umbilical Cord Blood Transplantation (dUCBT)

AbstractAbstract ▪358▪This icon denotes a clinically relevant abstractWe previously showed lower rates of relapse in recipients of dUCBT, compared to patients treated with single UCBT (Verneris, Blood, 2009). One unit predominates by D+100 in ∼90% of patients and recent studies show that single unit dominance is associated with T cell recognition of the rejected unit (Gutman, Blood, 2010). In addition, higher rates of acute graft-vs-host disease (aGVHD) have been observed after dUCBT compared to single UCBT, regardless of age (MacMillian, Blood, 2009). Thus, we hypothesized that graft vs. graft interactions that occur during dUCBT may drive both GVL and GVHD reactions. To test this hypothesis, we reviewed the records of patients with hematological malignancies transplanted with a dUCBT from 2000 to 2011 at the University of Minnesota. Myeloablative (MA) conditioning was with cyclophosphamide (Cy 120 mg/kg), fludarabine (Flu 75 mg/m2) and total body irradiation (TBI 13.2 Gy) (n=178) and non-MA conditioning was with Cy (50 mg/kg), Flu (200 mg/m2) and TBI (2 Gy) (n=282). Acute GVHD prophylaxis consisted of cyclosporine and mycophenolic acid. At D+100 316 patients were evaluable and had either BM (n=273) and/or PB (n=178) donor engraftment data. Engraftment was assessed using short tandem repeat analysis. “Mixed donor-recipient chimerism” was defined as >5% host cells and “dual chimerism” was defined as complete donor chimerism (donor 1 + donor 2), with a minimum contribution of 5% by each unit (5% is the lower limit of detection for this clinical test). As expected, patients with evidence of mixed donor-recipient chimerism in the blood or marrow at D+100 (n=35) had high rates of relapse (69% [95% CI, 49–89%] at 2 years) and were excluded from further analysis. Of the patients with evaluable peripheral blood chimerism data, 90.4% (n=161) had hematopoiesis derived from a single unit, while 9.6% (n=17) showed dual chimerism after dUCBT. In multivariate analysis, dual chimerism at D+100 was associated with higher relapse (HR=1.97 [95% CI, 1.08–3.59], p=0.01), less grade II-IV aGVHD (HR=0.36 [95% CI, 0.17–0.78], p=0.01) and a trend toward worse DFS (HR=1.68 [95% CI, 0.9–3.13], p=0.1). Dual chimerism was not associated with age (p=0.32), nor non-MA conditioning (p=0.15). Recipients of two HLA 6/6 matched units (with each other and the recipient), were more likely to have dual chimerism (p<0.01). Nearly identical results were obtained in a separate model examining BM engraftment (single (n=247) and dual chimerism (n=26)) and transplant outcomes. While these findings should be confirmed in a separate dataset, they suggest that an unexpected consequence of selecting well matched units for dUCBT is higher mixed donor-donor chimerim, less aGVHD and more relapse. Further study of dUCBT and GVL is needed. Disclosures:No relevant conflicts of interest to declare.

Fetal hemoglobin reactivation and cell engineering in the treatment of sickle cell anemia

The natural history of severe hemoglobinopathies like sickle cell disease (SCD) is rather variable, depending on the circumstances, but the main influence on such variability is the level of fetal hemoglobin (HbF) in the patient’s red cells. It is well known that a significant HbF level is associated with a milder course of disease and fewer complications. Therefore, attempts have been made to reactivate using various means the HbF production, which is normally switched off perinatally. A pharmacological approach has been attempted since the 1980s, ranging from drugs like 5-azacytidine and its derivative, decitabine, to a series of compounds like hydroxyurea and a number of histone deacetylase inhibitors like butyrate, which seem to act as epigenetic modifiers. Many other disparate agents have been tried with mixed results, but hydroxyurea remains the most effective compound so far available. Combinations of different compounds have also been tried with some success. Established treatments like bone marrow or cord blood transplantation are so far the only real cure for a limited number of patients with severe hemoglobinopathies. Improved chemotherapy regimens of milder toxicity than those employed in the past have made it possible recently to obtain a stable, mixed donor-recipient chimerism, with reversal of the SCD phenotype. However, great effort is directed to cell engineering, searching for an effective gene vector by which a desired gene can be transferred into new classes of vectors for autologous hemopoietic stem cells. Recent studies are also aiming at targeted insertion of the therapeutic gene into hemopoietic cells, which can also be “induced” human stem cells, obtained from somatic dedifferentiated cells. Attention in this area must be paid to the possibility of undesired effects, like the emergence of potentially oncogenic cell populations. Finally, an update is presented on improved HbF determination methods, because common international standards are becoming mandatory.

Allogeneic Hematopoietic Stem-Cell Transplantation for Sickle Cell Disease

Myeloablative allogeneic hematopoietic stem-cell transplantation is curative in children with sickle cell disease, but in adults the procedure is unduly toxic. Graft rejection and graft-versus-host disease (GVHD) are additional barriers to its success. We performed nonmyeloablative stem-cell transplantation in adults with sickle cell disease. Ten adults (age range, 16 to 45 years) with severe sickle cell disease underwent nonmyeloablative transplantation with CD34+ peripheral-blood stem cells, mobilized by granulocyte colony-stimulating factor (G-CSF), which were obtained from HLA-matched siblings. The patients received 300 cGy of total-body irradiation plus alemtuzumab before transplantation, and sirolimus was administered afterward. All 10 patients were alive at a median follow-up of 30 months after transplantation (range, 15 to 54). Nine patients had long-term, stable donor lymphohematopoietic engraftment at levels that sufficed to reverse the sickle cell disease phenotype. Mean (+/-SE) donor-recipient chimerism for T cells (CD3+) and myeloid cells (CD14+15+) was 53.3+/-8.6% and 83.3+/-10.3%, respectively, in the nine patients whose grafts were successful. Hemoglobin values before transplantation and at the last follow-up assessment were 9.0+/-0.3 and 12.6+/-0.5 g per deciliter, respectively. Serious adverse events included the narcotic-withdrawal syndrome and sirolimus-associated pneumonitis and arthralgia. Neither acute nor chronic GVHD developed in any patient. A protocol for nonmyeloablative allogeneic hematopoietic stem-cell transplantation that includes total-body irradiation and treatment with alemtuzumab and sirolimus can achieve stable, mixed donor-recipient chimerism and reverse the sickle cell phenotype. (ClinicalTrials.gov number, NCT00061568.)

Reduced-Intensity Conditioning Regimen Workshop: Defining the Dose Spectrum. Report of a Workshop Convened by the Center for International Blood and Marrow Transplant Research

During the 2006 BMT Tandem Meetings, a workshop was convened by the Center for International Blood and Marrow Transplant Research (CIBMTR) to discuss conditioning regimen intensity and define boundaries of reduced-intensity conditioning (RIC) before hematopoietic cell transplantation (HCT). The goal of the workshop was to determine the acceptance of available RIC definitions in the transplant community. Participants were surveyed regarding their opinions on specific statements on conditioning regimen intensity. Questions covered the “Champlin criteria,” as well as operational definitions used in registry studies, exemplified in clinical vignettes. A total of 56 participants, including transplantation physicians, transplant center directors, and transplantation nurses, with a median of 12 years of experience in HCT, answered the survey. Of these, 67% agreed that a RIC regimen should cause reversible myelosuppression when administered without stem cell support, result in low nonhematologic toxicity, and, after transplantation, result in mixed donor–recipient chimerism at the time of first assessment in most patients. Likewise, the majority (71%) agreed or strongly agreed that regimens including < 500 cGy of total body irradiation as a single fraction or 800 cGy in fractionated doses, busulfan dose < 9 mg/kg, melphalan dose <140 mg/m 2, or thiotepa dose < 10 mg/kg should be considered RIC regimens. However, only 32% agreed or strongly agreed that the combination of carmustine, etoposide, cytarabine, and melphalan (BEAM) should be considered a RIC regimen. These results demonstrate that although HCT professionals have not reached a consensus on what constitutes a RIC regimen, most accept currently used criteria and operational definitions. These results support the continued use of current criteria for RIC regimens until a consensus statement can be developed.

CD8+ T-Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti-CD154 mAb, nondepleting anti-CD4 combined to either depleting or nondepleting anti-CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long-term allograft survival in both combinations. Nevertheless, robust donor-specific tolerance, defined by the acceptance of a fresh donor-type skin allograft and simultaneous rejection of third-party grafts, required initial CD8(+) T-cell depletion. Mixed donor-recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL-2, IFN-gamma and TNF-alpha in MLC with donor antigen while significant alloreactivity persisted against third- party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor-recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8(+) T-cell depletion and costimulation/coreceptor blockade.

Feasibility of conditioning with thymoglobulin and reduced intensity TBI to reduce acute GVHD in recipients of allogeneic SCT

Murine studies using anti-T-cell antibodies for conditioning in allogeneic SCT demonstrate engraftment with low rates of GVHD. On the basis of this preclinical model, we conditioned 30 patients with advanced hematologic malignancies with rabbit antithymocyte globulin (ATG) and TBI, to reduce rates of fatal acute GVHD. Patients were enrolled in two sequential groups: cohort 1 received ATG 10 mg/kg in divided doses (days −4 to −1)+200 cGy TBI (n=16), and cohort 2 received ATG (days −10 to −7)+450 cGy TBI (n=14). Median donor blood chimerism for the combined group was 94, 93 and 93% in the first, second and third months after transplant. Only three developed grade II acute GVHD despite 43% of patients receiving unrelated donor transplants. One-year survival was 71±11 and 54±14%, respectively, in recipients of related and unrelated donor SCT. Donor lymphocyte infusions were needed in 12 patients for the management of relapse and for mixed donor–recipient chimerism in 4 patients. We conclude that 10 mg/kg ATG and TBI allows engraftment with a low risk of acute GVHD; however, further dose optimization of ATG is required to achieve a balance between GVHD and disease relapse.

Immunoablative Chemotherapy before Reduced-Intensity Allogeneic Stem Cell Transplantation: A Strategy to Provide Disease Control and to Enhance Early Graft-Versus-Lymphoma Effects.

Published data raise concerns about the efficacy of reduced-intensity allogeneic hematopoietic stem cell transplantation (RIST) in chemotherapy-resistant lymphomas. Reduced-intensity conditioning, compared with myeloablative regimens, has attenuated cytotoxic antitumor activity and can result in mixed donor-recipient chimerism, which may diminish potential graft-versus-lymphoma (GVL) activity after RIST. To compensate for these limitations, we prospectively studied a novel, conventional-dose chemotherapy regimen (EPOCH-F/R: etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and fludarabine; +rituximab for CD20+ lymphomas) designed to provide both disease control and host T-lymphocyte depletion before RIST, and to enhance early GVL effects by facilitating rapid and complete donor engraftment after RIST. The objectives of this study were to determine the toxicity, immune depletion, and antitumor activity of EPOCH-F/R in pts with relapsed/refractory lymphoid malignancies. EPOCH-F/R was administered until blood CD4 level < 100/μL to a maximum of 3 cycles. If disease progression occurred during EPOCH-F/R, the regimen was discontinued and pts proceeded to RIST, regardless of CD4 count. Twenty-one pts were enrolled (median age 52 years; range 31–71). Diagnoses included Hodgkin's lymphoma (n=4) and NHL (n=17): DLBC=5, follicular=4 (3 transformed), CLL/SLL=3, mantle cell=1, T-cell=4. Prior regimens (median 4, range 1–6) included anthracyclines (90%), fludarabine (29%), rituximab (67%), and autologous SCT (43%). Sensitive and refractory disease was present at enrollment in 62% and 29%, respectively. Pts received EPOCH-F/R for 1 (n=8), 2 (n=6), or 3 (n=7) cycles; 15 pts received rituximab with the regimen. Toxicity in 41 cycles of EPOCH-F/R included grade 4 thrombocytopenia (34%), grade 4 neutropenia (85%), and neutropenic fever/infection (21%). Grade 3/4 non-hematologic toxicity occurred in 28 cycles but was generally brief and reversible; no treatment-related deaths occurred. Median (range) CD4 before and after EPOCH-F/R were 165/μL (4–1,098) and 72/μL (4–380), respectively. Similarly, CD8 before and after EPOCH-F/R were 189/μL (1–1,763) and 42/μL (1–1,195), respectively. Overall response rate was 34% with 2 CR, 5 PR, 12 SD, and only 2 PD. Response to EPOCH-F/R was not associated with aggressive vs. indolent histology, sensitivity to prior treatment, specific prior therapies, or inclusion of rituximab with the induction regimen. All 22 pts proceeded as planned to reduced-intensity conditioning with fludarabine/cyclophosphamide and T-cell replete peripheral blood stem cell transplantation from a matched sibling. All pts engrafted, and 16/21 pts achieved > 95% donor myeloid and lymphoid chimerism by day 28 post RIST (median 100%). Thus, EPOCH-F/R achieved profound host immune depletion and provided disease stability with acceptable toxicity prior to RIST, setting the stage for enhanced potential GVL effects after RIST. Incorporation of an immunoablative chemotherapy regimen such as EPOCH-F/R into RIST strategies for resistant lymphomas may be considered an alternative to autologous SCT in preparation for RIST.

Follow-up of chimerism, including T- andB-lymphocytes and granulocytes in children more than one year after allogeneic bone marrow transplantation

In bone-marrow-transplanted children, early detection of graft failure, relapse, and other potentially treatable problems is facilitated by the use of polymerase chain reaction (PCR) assays that monitor whether blood and marrow cells are of recipient or donor origin. Presence of mixed donor-recipient chimerism (MC) within the first year after BMT frequently correlates with clinical problems. To study if MC detected one year or more post-BMT was also often associated with clinical problems, the chimeric status in 33 children surviving 1-11 yr (median: 2 yr) after BMT was investigated. A PCR with a sensitivity of 1-2%, using fluorescent primers analyzing DNA fragment length polymorphisms, was applied. T- and B-cells and granulocytes were immunomagnetically isolated and tested separately for all patients. Of the 33 patients, of whom 21 had received pretreatment including total body irradiation (TBI), 27 (82%) exhibited full donor chimerism. Six children (18%), four of whom had received pretreatment without TBI, had MC. In three of these children, all with aplastic anemia, isolated T-cell MC had not posed apparent clinical problems. In two patients, both with MC including B-cells, immune hemolytic anemia was observed. A sixth patient with AML presented with MC and relapse. In two of the six children MC was detected only by cell subset analysis. In conclusion, analysis of MC in leukocyte subsets is more informative than analysis of whole blood only and may reveal clinically important variations in the origin of different cell populations. The prevalence of MC is lower after the first year post-BMT, and when present is less often associated with clinical problems.