Durable Engraftment Research Articles

Matched Unrelated Allogeneic Stem Cell Transplantation for Patients with Congenital Amegakaryocytic Thrombocytopenia: Texas Children`s Hospital Experience

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by decreased or absent numbers of megakaryocytes and is not associated with congenital malformations. It is an autosomal recessive disorder with mutations in the thrombopoietin receptor c-MPL, presenting at birth with severe isolated thrombocytopenia. Given the increased risk of life threatening hemorrhage, close monitoring and supportive care with regular platelet transfusions is usually required. The severity of the MPL mutation may predict the clinical course of children with CAMT. Null mutations may rapidly progress to pancytopenia and aplastic anemia, while more modest functional loss of receptor function cause a transient increase in platelet count to > 50,000/µL during the first year of life with later progression to pancytopenia. Moreover, like other IBMFSs, CAMT has been referred to as a cancer predisposition syndrome. Allogeneic hematopoietic stem cell transplant (HSCT) offers the only curative option.We present our institutional experience of three patients with CAMT who underwent matched unrelated HSCT early in the course of the disease when their presenting problem was isolated thrombocytopenia without pancytopenia, marrow failure or clonal evolution. We have used a fully ablative regimen with busulfan 1 mg/kg/dose for 16 doses (days -9, -8, -7, and -6), cyclophosphamide 50 mg/kg/dose for 4 doses (days -5, -4, -3, and -2) and alemtuzumab 3 mg/day (weight-based dosage) for 4 doses (days -5, -4, -3, and -2). Cyclosporine and mini methotrexate (on days +1, +3, +6 and +11) were given for GVHD prophylaxis.The first two patients were siblings with persistent thrombocytopenia at birth, the first of whom had compound heterozygous mutations (c.256dupC and c.391+5 G>C) in the MPL gene. Both parents were carriers and the second sibling was diagnosed prenatally with the same mutations. No other phenotypic abnormalities were noted and testing for Fanconi anemia was negative. The siblings were transplanted with matched unrelated donors at 12 months and 14 months respectively.Our third patient was diagnosed prenatally with germinal matrix hemorrhage and vetriculomegaly. He was noted to have thrombocytopenia after birth. He was treated initially for presumed neonatal alloimmune thrombocytopenia. Sequencing of the MPL gene revealed two compound heterozygous missense mutations (R257C and R102P). The patient was transplanted with a matched unrelated donor at the age of 11 months.All patients tolerated the transplant with minimal toxicity, durable engraftment, and no acute or chronic GVHD. The first two siblings are approximately 4 years and 2 years post HSCT and the third patient is past day 100.Previous reports of HSCT for CAMT that used matched unrelated donors recorded poor outcomes, with a high rate of graft failure. Although our current study is small in size, the results suggest that a HSCT following a fully ablative regimen containing alemtuzumab and performed early in the course of the disease may produce better outcomes, and will avoid the complications associated with marrow failure and clonal abnormalities. DisclosuresAllen:Roche: Consultancy, Other: unpaid; NovImmune: Consultancy, Other: unpaid. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Brenner:Celgene: Other: Collaborative Research Agreement; Cell Medica: Other: Licensing Agreement; Bluebird Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Excellent Engraftment and Long-Term Survival in Patients with Severe Aplastic Anemia (SAA) Undergoing Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) with Haplo-Identical CD34+ Cells Combined with a Single Umbilical Cord Blood Unit

For SAA patients (pts) lacking an HLA identical donor, outcomes of HSCT using single or dual unrelated cord blood (UCB) transplants or haplo-identical donors have been associated with high graft failure rates and poor survival. We investigated whether co-infusion of a single UCB unit with CD34+ selected cells from a haplo-identical relative following a highly immunosuppressive conditioning could improve transplant outcome for this high-risk population.Methods: Eligible pts for HSCT were required to be between the ages of 4-55, have a diagnosis of SAA or SAA evolved to MDS, be unresponsive to immunosuppressive therapy, have severe neutropenia (ANC<500 cells/µL), lack an HLA-matched donor, have an available haploidentical family member, and at least one ≥ 4/6 HLA-matched UCB unit with a minimum TNC dose ≥1.5x107 cells/kg. Pts received conditioning with cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine-ATG (160 mg/kg) and 200 cGy of total body irradiation. On Day 0, pts received a CD34-selected (Miltenyi CliniMacs) G-CSF mobilized haplo-donor allograft combined with a single UCB. Tacrolimus and MMF were used for GVHD prophylaxis. Results: 21 pts (median age: 19.6 yrs, range: 4.5-48.6) including 14 with SAA and 7 with SAA that evolved to early stage MDS (< 5% blasts) underwent haplo-cord transplant. 14 pts received 4/6, and seven pts received a 5/6 HLA-matched UCB unit. UCB units contained a median 3.77x107 TNCs/kg (range: 1.96-6.93), a median 1.54x105 CD34+ cells/kg (range 0.46-3.45), and the haplo-graft contained a median 3.2x106 CD34+ cells/kg (range: 3.0-4.1) and a median 0.95x103 CD3+ cells/kg (range 0.3-5.04). All 21 pts engrafted, which was sustained and associated with transfusion independence in 20/21 pts. At a median follow-up of 3 yrs (1116 days, range: 226-2572), 19/21 pts survive for an overall survival rate of 90%. 2 pts died at day 414 and 402 post-HSCT from viral related complications (CMV pneumonitis and limbic encephalopathy). Neutrophil and platelet recovery occurred at a median 10 (range: 9-22), and 30 (range: 16-129) days respectively. Cord myeloid engraftment (cord ANC>500 cells/µl, calculated from chimerism data) occurred in 18/21 pts at a median 42 days while 3/21 did not achieve a cord ANC>500 but had haplo-donor engraftment which was sustained in 2 cases. The cumulative incidence of grade II-IV acute GVHD (aGVHD) was 28.6%, with only 1 pt (4.8%) developing grade III and no cases of grade IV or steroid refractory aGVHD. The cumulative incidence of chronic GVHD (cGVHD) was 47.8%, with 37% mild, 11% moderate, and no severe cGVHD. Engraftment profiles showed 16 achieved full cord chimerism in all cell lineages, 2 remained mixed haplo-cord chimeras, and 2 failed to have UCB engraftment but had sustained 100% haplo-donor myeloid chimerism. Higher degrees of HLA matching between recipient and UCB unit were associated with faster rates of cord engraftment (P =0.012) and the achievement of full cord chimerism (P= 0.004). Among the 16 pts who had lower degrees of HLA matching (≤5/8 HLA alleles) between the recipient and UCB unit, 9 received grafts with KIR ligand incompatibility in the haplo vs. cord direction ( defined as the presence of a KIR ligand in the haplo-donor graft that was absent in the UCB unit at HLA epitopes Bw4, HLA-C Groups 1 & 2, HLA-A3, and HLA-A11); KIR ligand incompatibility had a negative impact on cord myeloid engraftment as 6/9 (67%) of these pts failed to achieve full cord myeloid chimerism by day 400 and 5/9 (56%) pts failed to achieve a cord ANC>500 by day 250 post-HSCT. In contrast, cord myeloid engraftment occurred quicker in the 12 pts who did not have haplo vs. cord KIR ligand incompatibility including 7 pts with lower degrees of HLA matching (≤5/8 HLA alleles between the recipient and UCB) and 5 pts with higher degrees of HLA matching, where the median times to achieve a cord ANC>500 and full cord myeloid chimerism was 42 days (range: 28-58) and 60 days (range: 14-230) respectively ( both P<0.01 compared to the pts that received grafts with KIR ligand incompatibility). Conclusion: Transplantation of haplo-identical CD34+ cells with a single UCB unit in neutropenic pts with SAA and SAA evolved to MDS was associated with rapid neutrophil recovery, durable donor engraftment and excellent long-term survival. These results suggest that haplo-cord HSCT is an effective treatment option associated with excellent survival in pts with SAA who lack an HLA-matched donor. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Improved Outcomes of Haploidentical Blood and Marrow Transplantation in Hematologic Malignancies: A Single Center Study of 514 Cases

Introduction: With GIAC regimen, haploidentical blood and marrow transplantation (haplo-BMT) has achieved comparable outcomes with identical sibling transplant (Dao-Pei Lu et al., Blood 2006; 107:3065). Our previous study has shown that the third party cell co-infusion in haplo-BMT (GIAC-3 regimen) could significantly reduce aGVHD and transplant-related mortality (TRM). We have also demonstrated that individualized chemotherapy to decrease leukemia burden followed by conditioning could improve disease-free survival (DFS) in refractory/relapsed AML.Objective: To learn the outcomes of our haplo-BMT with these integrated approaches, all patients who received haplo-BMT for hematologic malignancies in our center were analyzed retrospectively.Methods: Between April 2012 and December 2014, consecutive 514 patients with hematologic malignancies who underwent haplo-BMT were included. The median age was 20 (1.8 to 64) years old. The diagnosis included AML 232 (45.1%), ALL 207 (40.3%), MDS 27(5.3%), CML 14 (2.7%), lymphoma 13 (2.5%) and others 21 (4.1%). Transplants at CR1, ≥CR2 or advanced disease were 216 (42.0%), 114 (22.2%), 184 (35.8%), respectively. All patients received unmanipulated bone marrow (BM) and peripheral blood stem cells as graft after myeloablative conditioning plus ATG. Majority of the patients with AML received BuCy-based conditioning, while most ALL patients received TBICy-based regimen. Fludarabine was substituted for cyclophosphamide in some patients due to impaired organ function or high tumor burden. For refractory/relapsed diseases, individualized chemotherapy followed by conditioning was administered. Cyclosporine/tacrolimus, short-term Methotrexate, and Mycophenolate mofetil were employed for GVHD prophylaxis. Either 1ml/kg (recipient's body weight) haploidentical BM from the second haploidentical donor or one unit of unrelated cord blood was infused right after haplo-BMT as the third party cells. Minimal residual disease (MRD) was monitored routinely by quantitative PCR or flow cytometry. The patients with persistent MRD were interfered by immunosuppressant withdrew, adoptive immunotherapy with cytokine induced killer or NK cells or donor lymphocyte infusion.Results: All patients but 5 achieved durable engraftment. The cumulative incidences of grade II to IV aGVHD and grade III to IV aGVHD were 32.2%, 19.8%, respectively. The cumulative incidences of cGVHD and extensive cGVHD were 48.3%, 18.4%, respectively. 100-day TRM and 2-year TRM were 4.1%, 14.9%, respectively. Two-year relapse rate was 22.8%. With the median follow up 17 (6 to 38) months, overall 2-year DFS rates in CR1, ≥CR2 and advanced disease were 75.6%, 70.9%, 49.2%, respectively. For AML, two-year DFS rates in CR1, ≥CR2 and advanced disease were 74.1%, 76.9%, 48.2% (CR1 vs. ≥CR2, p=0.84; CR vs. advanced disease, p=0.000). For ALL, two-year DFS rates in CR1, ≥CR2 and advanced disease were 78.9%, 56.6%, 38%, respectively (CR1 vs. ≥CR2, p=0.018; CR1 vs. NR, p=0.000; ≥CR2 vs. NR P=0.02 ).Conclusions: With our strategies, overall outcomes of haplo-BMT have been improved remarkably and very encouraging. Therefore, haplo-BMT should be an important way to save life for the patients with hematologic malignancies who need urgent BMT but without matched either sibling or unrelated donor. DisclosuresNo relevant conflicts of interest to declare.

Unrelated donor hematopoietic stem cell transplantation for the treatment of non-malignant genetic diseases: An alemtuzumab based regimen is associated with cure of clinical disease; earlier clearance of alemtuzumab may be associated with graft rejection.

Hematopoietic stem cell transplantation (HSCT) with matched unrelated donors (MUD), offers potentially curative therapy for patients with non-malignant genetic diseases. In this pilot study conducted from 2006 to 2014, we report the outcomes of 15 patients with non-malignant genetic diseases who received a myeloablative regimen with a reduced cyclophosphamide dose, adjunctive serotherapy and MUD HSCT [intravenous alemtuzumab (52 mg/m(2) ), busulfan (16 mg/kg), fludarabine (140mg/m(2) ), and cyclophosphamide (105 mg/kg)]. Graft-versus-host-disease (GVHD) prophylaxis consisted of tacrolimus/cyclosporine and methylprednisolone. Median (range) time to neutrophil engraftment (>500 cells/µL) and platelet engraftment (>20,000/mm(3) ) were 15 (12-28) and 25 (17-30) days, respectively. At a median follow-up of 2 (0.2-5.4) years, the overall survival (OS) was 93.3% (95% CI: 0.61-0.99) and disease-free survival (DFS) was 73.3% (95% CI: 0.44-0.89). Among this small sample, earlier alemtuzumab clearance was significantly associated with graft rejection (P = 0.047), earlier PHA response (P = 0.009) and a trend toward earlier recovery of recent thymic emigrants (RTE) (P = 0.06). This regimen was associated with durable donor engraftment and relatively low rates of regimen related toxicity (RRT); future alemtuzumab pharmacokinetic studies may improve outcomes, by allowing targeted alemtuzumab clearance to reduce graft rejection and promote more rapid immune reconstitution.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium.

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell.

Low-dose total-body irradiation and alemtuzumab-based reduced-intensity conditioning regimen results in durable engraftment and correction of clinical disease among children with chronic granulomatous disease.

HSCT with MAC is associated with durable donor engraftment for patients with CGD; however, MAC is limited by high rates of RRT. We used a novel RIC regimen with LD-TBI (200 cGy × two doses), fludarabine (30 mg/m(2) × three doses), and proximal alemtuzumab (0.5 mg/kg/dose × one dose) and unrelated donor grafts for consecutive patients with high-risk CGD who were not candidates for MAC at our institution. Among four children with CGD transplanted at our institution, three PBSC recipients are alive with sustained donor engraftment (median follow-up: two yr) and resolution of pre-HSCT active infections while one patient with bone marrow graft is alive after graft failure and autologous recovery. RIC may be a curative option for children with high-risk CGD.

Successfull Engraftment and Clearance of Donor Specific Antibodies after Haploidentical Related Stem Cell Transplantation for an Adult Patient with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is one of the most common genetic disorders in the world and despite advances in best supportive care it remains a disease with high risk of morbidity and mortality. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only curative treatment modality. Reports of adults are limited because of the lack of suitable matched donors and the high treatment related toxicity (TRT) after conventional myeloablative conditioning regimen resulting from the accumulated disease specific end organ damage. Initial reports of non myeloablative conditioning regimen showed disappointing results because of the high risk of graft failure (GF) and disease recurrence. Recently, the development of reduced toxicity conditioning regimen (RTC) with lower TRT and the use of haploidentical family donors has widened the applicability of alloHSCT. Since SCD patients (pt) are highly alloimmunosized, donor HLA-specific antibodies (DSA) are often detectable and might compromise engraftment in HLA mismatched transplants.Material and Methods: We report a twenty two year old male pt with severe sickle cell-ß thalassemia and chronic blood transfusion related alloimmunization who received a family donor bone marrow alloHSCT from his haploidentical father. To reduce the risk of graft rejection, the preparative regimen consisted in a prephase-conditioning sequential immunoablation using two courses of a four day immunosuppressive treatment with fludarabine 40 mg/m2/d and dexamethasone 40 mg/d, six and three weeks before the start of the RIT conditioning regimen consisting of antithymocyte globuline (ATG, rabbit) 1.5 mg/kg from days -12 to -10; thiotepa 5 mg/kg on day -9; fludarabine 40 mg/m2 and intravenous busulfan 100 mg/kg from days -6 to -3. Graft versus host disease (GVHD) prophylaxis was provided by posttransplantation cyclophosphamide 50 mg/kg on day +3 and +4; cyclosporine and mycofenolate mofetil were started at day +5. The transplant work-up program included routine HLA antibody screening and prophylactic desensitization treatment with Rituxan combined with high dose immunoglobulin infusions after each cycle of immunoablation; additionally, plasmapheresis were performed at day -13 and -1, as well as one cycle of red blood cell exchange before the start of the RIT. Disease response was weekly measured by HbS levels using hemoglobin electrophoresis. Chimerism studies were done monthly by peripheral blood isolated CD3+ cells PCR analysis of variable number of nucleotide tandem repeats. DSA were examined using the luminex-based single antigen assay and MFI>=1000 were considered positive.Results: The time to neutrophil recovery over 500 x 103/mm3 and platelet recovery over 50 x 103/mm3 were 29 and 43 days respectively; transfusion independency was reached at day +36. No GF occurred with predominantly donor mixed chimerism (82%) at last follow-up. HbS levels (baseline 68%) continuously decreased after the start of the preparative regimen to < 1% at day +77. DSA were substantially reduced after desensitization treatment and importantly dropped down further after alloHSCT to undetectable levels. Of note, no increase of DSA posttransplantation was observed. With a follow up of 140 days, the pt developed no GVHD or early TRT.Conclusions: We presume that our preparative regimen approach in association with posttransplantation cyclophosphamide provides sufficient T-cell depletion and tolerance induction to enhance durable donor engraftment. The presence of DSA should not be a barrier to HLA haploidentical related alloHSCT. DisclosuresNo relevant conflicts of interest to declare.

A Reduced-Toxicity Regimen Is Associated with Durable Engraftment and Clinical Cure of Nonmalignant Genetic Diseases among Children Undergoing Blood and Marrow Transplantation with an HLA-Matched Related Donor

Blood and marrow transplantation (BMT) is a standard curative therapy for patients with nonmalignant genetic diseases. Myeloablative conditioning has been associated with significant regimen-related toxicity (RRT), whereas reduced-intensity conditioning regimens have been associated with graft failure. In this prospective pilot trial conducted at 2 centers between 2006 and 2013, we report the outcome of 22 patients with nonmalignant genetic diseases who were conditioned with a novel reduced-toxicity regimen: i.v. busulfan (16 mg/kg), alemtuzumab (52 mg/m2), fludarabine (140 mg/m2), and cyclophosphamide (105 mg/kg). The median age of the study population was 3.5 years (range, 5 months to 26 years). No cases of sinusoidal obstruction syndrome, severe or chronic graft-versus-host disease (GVHD), or primary graft failure were reported. Median time to neutrophil engraftment (>500 cells/μL) and platelet engraftment (>20K cells/μL) were 19 (range, 12 to 50) and 23.5 (range, 14 to 134) days, respectively. The median length of follow-up was 3 years (range, .2 to 6.3). The overall survival rates were 95% at 100 days (95% confidence interval, .72 to .99) and 90% at 6 years (95% confidence interval, .68 to .98). RRT and chronic GVHD are significant barriers to BMT for patients with nonmalignant genetic diseases. This alemtuzumab-based reduced-toxicity regimen appears to be promising with durable engraftment, effective cure of clinical disease, low rates of RRT, and no observed chronic GVHD.

Donor hematopoiesis in mice following total lymphoid irradiation requires host T-regulatory cells for durable engraftment

AbstractTotal lymphoid irradiation (TLI) with antithymocyte globulin (ATG) is a unique regimen that prepares recipients for allogeneic hematopoietic cell transplantation by targeting lymph nodes, while sparing large areas of the bone marrow. TLI is reported to increase the frequency of CD4+CD25+FoxP3+ T-regulatory cells (Treg) relative to conventional T cells. In this study, barriers to hematopoietic stem cell (HSC) engraftment following this nonmyeloablative conditioning were evaluated. TLI/ATG resulted in profound lymphoablation but endogenous host HSC remained. Initial donor HSC engraftment occurred only in radiation exposed marrow sites, but gradually distributed to bone marrow outside the radiation field. Sustained donor engraftment required host lymphoid cells insofar as lymphocyte deficient Rag2γc−/− recipients had unstable engraftment compared with wild-type. TLI/ATG treated wild-type recipients had increased proportions of Treg that were associated with increased HSC frequency and proliferation. In contrast, Rag2γc−/− recipients who lacked Treg did not. Adoptive transfer of Treg into Rag2γc−/− recipients resulted in increased cell cycling of endogenous HSC. Thus, we hypothesize that Treg influence donor engraftment post-TLI/ATG by increasing HSC cell cycling, thereby promoting the exit of host HSC from the marrow niche. Our study highlights the unique dynamics of donor hematopoiesis following TLI/ATG, and the effect of Treg on HSC activity.

Durable Engraftment, Correction of Genetic Defects and Prevention of Veno-Occlussive Disease, Following Blood and Marrow Transplantation with an HLA-Matched Sibling DONOR, Using a Reduced Toxicity Conditioning Regimen with Busulfan, Reduced Dose Cyclophos

Durable Engraftment, Correction of Genetic Defects and Prevention of Veno-Occlussive Disease, Following Blood and Marrow Transplantation with an HLA-Matched Sibling DONOR, Using a Reduced Toxicity Conditioning Regimen with Busulfan, Reduced Dose Cyclophos

Reduced-Intensity Conditioning and Umbilical Cord Blood Transplantation in Children. Experience of a Pediatric Hospital in Colombia

Umbilical cord blood (UCB) has been shown to contain sufficient progenitor cells to provide durable engraftment in children, and it provides an alternative stem cell source for patients without matched related or unrelated donors. We describe the preliminary results of a cohort of 16 pediatric patients with a median age of 9 years (range 3 -18) with haematological malignancies undergoing to a unrelated umbilical cord blood transplant with a reduced-intensity conditioning regimen of fludarabine, melphalan, and antithymocyte globulin. All patients achieved hematologic recovery, the median time to an absolute neutrophil count > 0.5 x 109/L was 20 days, and the median time to an unsupported platelet count > 20 x 109/L was 32 days. Acute graft-versus-host disease (GVHD) grade III-IV occurred in 37,5% of patients. The 100-day TRM was 25%, and the 1 -year disease-free survival was 58%. Our findings support the use of reduced-intensity regimen in pediatric patients with hematologic malignancies, however the incidence of acute GVHD is a little higher than that reported in other series so a better strategy for prophylaxis against acute GVHD should be implemented.

Allo-Reactivity of Mesenchymal Stem Cells in Rhesus Macaques Is Dose and Haplotype Dependent and Limits Durable Cell Engraftment In Vivo

The emerging paradigm that MSCs are immune privileged has fostered the use of “off-the-shelf” allogeneic MSC-based therapies in human clinical trials. However, this approach ignores studies in experimental animals wherein transplantation of MSCs across MHC boundaries elicits measurable allo-immune responses. To determine if MSCs are hypo-immunogeneic, we characterized the immune response in rhesus macaques following intracranial administration of allogeneic vs. autologous MSCs. This analysis revealed unambiguous evidence of productive allo-recognition based on expansion of NK, B and T cell subsets in peripheral blood and detection of allo-specific antibodies in animals administered allogeneic but not autologous MSCs. Moreover, the degree of MHC class I and II mismatch between the MSC donor and recipient significantly influenced the magnitude and nature of the allo-immune response. Consistent with these findings, real-time PCR analysis of brain tissue from female recipients administered varying doses of male, allogeneic MSCs revealed a significant inverse correlation between MSC engraftment levels and cell dose. Changes in post-transplant neutrophil and lymphocyte counts also correlated with dose and were predictive of overall MSC engraftment levels. However, secondary antigen challenge failed to elicit a measurable immune response in allogeneic recipients. Finally, extensive behavior testing of animals revealed no main effect of cell dose on motor skills, social development, or temperament. Collectively, these data indicate that allogeneic MSCs are weakly immunogenic when transplanted across MHC boundaries in rhesus macaques and this negatively impacts durable engraftment levels. Therefore the use of unrelated donor MSCs should be carefully evaluated in human patients.

A Novel Reduced-Intensity Conditioning Regimen for Unrelated Umbilical Cord Blood Transplantation in Children with Nonmalignant Diseases

Reduced-intensity conditioning (RIC) regimens have the potential to decrease transplantation-related morbidity and mortality. However, engraftment failure has been prohibitively high after RIC unrelated umbilical cord blood transplantation (UCBT) in chemotherapy-naïve children with nonmalignant diseases (NMD). Twenty-two children with a median age of 2.8 years, many with severe comorbidities and prior viral infections, were enrolled in a novel RIC protocol consisting of hydroxyurea, alemtuzumab, fludarabine, melphalan, and thiotepa followed by single UCBT. Patients underwent transplantation for inherited metabolic disorders (n = 8), primary immunodeficiencies (n = 9), hemoglobinopathies (n = 4) and Diamond Blackfan anemia (n = 1). Most umbilical cord blood (UCB) units were HLA-mismatched with median infused total nucleated cell dose of 7.9 × 107/kg. No serious organ toxicities were attributable to the regimen. The cumulative incidence of neutrophil engraftment was 86.4% (95% confidence interval [CI], 65% to 100%) in a median of 20 days, with the majority sustaining > 95% donor chimerism at 1 year. Cumulative incidence of acute graft-versus-host disease (GVHD) grades II to IV and III to IV by day 180 was 27.3% (95% CI, 8.7% to 45.9%) and 13.6% (95 CI, 0% to 27.6%), respectively. Cumulative incidence of extensive chronic GVHD was 9.1% (95% CI, 0% to 20.8%). The primary causes of death were viral infections (n = 3), acute GVHD (n = 1) and transfusion reaction (n = 1). One-year overall and event-free survivals were 77.3% (95% CI, 53.7% to 89.8%) and 68.2% (95% CI, 44.6% to 83.4%) with 31 months median follow-up. This is the first RIC protocol demonstrating durable UCB engraftment in children with NMD. Future risk-based modifications of this regimen could decrease the incidence of viral infections. (www.clinicaltrials.gov/NCT00744692).

Treosulfan Based Myeloablative Regimen Provides High Rate Of Allogeneic Engraftment and Low Toxicity In Patients With Advanced Myelofibrosis,

AbstractAbstract 5504 BackgroudAllogeneic transplantation (HSCT) is the only potentially curative therapy for myelofibrosis, even in the era of new drugs that only impact on selected manifestations of disease. The time to evolution of myelofibrosis in advanced phases is variable (months to several years). The choice to proceed to allogeneic transplantation is based on several clinical variables; conditioning regimen and time to transplantation may influence the outcome of HSCT. Here we present our experience in a group of patients with myelofibrosis transplanted in Our Center with a myeloablative regimen based on full dose of treosulfan. MethodsWe retrospectively evaluated 20 patients (15 male) with primary myelofibrosis (14) and post-ET (essential thrombocythemia) myelofibrosis (6) who underwent HSCT at our institute from July 2005 to February 2013. In this cohort we included 4 patients in blast phase, 3 of which received a brief course of chemotherapy prior to transplant; 4 patients who underwent a second allogeneic transplantation for disease relapse (2) or graft failure (1); 1 patient referred at our Institute in graft failure 3 months after first HSCT. Only 1 patient had splenectomy before HSCT. ResultsThe median age at diagnosis was 59 years (range 41-76). JAK2 V617F mutation was positive in 7 and unknown in 3 patients. In post-ET MF patients, the median time to transformation in MF was 134 months (60-144). The median time from diagnosis to transplant was 14 months (4-101 months). The median age at transplant was 62 years. DIPSS score at transplant was low in 1, int-1 in 3, int-2 in 13 and high in 3 patients. We adopted myeloablative regimen based on treosulfan 42 gr/ms and fludarabine 150 mg/ms, plus TBI 4 Gy in 7 patients. GvHD prophylaxis was based on ATG, Rituximab and CSA/MTX (8 pts) or rapamycin (11 pts). The graft was PBSCs (19) and BM (1) from related HLA-identical donor (7 pts), matched unrelated donor (5 pts) and haploidentical donor (8 pts). The incidence of graft failure and poor graft function was 5% and 20% respectively. The median time to neutrophils engraftment was 20 days. The incidence of acute and chronic graft versus host disease was 50% and 40% respectively. Cumulative incidence of TRM (transplant related mortality) was 45 %; day 100 OS (overall survival) was 70% while 2 year OS was 40%. In multivariate analysis there was no statistically significant correlation between individual factors (including WBC, hemoglobin, platelets, ferritin, beta2-microglobulin, IgA, IgG, IgM, LDH, psuedocholinesterase levels, age, Sorror comorbidity score, donor match, bone marrow kariotype) and survival. However, we found that a linear composition of level of peripheral blasts (0%, ≥1%, ≥5%), bone marrow CD34+ cells (<5%, ≥5%, ≥20%), spleen size and number of red blood cells units transfused pre-HSCT is able to predict the outcome (p-value 0.03), even if each factor alone is not strongly correlated with survival. There was no statistical significant correlation between the outcome and DIPSS score, Bacicalupo score and Lille score. ConclusionsA myeloablative regimen based on full dose treosulfan provide a 95% rate of primary allogeneic engraftment and 40% overall survival in myelofibrosis at advanced stage, with low extra-hematological toxicity in frail patients. Allogeneic transplantation after myeloablative low-toxicity regimens may be offered in myelofibrosis in early chronic phase of disease and for younger patients, two conditions usually linked with durable engraftment, low toxicities and low rate of relapse. Disclosures:No relevant conflicts of interest to declare.

A Novel Reduced-Toxicity Myeloablative Conditioningusing Full-Dose Busulfan and Melphalan For Cord Blood Transplantation Provides Durable Engraftment and Remission Without Increasing Non-Relapse Mortality In Advanced Myeloid Malignancies

IntroductionAlthough cord blood transplantation (CBT) using reduced-intensity regimens has emerged as an effective therapy for elderly or patients with comorbidities, high relapse rate and non-relapse mortality (NRM) still remain to be resolved. Tointensify antitumor effects without increasing NRM, we have conducted a pilot study of a novel reduced-toxicity myeloablative conditioning for CBT in our institute. MethodsThe conditioning consisted of intravenous busulfan 12.8 mg/kg, fludarabine 180 mg/m2 and melphalan 80 mg/m2 followed by single CBT. Fifty-one patients with myeloid malignancies not in remission were included in this study between June 2007 and November 2012. All patients provided written informed consent, andmedical records were retrospectively reviewed. ResultsTheir median age was 59 years (range, 19-70), with a median HCT-CI score of 3 (0-6). Underlying diseases were AML in 44, MDS (RAEB-2) in 3, and CML (AP/BC) in 4. All patients were not in remission including primary induction failure (n=19), relapse 1 (14), relapse>2 (7), and untreated (11). Tacrolimus (TAC) plus mycophenolate mofetil were used in 38 cases as GVHD prophylaxis, while TAC alone in 13. All patients received single cord blood unit with 2-mismatches (MM) to the recipient (n=47) and 1-MM (4). The median number of total nucleated cell and CD34+ cells infused at cryopreservation was 2.61 (range, 1.67-5.09) x 107/kg and 0.94 (range, 0.28-2.97) x 105/kg, respectively. Median observation period of survivors was 18.3 (range, 9-75.5) months. Overall survival (OS) and disease free survival (DFS) at 2 years were 62.4% and 55.1%, respectively (Figure 1). Eleven patients relapsed at a median of 5.2 months (range; 0.5-26.7). Cumulative incidences of relapse at 100 days and 2 years were 7.8% and 20.1%, respectively. Cumulative incidences of NRM at 100 days and 2 years were 11.8% and 21.7%, respectively. Causes of NRM were Infection (n=8), GVHD (2) and idiopathic pneumonia syndrome (1). Forty-six out of 51 achieved neutrophil engraftment at median of 19.5 days (range, 13-38) post-transplant, with a cumulative incidence of 90.2%. All patients who achieved engraftment showed complete remission with complete donor chimerism in bone marrow analysis performed at around 30 days post-transplant. Among 5 who failed to achieve neutrophil recovery, 3 experienced early disease progression and 2 died before engraftment, while no patients developed graft rejection. Cumulative incidence of grade II-IV and III-IV acute GVHD were 52.9% and 13.7%, respectively. Of the 42 patients who achieved engraftment and survived longer than 100 days post-transplant, 13 developed limited type of chronic GVHD, and 5 developed extensive type. ConclusionThis study demonstrated that the novel conditioning regimen using full-dose busulfan and melphalan for CBT provides reliable engraftment without graft rejection and high OS and DFS rates without increasing NRM, even for relatively higher age population with myeloid malignancies not in remission. The promising results using the regimen deserve further evaluation in a multicenter prospective study. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Fludarabine and 2-Gy TBI is Superior to 2 Gy TBI as Conditioning for HLA-Matched Related Hematopoietic Cell Transplantation: A Phase III Randomized Trial

The risks and benefits of adding fludarabine to a 2-Gy total body irradiation (TBI) nonmyeloablative regimen are unknown. For this reason, we conducted a prospective randomized trial comparing 2-Gy TBI alone, or in combination with 90 mg/m2 fludarabine (FLU/TBI), before transplantation of peripheral blood stem cells from HLA-matched related donors. Eighty-five patients with hematological malignancies were randomized to be conditioned with TBI alone (n = 44) or FLU/TBI (n = 41). All patients had initial engraftment. Two graft rejections were observed, both in the TBI group. Infection rates, nonrelapse mortality, and graft-versus-host disease (GVHD) were similar between groups. Three-year overall survival was lower in the TBI group (54% versus 65%; hazard ratio [HR], .57; P = .09), with higher incidences of relapse/progression (55% versus 40%; HR, .55; P = .06), relapse-related mortality (37% versus 28%; HR, .53; P = .09), and a lower progression-free survival (36% versus 53%; HR, .56; P = .05). Median donor T cell chimerism levels were significantly lower in the TBI group at days 28 (61% versus 90%; P < .0001) and 84 (68% versus 92%; P < .0001), as was NK cell chimerism on day 28 (75% versus 96%; P = .0005). In conclusion, this randomized trial demonstrates the importance of fludarabine in augmenting the graft-versus-tumor effect by ensuring prompt and durable high-level donor engraftment early after transplantation.

Phase II Study of CD4+-Guided Pentostatin Lymphodepletion and Pharmacokinetically Targeted Busulfan as Conditioning for Hematopoietic Cell Allografting

One limitation of reduced-intensity preparative regimens is potential for graft failure. We have developed a regimen that targets CD4+ lymphodepletion to ensure early and durable engraftment. The primary endpoint was achievement of ≥50% CD3+ donor chimerism by day +28. Forty-two patients (median age, 53 years; range, 29 to 73 years) received pentostatin 4 mg/m2 i.v. on days −28, −21, and −14 when the CD4+ cell count was >100 cells/μL and on days −4 and −3 regardless of CD4+ level. Rituximab 375 mg/m2 was administered to patients with CD20+ malignancies on days −21, −14, −7, +1, and +8. Busulfan 200 mg/m2 i.v. was administered on days −4 and −2 at a dose to target a cumulative AUC dose of 16,000 (±10%) μmol·min/L. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus plus methotrexate in 86% of patients. Donors were matched-related (47%), matched unrelated (43%), or mismatched unrelated (10%). Chronic lymphocytic leukemia (45%) and follicular non-Hodgkin lymphoma (14%) were the most common diagnoses. Disease status at initiation of the preparative regimen was complete remission in 22%, partial response in 55%, and stable/progression in 24%. The median percent CD4+ cell count decrease from baseline (day −28) was 52% to day −21, 66% to day −14, 62% to day −7, and 91% to day 0. At day +28, all 42 patients (100%) had ≥50% CD3+ donor chimerism. No patient experienced graft failure. Overall response rate was 82% (complete remisson, 67%). The day +100 cumulative incidence of grade II-IV acute GVHD was 59% (grade III-IV acute GVHD, 19%), and the 2 year cumulative incidence of chronic GVHD was 69% (moderate/severe, 58%). Nonrelapse mortality was 2% at day +100 and 17% at 2 years. Two-year PFS was 55%, and OS was 68%. This regimen ensures durable engraftment, is effective against persistent disease, and results in relatively low mortality from causes other than relapse.

Virgin birth: engineered heart muscle from parthenogenetic stem cells

Cardiac muscle restitution, or true regeneration, is an unmet need in the treatment of myocardial infarction (MI), prompting a decade of study with stem cells of many kinds. Among key obstacles to effective cardiac cell grafting are the cost of autologous stem cell-derived cardiomyocytes, the ethical implications of using embryonic stem cell (ESC) products, immunological barriers to allogeneic cells, functional maturation beyond just the correct lineage decision, and the lack of durable engraftment. In this issue of the JCI, Didié and colleagues show that cardiomyocytes made from parthenogenetic stem cells (PSCs) and deployed as engineered heart muscle (EHM) may overcome all of these formidable barriers.

Engraftment of heavily transfused patients with severe aplastic anemia with a fludarabine‐based regimen

We have developed a practical conditioning regimen without anti-thymocyte globulin (ATG), irradiation, or other myeloablative alkylating agent for low-income countries in which patients with severe aplastic anemia (SAA), who usually have heavily transfused and a prolonged disease history. The application of ATG, Busulphan, and/or irradiation to cyclophosphamide (Cy) to avoid graft rejection has many short- and long-term complications. In this study, we focused on evaluating a fludarabine-based conditioning regimen, among 83 patients with SAA. Patients were treated with fludarabine (40 mg/m(2) /d; day [-5 to -2]) and cyclophosphamide (50 mg/kg/d; day [-5 to -2]). Altogether, 81 patients indicated initial engraftment, whereas two cases showed primary graft failure. And four of the 81 cases indicated graft rejection during follow-up. Regardless of a high cumulative incidence of acute (55/83; 66.2% grade II-IV; 47/83; 56.6% III-IV) and chronic graft-versus-host disease (50/83; 60.2%), in total, 77 patients showed durable engraftment and transfusion independence, and 64 are alive at a median time of 49 months with an overall survival rate of 66%. In conclusion, this conditioning indicated well toleration, mild toxicity, durable engraftment, excellent survival as well as less cost. Its application might shed new light on SAA at high risk of graft rejection in resource-limited countries.