Low Incidence Of GVHD Research Articles

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.

Low Incidence of Chronic Gvhd after Haploidentical T-Cell Replete Peripheral Blood Stem Cell Transplantation with Post Transplantation Cyclophosphamide (PT-Cy)

Background:Since the first publication at the John Hopkins Hospital, (Luznik et al. BBMT 2008), Haploidentical T-cell replete Stem Cell Transplantation (HaploSCT) with post transplantation cyclophosphamide (PT-Cy) has become a reproducible and feasibility therapeutic option for many patients (pts) with hematologic malignancies , notably because of the low incidence of GVHD and infections, without increased graft failure. Initially, bone marrow (BM) was considered as the favorite source of hematopoietic stem cells in order to minimize the risk of GVHD. In a retrospective study, we previously showed no difference in terms of increased events (GVHD, NRM), regardless of the hematopoietic stem cell source (PBSC or BM) (Castagna et al. BMT 2014). Some recent publication seems to confirm this observation (Sugita J. BBMT 2015 , Solomon R. Adv in Hem. 2016), although no randomized study so far has been conducted.Here, we retrospectively analyze the incidence and the characteristics of GVHD in the setting of HaploSCT using PT-Cy, after infusion of PBSC.Methods:Inclusion criteria were: adult pts with hematologic malignancies receiving a PBSC HaploSCT from 2012 to 2015 in 4 centers (3 in France and 1 in Italy) with PT-Cy as part of the GVHD prophylaxis.PBSC infusion at day 0 was followed by PT-Cy 50 mg/kg on days +3 and +4 in association with calcineurin inhibitors (cyclosporine A or Tacrolimus) and mycophenolate mofetil (MMF), started at day +5. All patients received G-CSF support from day+5 until neutrophil recovery.Study end points were the cumulative incidences of acute (a) and chronic (c) GVHD, with a specific organ grading evaluation, non-relapse mortality (NRM), relapse (CIR) as well as progression free (PFS) and overall survival (OS). Additionally, we analyzed the composite endpoint "GVHD and relapse free survival" (GFRS) for which the occurrence of relapse, death or severe chronic GVHD was considered as relevant events.Correlation between CD 34+ and CD 3+ and the incidence of aGVHD and cGVHD was studied by a linear continuous variable.Results:Between March 2012 and December 2015, 192 pts with a median age of 57 years (range: 16-73) received T-cell replete PBSC HaploSCT for hematologic malignancies (myeloid: n= 55%; lymphoid: n=45%) in 4 centers.Patient’s characteristics are shown in table 1. Pts received non myeloablative (according to Baltimore regimen) or busulfan-based reduced intensity conditioning, in 56% and 44% of cases, respectively. All, but 3 pts, engrafted, with a median time of 19 days (range, 14-47) to neutrophil recovery (ANC >500 x106/L) and 22 days (range, 14-252) to platelet recovery (PLT > 20 G x 109/L). The median CD34+ x 106/Kg and CD3+ x 106/Kg cells infused were 5.5 (range, 1.5-14.8) and 404 (range, 38-704), respectively. No relevant correlation was observed between the CD34+ and CD3+ infused cells and the incidence of GVHD, studied by linear continues variable. We noted only a trend to develop severe cGVHD with an increasing number of CD3+ cells infused. This result has to be considered with caution, because of the small events (6 pts affected by severe cGVHD).Complete donor T cell chimerisme was evaluable in 162 pts (83%) and achieved by day +30. The incidence of aGVHD was 38% at 100 days (all grades), whereas grade II-IV and III-IV were 24% and 10%, respectively. Concerning patients with aGVHD grade 2-4, the most affected organ was skin (19%), followed by gut (9%) and liver (2%). The incidence of 3-year cGVHD according to NIH classification was 15% (all grades).Three percent of pts developed severe cGVHD (lung n=1; liver, n=1). The most frequent involved organs were skin and mucosae (70%). No patient showed gut cGVHD. Finally, in univariate analysis, busulfan-based conditioning seems to negatively impact on severe cGVHD (p = 0.03; HR=3.37 [1.09 -10.46])After a median follow up of 20 (range, 4 - 52) months, NRM at 100 days and 1 year was 10% and 20%, respectively. Three-year OS, PFS, CIR and GRFS were 63%, 55%, 25% and 49%, respectively.Conclusion:This retrospective study shows a very low incidence of severe cGVHD after HaploSCT even with PBSC as stem cell source, suggesting that the use of PT-Cy may overcome the anticipated increased incidence of cGVHD, contrary to as previously reported in the HLA identical setting (Mohty et al. Leukemia 2003).Similar to HLA identical sibling and unrelated donor transplantation, the most frequent organs involved are skin and mucosae [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Haploidentical transplantation in children with unmanipulated peripheral blood stem cell graft: The need to look beyond post‐transplantation cyclophosphamide in younger children

Haploidentical transplantation with PTCY following marrow or PBSC graft has been associated with low incidence of GVHD in adults with similar data lacking in children. We report on the outcome of 25 patients <20 yr of age (median age 12 yr), undergoing a haploidentical PBSC transplantation for both malignant and non-malignant disorders. Engraftment was prompt and sustained. Cumulative incidences of acute GVHD and chronic GVHD were 40.3% and 16.7%, respectively. On subgroup analysis, it was evident that acute GVHD developed in 80% of patients <10 yr compared to only 13.3% in those between 10 and 20 yr [log-rank p = 0.001], despite similar graft composition with significantly higher NRM (60% vs. 0%; p = 0.001). The FFS was 63.5%; (79% in >10 yr and 40% in <10 yr, p = 0.01). Our data suggest that PTCY-based haploidentical PBSC transplantation is feasible in older children, but results in early and severe alloreactivity in younger children. These findings, despite being counterintuitive, could be explained by the variable metabolism of CY and oral mycophenolate in younger children indicating that PTCY-based approach as used in adults might not be adequate for younger children.

Low incidence of GvHD with T-cell depleted allografts facilitates further treatments for post-transplantation relapse in AML and MDS.

Low incidence of GvHD with T-cell depleted allografts facilitates further treatments for post-transplantation relapse in AML and MDS

Allogeneic Stem Cell Transplantation Using In Vivo T-Cell Depletion in Myeloid Disorders

Introduction:Alemtuzumab is used for in vivo T-cell depletion to reduce graft versus host disease in allogeneic Stem Cell Transplantation (SCT). Profound lymhotoxicity of this monocloncal antibody can potentially increase morbidity and mortality in SCT due to excessive viral infections and increased risk of graft rejection. We retrospectively analysed outcome of patients with myeloid disorders (acute myeloid leukaemia and myelodysplasia) who received in vivo T-cell depletion using Alemtuzumab based conditioning for allogeneic SCT over a period of 3 years in our centre.Methods:Patients were identified from department transplant data base. Data was collected for 73 consecutive patients over a period of three years using patient medical records, clinical work station and electronic patient records. The conditioning regimen included Fludarabine 30mg/m2 x5 (days -7 to -3), Alemtuzumab 10mgx5 (days -8 to -4) and Melphalan 140mg/m2 (day -2). Chimerism analysis was performed by polymerase chain reaction (PCR) to identify short tandem repeats within peripheral blood leucocytes and CD3 fraction. The quantification of donor chimerism was done by using gel photography system and LabWroks software. Viral testing was performed by PCR analysis.Results:Median age for the patients was 59 years (range 41-71). Median duration of follow up was 19 months (4-49 months). The majority of patients (67%) received SCT from a voluntary unrelated donor. Sixty four patients (88%) had AML whilst 9 had myelodysplasia. Sixty five (89%) patients were in morphological complete remission (<5% blasts) at the time of SCT. Median HCT-Comorbidity index was 2 (range 1-5). Median time to neutrophil engraftment was 13 days (range 9-23) and platelet engraftment 14 days (median 8-48). During the course of transplantation, 50 patients were treated for neutropenic fever with broad spectrum antibiotics and 16 for presumed fungal infection. Forty three patients (59%) reactivated cytomegalovirus (CMV), 10 (13%) had Ebstein Barr Virus (EBV) viremia and 4 (5%) were found to have adenovirus on peripheral blood PCR analysis after SCT. Denovo acute graft versus host disease (grade 2-4) required treatment in 12 patients (16%) whilst chronic GVHD was seen in 14% (n=10). 20 patients required donor lymphocyte infusion due to mixed chimerism after SCT. Total incidence of acute GVHD (grade 2-4) was 23% (n=17) and that of chronic GVHD was 15%. Median donor chimerism in peripheral blood leucocyte fraction was 100% at day 30, 60, 90 and 180 after SCT. Chimerism analysis on CD3 compartment showed 100%, 96%, 95.5% and 95% donor fraction respectively at these time points. Predicted overall survival at 2 years was 53% and Event Free Survival 49%. Non relapsed mortality in this cohort was 18% at one year. One patient had primary graft rejection with no secondary graft rejections. Overall survival was not statistically different between those who were treated for viremia (CMV, EBV, adenovirus) when compared to those who did not have viremia (p=0.31). No deaths were attributed to either CMV or EBV.Conclusion:Our retrospective data show that Alemtuzumab based conditioning regimen in myeloid disorders have low incidence of GVHD, very low risk of graft rejection and comparable overall survival to those conditioning regimens which utilize in vivo T-cell depletion strategies other than Alemtuzumab. Moreover, a high incidence of viremia, in our cohort, did not translate into worse overall survival. DisclosuresSaif:Novartis: Honoraria; Alexion: Honoraria. Dignan:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Therakos: Honoraria, Speakers Bureau. Tholouli:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Effect of Transfusion of the Third Party Umbilical Cord Blood on Haplo-Identical Hematopoietic Stem Cell Transplantation

Objective To retrospectively evaluate the effect of the third party umbilical cord blood on haplo-identical hematopoietic stem cell transplantation.Methods From June 2012 to May 2015, 125 leukemia patients were enrolled, including 41 cases of ALL, 62 cases of AML, 12 cases of MDS, 7 cases of CML-BP, 2 cases of acute mixed leukemia and 1 case of Mother cell dendritic cell tumor. Inclusion criteria: 1) AL patients; 2) halpo-identical HSCT; 3) 3/6 matched cord blood was available. Patients were divided into two groups, ie. group A (HSCT, n=65) and group B (HSCT plus umbilical cord blood transfusion group, n=60). Myeloablative conditioning regimens consisted of BuCy, TBI/FLAG, TBI/Cy, and FLAG that followed by reduced-intensified BUCY. The median dose of mononuclear cells in group A and B were 8.58×108/kg and 9.01×108/kg, respectively. The median dose of CD34+ cells for transfusion in each group were 3.67×106/kg and 2.94×106/kg, respectively. The dose of grafted UCB MNCs and CD34+ cells for group B were 3.5×107/kg and 2×105/kg, respectively. All patients received cyclosporineA, MMF and methotrexate for GVHD prophylaxis. The endpoints of this study were hematological engraftment, incidence of acute and chronic GVHD, incidence of relapse, transplant-related mortality(TRM), non-relapse mortality(NRM), Overall survival(OS) and Disease-free survival(DFS) in each group.Results The median follow-up time was 17(3-29) months in group A and 18(3-35) months in group B. Patients in group A reached a sustained ANC of more than 0.5*109/L at a median of 11 days, whereas 14 days in group B. Platelet more than 20*109/L occurred at a median of 19 days in group A, whereas 17 days in group B (P= .4). The rate of aGVHD was not significantly different in the two groups, 56.9% in group A and 48.3% in group B (P= .21). The accumulative incidence of II-IV grade aGVHD was 35.4% in group A and 30% in group B (P= .42). The incidence of chronic GVHD was 79.2% in group A and 71% in group B (P= .47). The incidence of extensive type was lower in group B, 69.2% Vs 35%, P=0.09. The incidence of CMV was lower in group B, 80% Vs 60% (P= .01). The accumulative incidence of EBV was lower in group B, 35.4% Vs 3.3% (P<0.01). At two years, the accumulative incidence of relapse was 24.4% in group A and 17.2% in group B, P=0.13. The two-year accumulative incidence of OS was 72.9% in group A and 84.8% in group B, P=0.07. The one-year accumulative incidence of DFS in each group were 65.7% and 79.4%, respectively, P=0.03. Conclusion Our clinical results have shown that HSCT with transfusion of the third party umbilical cord blood is a promising modality for induction of immunity reconstitution. Better survival results may benefit from lower incidence of GVHD and virus infection. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic hematopoietic cell transplantation after failed autologous transplant for lymphoma using TLI and anti-thymocyte globulin conditioning.

We describe 47 patients with lymphoma and failed prior autologous hematopoietic cell transplantation (HCT) who received TLI-ATG conditioning followed by allogeneic HCT. Thirty-two patients had non-Hodgkin lymphoma (NHL; diffuse large B cell lymphoma [n=19], T-cell NHL [n=6], mantle cell lymphoma [n= 4], or other B-cell subtypes [n=3]), and 15 had Hodgkin lymphoma. The median follow-up was 4.9 (range, 2.1–11.9) years. The cumulative incidence of grade II–IV acute GVHD at day +100 was 12%, and the cumulative incidence of extensive chronic GVHD at 1 year was 36%. The 3-year cumulative incidences of overall survival, progression-free survival (PFS), and non-relapse mortality (NRM) were 81%, 44%, and 7%, respectively. Fifteen patients died (relapse, n=10; NRM, n=5). Among the 25 patients with relapse after allogeneic HCT, 11 (44%) achieved durable (>1 year) complete remissions following donor lymphocyte infusion or chemoradiotherapy. The majority of surviving patients (75%; n=24) were able to discontinue all immunosuppression. For patients with relapsed lymphoma after autologous HCT, allogeneic HCT using TLI-ATG conditioning is a well-tolerated, predominantly outpatient therapy with low NRM (7% at 3 years), a low incidence of GVHD, durable disease control, and excellent overall survival (81% at 3 years).

King’s College Hospital FCC Conditioning for Severe Aplastic Anemia Induces Tolerance with Mixed T-Cell Chimerism and Extremely Low Incidence of Gvhd

We have previously shown that HSCT conditioning regimen for severe aplastic anemia (SAA) using fludarabine, cyclophosphamide and alemtuzumab (FCC), is associated with very low rate of GvHD (especially chronic, cGVHD), and excellent overall survival (OS) of 83% and 95% for matched unrelated (MUD) and matched sibling donor (MSD) HSCT, respectively. Sustained full donor myeloid chimerism occurred most frequently with mixed T-cell chimerism, and this persisted despite discontinuation of post graft immunosuppression with ciclosporin (CSA). We proposed that prolonged mixed T-cell chimerism with alemtuzumab conditioning may facilitate tolerance and hence a low risk of GVHD.We have investigated the basis for the mixed T-cell chimerism in a cohort of 35 patients, median age 32y (range 15-63), transplanted for acquired idiopathic SAA from 2007 - 2014 at King’s College Hospital. FCC conditioning comprised fludarabine 30 mg/m2 iv (day -7 to -4), cyclophosphamide 300 mg/m2 iv (day -7 to -4) and alemtuzumab (median dose 70mg, range 45-100). CSA was used as post graft immune suppression for 9 months, followed by dose tapering to zero over the next 3 months depending on PB chimerism. Stem cell source was BM in 7(20%) patients and 28(80%) PBSC, with a median cell dose of 6.8 (1.9-12.4) CD34+ cells x 106/Kg. Donor type was MSD in 9(26%) pts and UD in 26(74%), of whom 20 were matched (MUD) and 6 were 9/10 match ( received FCC with 2 Gy TBI). Median time to neutrophils >0.5x109/l was 12(10-20) days and to platelets >50x109/l was 10(9-61) days. Primary graft failure occurred in only one patient (2%). Acute GvHD was seen in 6(17%) pts (all grade I-II), and chronic GvHD in 5(14%) pts (3 mild, 1 moderate, 1 severe, all with only skin involvement). On univariate analysis with Cox regression model, factors significantly predicting cGVHD were previously diagnosed acute GVHD (p=0.035) and CD3+ chimerism at day+100 ≥90% (p=0.006). Four patients developed autoimmune anemia post HSCT (2 hemolytic anemia, 2 PRCA). Median follow-up was 30 months (4.3-86.8), OS 94% and event free survival 90%. One year TRM was 6%.Of 32(91%) patients evaluable for chimerism, 8 (23%) were fully donor chimeric for unfractionated BM, PB CD15+ and PB CD3+, while 24 (77%) patients had persistent mixed CD3+ chimerism. Median CD3+ chimerism values were 77% at day+30, 60% at day+60, 41% at day +100,57% at +day180, 70,5% at 1 year, 77% at 2 years and 80% at 3 years. The proportion of evaluable patients no longer taking CSA was 0/23 at 1yr, 9/19 at 1.5yr, 12/16 at 2yr, 12/12 at 2.5yr and 4/4 at 3yr. PB immunophenotyping was performed in 19 patients. During the first year post HSCT, there was profound and prolonged lymphopenia, NK cells recovered first but numbers remained below normal. T cells comprised only 2.6% of lymphocytes at day 30, rising slowly to 41% at day 360 but still significantly below normal (66% in 11 aged-matched healthy individuals), with deficiency greatest for CD4+ T cells. Despite slow recovery of CD4+ T cells, percentage of T-regulatory cells (CD4+ CD25high CD27+ FoxP3+) within this population was normal (range 4.2-7.4% for all time points compared to 5.3% for healthy individuals). Patients aged <50y showed recovery of naïve T cells that expressed markers of recent thymic emigrants (CD31+ CD62L+ CD45RA+), indicating renewed thympoiesis and implementation of central tolerance B cell recovery was notably robust, with numbers comparable to healthy individuals for most patients by day 100. Subset analysis showed absence of memory B-cells but significantly increased proportion of immature transitional (T1 and T2) B cells (CD24+ CD38+, CD27-, IgM high IgD high) for 6 months after FCC HSCT. Cells within this subset are known to possess immune suppressive activity.The mechanism for prolonged mixed T-cell chimerism and associated low incidence of chronic GVHD following FCC conditioning for SAA HSCT may be multifactorial, but we here report novel findings: apart from a markedly low proportion of T cells, we show for the first time in SAA post HSCT the presence of T and B cell subsets with potential regulatory properties and establishment of central tolerance of naïve T-cells in younger patients. Our results also confirm the excellent outcomes of UD HSCT using alemtuzumab with reduced intensity conditioning in SAA, and we propose it is now time to re-think the up-front MUD transplant at all ages in eligible patients. DisclosuresNo relevant conflicts of interest to declare.

Development of an Alpha/Beta T-Cell Depleted Allogeneic Stem Cell Transplantation Protocol Form Matched Related and Unrelated Donor Grafts in Patients with Poor Risk Leukemia

▪Introduction: The outcome of allo-SCT in patients with poor risk leukemia is still hampered by GVHD and relapse. The innate immune system has been reported to contribute to tumor control, with lower incidence of GVHD. Specific depletion of αβ T- cells – key players in the development of GVHD – will render NK cells and γδ T cells within the allograft. Recently reported results have shown the great promise of this approach in haploindentical transplantations. Within this study, we aim to extend αβT- cell depleted allo-SCT to patients with a MRD or MUD.Methods: Patients with either ‘poor-risk’ or ‘very poor-risk’ leukemia were included in this phase I study. Either HLA matched siblings (MRD) or fully matched HLA matched (10/10) unrelated donors (MUD) were eligible. abT-cell reduction was performed by negative selection with anti-abTCR antibodies in combination with magnetic microbeads, using the automated CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). The maximal contamination with αβT-cells for all dose levels was 5x105/kg. Three conditioning regimens have been investigated (I): fludarabine 120 mg/m2 + cyclophosfamide 4800 mg/m2, (II): fludarabine 120 mg/m2 + busilvex AUC=90 and (III): ATG (Genzyme®) 4 mg/m2 + fludarabine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. Within cohort II and III, no additional immune suppression was given after allo-SCT.Results: Products for 14 patients have been successfully processed and used for αβT-cell depleted allo-SCT between 2011 and 2013. A ~4 log depletion of αβT-cells has been observed in the product with a recovery of ~75% of CD34+ cells. In cohort I and cohort II, 60% and 25% primary graft failures were observed, whereas in cohort III primary engraftment (chimerism > 95%) was observed in all patients. The combination of ATG/fludarabine/busilvex was well tolerated with a hematological recovery of within 3 weeks. In all 14 patients immune reconstitution primarily consisted of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. In addition, no increase in CMV or EBV reactivations has been observed so far under the profound “innate control”.Conclusion: ATG Busulfan Fludarabine is a low toxicity platform for abTCR-depleted transplantations, resulting in a swift reconstitution of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. This transplantation strategy can serve as a tool for future immunological interventions such as a low dose DLI or genetically modified T cells. DisclosuresNo relevant conflicts of interest to declare.

Reduced toxicity, myeloablative conditioning with BU, fludarabine, alemtuzumab and SCT from sibling donors in children with sickle cell disease

BU and CY (BU/CY; 200 mg/kg) before HLA-matched sibling allo-SCT in children with sickle cell disease (SCD) is associated with ~85% EFS but is limited by the acute and late effects of BU/CY myeloablative conditioning. Alternatives include reduced toxicity but more immunosuppressive conditioning. We investigated in a prospective single institutional study, the safety and efficacy of a reduced-toxicity conditioning (RTC) regimen of BU 12.8-16 mg/kg, fludarabine 180 mg/m(2), alemtuzumab 54 mg/m(2) (BFA) before HLA-matched sibling donor transplantation in pediatric recipients with symptomatic SCD. Eighteen patients, median age 8.9 years (2.3-20.2), M/F 15/3, 15 sibling BM and 3 sibling cord blood (CB) were transplanted. Mean whole blood and erythroid donor chimerism was 91% and 88%, at days +100 and +365, respectively. Probability of grade II-IV acute GVHD was 17%. Two-year EFS and OS were both 100%. Neurological, pulmonary and cardiovascular function were stable or improved at 2 years. BFA RTC and HLA-matched sibling BM and CB allo-SCT in pediatric recipients result in excellent EFS, long-term donor chimerism, low incidence of GVHD and stable/improved organ function.

Enhancement Of Human Regulatory γδ T Cells In Vitro Induced By Granulocyte Colony-Stimulating Factor

Background and ObjectiveGraft versus host disease (GVHD) is the main complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Regulatory γδ T cells (γδ Tregs), which express Foxp3 and primarily belong to CD27+CD25high phenotype, are a novel subset of cells with immunosuppressive function and have the potential application prospect in GVHD therapy. Peripheral blood mononuclear cells (PBMCs) could be induced to generate γδ Tregs in vitro by stimulating with anti-TCR γδ and transforming growth factor-β (TGF-β). Our previous studies demonstrated that granulocyte colony-stimulating factor (G-CSF) had immunomodulatory effect on T cells; G-CSF mobilization influenced the distribution and clonality of TRGV and TRDV repertoire (T cell receptors of γδ T cells), and significant positive correlation was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients. To further characterize the immunoregulatory functions of G-CSF, this study explored the possibility of γδ Tregs induced by G-CSF in vitro. MethodsPBMCs of healthy donors were cultured in vitro by stimulating with anti-TCR γδ and different cytokines for 9-12 days. The culture system was grouped based on the difference of added cytokines, including: (1) TGF-β (2) G-CSF (3) TGF-β+G-CSF (4) blank group. The induced cells were used as effector cells in the carboxylfluorescein diacetate succinimidyl ester (CFSE) assays and cell immunophenotyping was analyzed by flow cytometry. Autologous CD4+T cells were purified by microbeads, labeled with CFSE, used as responder cells, and finally co-cultured with effector cells. After 5 days incubation, cells were harvested and analyzed for flow cytometry by gating on the CFSE-labeled cells. The expression levels of Foxp3 and CD25 genes were detected by real-time polymerase chain reaction. ResultsAfter 9 days' culture in vitro, the proportion of Foxp3+ γδ T cells was 7.87% in the blank group, which was significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (62.3%, 52.9% and 63.5%) (P<0.001). The proportions of CD25+γδ T and CD27+γδ T cells were 3.9% and 3.5% in the blank group, which were also significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (CD25+γδ T cells: 43.5%, 43.4% and 44.4%; CD27+γδ T cells: 41.5%, 41.9% and 44.0%, respectively) (P<0.001, P<0.001). The effector cells induced by anti-TCR γδ with TGF-β, G-CSF and TGF-β + G-CSF all manifested a more significant suppressive effect on responder cells than cells induced only by anti-TCR γδ. However, there was no significant difference in the effect of cell proliferation suppression in the TGF-β, G-CSF and TGF-β + G-CSF groups. The expression level of Foxp3 gene was 0.384% in the blank group, which was significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (0.623%, 0.639% and 0.843%, respectively) (P<0.001). The expression level of Foxp3 in TGF-β+ G-CSF group was significantly higher than that in the TGF-β and G-CSF group (P=0.001, P=0.007). The expression level of CD25 gene was similar among the four groups (P=0.457). ConclusionsThe generation of γδ Tregs could be enhance in vitro induced by G-CSF. This may be one of the reasons that G-CSF play an immunoregulatory role on decreased GVHD onset during allo-HSCT. Disclosures:Wu:National Natural Science Foundation of China (No. 81200388); the Technology Plan of Guangdong Province of China (No. 2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (No. 2013027).: Research Funding.

Feasibility Of αβT- Cell Depleted Allogeneic Stem Cell Transplantations From Matched Related and Unrelated Donor Grafts and Engraftment In Patients With Poor Risk Leukemia

RationaleαβT- cell depleted allogeneic haematopoietic stem cell transplantations (allo-SCT) have so far been reported in haploidentical stem cell transplantations. First results have been promising with a good engraftment and low incidence of GVHD. However, high rates of life-threatening GVHD are still observed within the context of matched related (MRD) and unrelated (MUD) donors. Therefore we aimed to assess the feasibility of αβT-cell depletion in the context of MUD and MRD and aimed to establish a transplantation regimen for patients with MRD and MUD undergoing an αβT- cell depleted allo-SCT. ObjectiveTo test feasibility of the generation of αβT- cell depleted grafts from MRD and MUD and assess the engraftment in the context of different conditioning regimens after allo-SCT in patients with poor risk leukemia. MethodInitial proof runs for the generation of αβT-cell and CD19 depleted grafts have been performed in 4 healthy donors. Grafts for transplantation of the first 5 patients (cohort I) have been depleted with GMP-grade anti-αβTCR and anti-CD19 antibodies. The subsequent grafts for patients in cohort II (n=4) and III (n=2) have been selectively depleted with GMP-grade anti-αβTCRs antibodies. Three conditioning regimens have been investigated (I): fludarabine 120 mg/m2 + cyclophosfamide 4800 mg/m2, (II): fludarabine 120 mg/m2 + busilvex AUC=90 and (III): ATG (Genzyme®) 4 mg/m2 + fludarabine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. No additional immune suppression was given after allo-SCT.Main study parameters/endpoints: (1) Feasibility to generate an αβT-cell depleted graft form MRD and MUD. (2) Engraftment and reconstitution of T-cells within the context of different transplantation regimens. ResultsProducts for 11 patients have been successfully processed and used for αβT-cell depleted allo-SCT between 2011 and 2013. A ∼4 log depletion of αβT-cells has been observed in the product with a recovery of ∼75% of CD34+ cells. In cohort I, primary engraftment (chimerism > 95%) was 40%. Engrafted patients showed a rapid reconstitution of γδT-cells and αβT-cells with a broad αβT-cell repertoire as determined by spectratyping. Therefore the next cohort was dose-intensified and the CD19-depletion omitted (cohort II). 75% of cohort II showed a swift engraftment. Again a dominance of γδT-cells was observed which associated with a rapidly reconstituting αβT-cell repertoire. Omitting CD19-depletion did not result in severe EBV-reactivations. One patient had an EBV reactivation under a short course of prednisone; however EBV was rapidly cleared after tapering steroids. In order to further increase engraftment cohort III was additionally treated with an early application of ATG (day -10/-9) and no graft failure has been observed so far. ConclusionαβT-cell depletion is feasible in the context of MRD and MUD. An intensified conditioning with additional host T-cell depletion seems to be beneficial for a profound engraftment. αβT-cell depletion associates with a swift and dominant reconstitution of γδT-cells as well as a rapidly restoring αβT-cell repertoire which is able to control viral re-activations. Disclosures:Kuball:Miltenyi: GMP product development Other.

Haploidentical Stem Cell Transplantation (HAPLO-HSCT) With High Dose Cyclophosphamide Post-Transplant (HD-CY) As Gvhd Prophylaxis In High Risk Hematologic Malignancies: Multicentric Spanish Experience

IntroductionAllogeneic transplantation is the only curative option for patients with high risk hematologic malignancies. Only one third of them have an HLA identical sibling donor and around 60-70% will find an unrelated donor, that´s why HAPLO-HSCT offers a therapeutic option to most of these patients with the advantages of quick availability, easy programation and logistics, and a committed donor. Patients and methodsWe retrospectively evaluate the results of HAPLO-HSCT with reduced conditioning or myeloablative regimens and GVHD prophylaxis based on HD-CY (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5 performed in GETH centers. ResultsFrom Dec-2007, 80 HAPLO-HSCT have been done in 14 centers. Median age was 37 years (16-66), 67.5% were males and all were in advanced phases of their disease or presented high risk features (29 Hodgkin´s, 22 AML, 9 ALL, 8 MDS, 5 NHL, 4 myeloma and 2 myelofibrosis). Previous HSCT has been employed in 65%, autologous in 38 and allogeneic in 15 (5 siblings, 3 unrelated and 7 cord blood transplants), and in 35% the HAPLO-HSCT was their first transplant. Disease status at HAPLO-HSCT was CR in 45%, with persistent disease in 55%. Bone marrow was the graft source for 51% and peripheral blood for 49%, non T-cell depleted in all cases. The haploidentical donor was the patient´s mother (21), father (7), brother/sister (35) or offspring (17). Non-myeloablative conditioning was employed in 77.5% and myeloblative in 22.5%. Median neutrophils engraftment was reached at day +18 (13-45) and platelets >50K at day +27 (11-150). Main toxic complications were grade II-III muchositis in 36%, febrile neutropenia in 75% and CMV reactivations in 62%, with a transplant related mortality rate of 12.5% at day +100 and 19% at 6 months post-transplant. Acute GVHD grade II-IV affected to 24/73 patients at risk (33%), with grade III-IV in 10/73 (14%). Chronic GVHD was present in 12/51 (24%), being extensive in 6/51 (12%). After a median follow-up of 9 months (0.3-49), 26/80 patiens have died due to relapse in 13, infections in 10 and GVHD in 3 cases. Event-free survival and overall survival at 1 year were 48% and 60% respectively. Immune reconstitution was fast and complete in those evaluated. ConclusionsHAPLO-HSCT with HD-CY is a useful tool in the treatment of high risk hematologic malignancies, rendering long-lasting remissions with limited toxicity, low GVHD incidence and early immune reconstitution. Disclosures:No relevant conflicts of interest to declare.

Haploidentical Stem Cell Transplantation (HAPLO-HSCT) With Reduced Intensity Conditioning (RIC) Regimens and High Dose Cylophosphamide Post-Transplant (HD-CY) As Gvhd Prophylaxis In Patients With Relapsed Or Refractory Hodgkin´s Disease: Multicentric Spanish Experience

IntroductionAllogeneic transplantation is the only curative option for patients with high risk hematologic malignancies. HAPLO-HSCT offers a therapeutic option to most of these patients with the advantages of quick availability, easy programation and logistics, and a committed donor. This procedure has shown promissing results in patients diagnosed with relapsed or refractory Hodgkin´s disease (Burroughs LM et al. Biol Blood Marrow Transplant 2008; 14:1279-1287). Patients and MethodsWe retrospectively evaluate the results of HAPLO-HSCT with RIC regimens (Fludarabine 30 mg/m2 x5 days (-6 to -2), Cyclophosphamide14,5 mg/kg x2 days (-6 to -5), Busulfan IV 3,2 mg/kg x 1-2 days (BUX, days -3 to -2) or 200 cGy TBI on day -1) and GVHD prophylaxis based on HD-CY (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5 performed in GETH centers to patients diagnosed with relapsed or refractory Hodgkin´s disease. ResultsFrom March-2009, 29 HAPLO-HSCT have been performed in patients diagnosed with relapsed or refractory Hodgkin´s disease in 11 GETH centers. Median age was 31 years (18-53), 19 were males and all were in advanced phases of their disease. Autologous HSCT was previously employed in 90% of them, and allogeneic HSCT in 10%. Disease status at HAPLO-HSCT evaluated by PET was complete remission in 8 (28%) and persistent disease in 21 (72%). Bone marrow was the stem cell source in 15 (52%) and peripheral blood in 14 (48%), without T-cell depletion in all cases. The haploidentical donor was the patient´s mother (13), father (2), brother (8), sister (5) or daughter (1). The RIC regimens employed included 1 dose BUX (11), 2 doses BUX (14) or 200cGy TBI (4). Median neutrophils engraftment was day +17 (11-44) and platelets >20K was day +26 (11-150). Main toxic complications were grade II-III muchositis in 50%, febrile neutropenia in 75% and CMV reactivations in 58% with a transplant related mortality rate of 7% (2/29) at day +100 and 17% (5/29) at 6 months post-transplant. Acute GVHD grade II-IV affected to 7/28 patients at risk (25%), with grade III-IV in 3/28 (11%). Chronic GVHD was present in 3/19 (16%), being extensive in 1/19 (5%). After a median follow-up of 9 months (0.3-49), 13/22 (59%) remain alive and in complete remission. Relapse or progression occured in 6/28 (21%). Immune reconstitution was fast and complete in those evaluated. ConclusionsHAPLO-HSCT with HD-CY is a useful tool in the treatment of patients with relapsed or refractory Hodgkin´s disease, rendering long-lasting remissions with limited toxicity, low GVHD incidence and early immune reconstitution. Disclosures:No relevant conflicts of interest to declare.

HLA Haplo-Identical Peripheral Blood Stem Cell Transplantation Using High-Dose Cyclophosphamide Post-Transplantation For Poor Prognosis Or Refractory Acute Leukemia and Myelodysplastic Syndrome: A Prospective Pilot Study At a Single Center

Recently nonmyeloablative, haplo-identical T-cell replete bone marrow transplantation using high-dose cyclophosphamide (CY) post-BMT to control GVHD and prevent graft rejection by inducing bi-directional tolerance was reported. This strategy resulted in promising outcomes with low transplantation-related mortality (TRM) due to a low incidence of GVHD and infectious disease. However, the high relapse rate remained a major problem in high-risk hematological disease. We therefore planned a prospective pilot study of myeloablative or reduced intensity HLA haplo-identical allogeneic hematopoietic cell transplantation (HCT) using reduced doses of CY post-transplantation, followed by administration of peripheral blood stem cells (PBSC) instead of bone marrow, for those with a poor-prognosis or refractory leukemia and MDS.As of 30 June 2013, 17 patients had been enrolled in this prospective pilot study. Patients with a poor prognosis or refractory leukemia and MDS requiring prompt HCT were eligible for this study if they lacked a donor who was serologically HLA-identical or had a single antigen mismatch.The conditioning regimen consisted of fludarabine 15 mg/m2 and cytarabine 2.0 g/ m2 twice a day on days -11 and -10, thymoglobulin 2.0 mg/kg on days -8 and -7, and fludarabine 30 mg/ m2/day with intravenous busulfan 3.2 mg/kg/day on days -6 to -3 (n=11) or melphalan 100 mg (n=6) on day -2. Nine patients received a single dose of 25 mg/kg CY on day 3 and eight patients, double doses of 25 mg/kg CY on days 3 and 4 post-peripheral blood stem cell transplantation (PBSCT). Tacrolimus and oral mycophenolate mofetil was started after the completion of high-dose CY post-HCT.The median age of patients was 42 years (range 18–65). Disease diagnoses included AML (n=14), ALL (n=2) and MDS (n=1). In 12 (71%) of 17 patients, disease status was non-remission, with active disease at the time of PBSCT. Eight (47%) of 17 patients had a history of prior HCT. Donors were partially HLA-mismatched (haplo-identical) first degree relatives of the patients and differed from the patients at a median of 3/8 HLA loci in both the HVG and GVH directions. All patients received G-CSF-mobilized, unmanipulated PBSC containing a median of 3.0 x 106 CD34+cells/kg and 1.4 x 108CD3+T-cells/kg.Non-infectious fever due to PBSC infusion occurred in 14 (82%) patients and persisted until day 2 in most patients. Except for one patient who died soon after PBSCT due to disease progression, full donor T-cell chimerism was achieved at day 30 following PBSCT in all patients. Nine of 17 developed grade II-III acute GVHD. None developed grade IV acute GVHD. Three of nine patients receiving a single dose of CY, but only one of eight patients receiving double doses of CY developed grade III acute GVHD. In 12 of 16 evaluable patients, CMV reactivation was observed by day 100. Three patients developed CMV disease. BK virus cystitis occurred in four of eight patients receiving double doses of CY, however, none receiving a single dose of CY post-PBSCT developed BK virus cystitis. The cumulative incidence of TRM at one year was 13%. At one year, the overall survival of all patients, and the percentages of patients in remission and those with active disease were 43%, 75% and 31%, respectively.The results of our study demonstrated that our HLA haplo-identical transplantation using high-dose CY post-PBSCT resulted in a low incidence of TRM and was feasible even in patients with high-risk acute leukemia and MDS as an alternative stem cell source. Of note, the dose of CY post-PBSCT affected the incidence of both BKV cystitis and severe GVHD. The optimal dose of CY following HLA haplo-identical PBSCT and preparative regimen should be further explored to establish a standard regimen. Disclosures:Nakamae:Otsuka Pharmaceutical Co., Ltd.: Honoraria, Travel/accommodations/meeting expenses, Travel/accommodations/meeting expenses Other; Kyowa Hakko Kirin Pharma, Inc.: Research Funding, Speakers Bureau, Travel/accommodations/meeting expenses Other. Off Label Use: Mycophenolate mofetil was used as one of drugs for acute GVHD prophylaxis. Koh:Kyowa Hakko Kirin Pharma, Inc.: Travel/accommodations/meeting expenses Other. Nishimoto:Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Pharma, Inc.: Travel/accommodations/meeting expenses Other. Nakashima:Kyowa Hakko Kirin Pharma, Inc.: Travel/accommodations/meeting expenses Other. Nagasaki:Kyowa Hakko Kirin Pharma, Inc.: Travel/accommodations/meeting expenses Other. Nakane:Otsuka Pharmaceutical Co., Ltd.: Honoraria, Speakers Bureau; Kyowa Hakko Kirin Pharma, Inc.: Speakers Bureau, Travel/accommodations/meeting expenses Other. Nakamae:Kyowa Hakko Kirin Pharma, Inc.: Research Funding, Travel/accommodations/meeting expenses Other. Hino:Sanofi K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinyaku Co.,Ltd.: Honoraria, Research Funding; Kyowa Hakko Kirin Pharma, Inc.: Honoraria, Research Funding, Travel/accommodations/meeting expenses Other.

An Alternative Method for Myeloablative Allogenic Stem Cell Transplantation in Hematological Malignancies: Reduced BUCY2 and GCS-F Primed Bone Marrow Stem Cells

AbstractAbstract 4138 Background:Allogeneic stem cell transplantation (ASCT) is the standard treatment for several diseases. However, the morbidity and mortality associated with the procedure limit its widespread use in developing countries. We implemented a novel conditioning method to reduce complications and improve survival. We hypothesized that the dose reduction in this regimen (about 20–25%), would preserve its myeloablative and immunosupressive effect, but with significantly reduced toxicity and mortality. Objectives:Describe clinical characteristics, toxicity, frequency of GVHD and survival of adult mexican patients undergoing ASCT using reduced BUCY2 and G-CSF primed bone marrow as a conditioning method. Material and Methods:Prospective cohort study of patients with matched related donor allotransplant using this new method from November 1999 to December 2011. Stem cell collection was obtained from iliac crests by multiple aspiration. The donor received GCS-F (10μg/kg/day) 3 to 5 days prior to harvest. The conditioning included: Busulfan 12mg/kg PO (3mg/kg/d in -7, -6, -5, -4), Cyclophosphamide 80mg/kg IV (40mg/kg/d in −3, −2), and GVHD prophylaxis with Cyclosporine A (1.5mg/kg bid, beginning at -1: target 200–300 ng/μL) and a short course of Methotrexate. All patients received antimicrobial prophylaxis once the neutrophil count reached < 500/μL. Weekly CMV antigenemia was performed until the 4th month post-transplant. Neutrophil and platelet engraftment were defined as the first of three consecutive days with a neutrophil count □ 0.5 × 109/L and □ 20 × 109/L without transfusion, respectively. Transplant related mortality was defined as any death directly attributed to the conditioning regimen, to aplasia during transplant and/or to infectious or GVHD complications. Engraftment failure was defined as the inability to achieve neutrophil and platelet engraftment during the first 28th days postransplant. Statistical Analysis:Descriptive analysis was used for continuous and categorical variables. Kaplan–Meier curves to asses overall survival, and central tendency measures to analyze: general features, time of engraftment and hospitalization days (SPSS 17.0). Results:29 patients undergoing ASCT with reduced BUCY2 and GCS-F primed-bone marrow were included. All had a HLA-matched related donor, median age of 29 years. 19 patients (65.5%) were male. The diagnosis was: MDS in 10 patients (34.5%), CML in 9 (31%), ALL in 6 (20.7%), AML in 2 (6.9%) and PNH in 2 (6.9%). The median CD34+ transfused cells: 1.9 × 106/kg. Median time to neutrophil and platelet recovery: 20 days (range 14–29) and 15 days (range 7–36), respectively. All patients engrafted. The most common toxicity was mucositis (79.3%) with grade III-IV in 48.3% of cases. Hepatic (41.4%) and renal (48.3%) toxicities, were mild, transient and easily managed with support measures (table 1).Acute and chronic GVHD developed in 6.9% and 34.5% of patients, respectively. There was no mortality at 30 days, and 100-day mortality was 7% (2 patients), attributed to infectious complications. Transplant related mortality was 10% (3 patients) and 5-year overall survival was 70.3%.Table 1Grade IGrade IIGrade IIIGrade IVTotalMucositis3 (10.3%)6 (20.7%)10 (34.5%)4 (13.8%)23 (79.3%)Hepatic toxicity4 (13.8%)3 (10.3%)5 (17.2%)012 (41.4%)Nausea11 (37.9%)5 (17.2%)1 (3.4%)017 (58.6%)Vomit11 (37.9%)2 (6.9%)0013 (44.8%)Diarrhea5 (17.2%)3 (10.3%)1 (3.4%)09 (31.0%)Renal toxicity11 (37.9%)3 (10.3%)0014 (48.3%) Discussion and conclusions:Our results show some advantages compared with standard regimens: noteworthy 5-year OS (70%), similar time to engraftment as for HSC from peripheral source and low GVHD incidence. There was a low TRM (10%), compared to myeloablative regimens, and probably lower than reduced intensity transplants. Main toxicities were grade II-III mucositis and hepatic toxicity, without serious clinical significance. Also, there was a low incidence of acute GVHD (6.9%) probably related to reduced tisular toxicity associated to the conditioning dose reduction, as well as the use of primed bone marrow. In conclusion, this strategy preserves an immunosuppressive and cytotoxic effect allowing eradication of the malignant clone with adequate bone marrow engraftment, acceptable toxicity, low incidence of GVHD and low TRM, representing a new alternative for ASCT. Disclosures:Armengol-Alonso:European Society of Clinical Oncology: Fellowship Other. Neme-Yunes:Agrupación Mexicana para el Estudio de la Hematología: Employment.

Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML)

AbstractStrategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic stem cell transplantation (SCT). Azacitidine (AZA) up-regulates the expression of tumor Ags on leukemic blasts in vitro and expands the numbers of immunomodulatory T regulatory cells (Tregs) in animal models. Reasoning that AZA might selectively augment a GVL effect, we studied the immunologic sequelae of AZA administration after allogeneic SCT. Twenty-seven patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia were treated with monthly courses of AZA, and CD8+ T-cell responses to candidate tumor Ags and circulating Tregs were measured. AZA after transplantation was well tolerated, and its administration was associated with a low incidence of GVHD. Administration of AZA increased the number of Tregs within the first 3 months after transplantation compared with a control population (P = .0127). AZA administration also induced a cytotoxic CD8+ T-cell response to several tumor Ags, including melanoma-associated Ag 1, B melanoma antigen 1, and Wilm tumor Ag 1. These data support the further examination of AZA after transplantation as a mechanism of augmenting a GVL effect without a concomitant increase in GVHD. The trial was registered at http://isrctn.org as #ISRCTN36825171.

Haploidentical Transplant with High Dose of Unmanipulated CD34, in Vivo T Cell Depletion with Alemtuzumab and Ric Regimen. Good Engraftment Rate, Low GVHD Incidence and Encouraging Survival. Single Center Experience in Colombia

Haploidentical Transplant with High Dose of Unmanipulated CD34, in Vivo T Cell Depletion with Alemtuzumab and Ric Regimen. Good Engraftment Rate, Low GVHD Incidence and Encouraging Survival. Single Center Experience in Colombia

High Absolute T Regulatory Cell Counts and Resistance of CD8 Central Memory Cells to Killing by Fludarabine Predicts for Poor Responses to DLI in the Context of a Pre-DLI Lymphoreduction Strategy

Abstract 1912The response to donor leukocyte infusions (DLI) can be improved by the use of pre-DLI lymphoreduction. The mechanism underlying this is poorly explained. In order to investigate this further we examined the T cell subsets in patients entered into a clinical trial testing the utility of pre-DLI lymphoreduction using fludarabine.Inclusion criteria were: 1. mixed chimerism (MC) in whole blood (< 95% donor), 2. previous reduced intensity transplant for a haematological malignancy or failure to respond to a previous dose of DLI and 3. a lymphocyte count of >1.0 × 109/l. Patients consenting to the trial received three doses of oral fludarabine as an outpatient at 25mg/m2 on day −7, −6 and −5. DLI was given on day 0. The starting dose of DLI for patients with MC alone was 5 × 105 CD3/kg (unrelated donor) or 1 × 106 CD3/kg (sibling donor). Higher doses were used in those with evidence of both disease and MC. Samples were collected at D-7, D0, D+7, D+28, D+60 and D90 for T cell subset analysis.Samples were analysed for a combination of markers for CD4, CD8 and T regs using multicolour flow cytometry. CD4 and CD8 were analysed for subset: central memory (CM 62L+RA-), effector memory (EM 62L-RA-), naïve (N 62L+RA+) and effectors (EFF 62L-RA+). Tregs were characterised as CD4+25hi127loFOXP3+.The study was a phase II pilot study with 18 patients required. At the end of the trial, 17 patients were eligible for analysis (1 early death due to leukaemia relapse). The disease categories were: AML (7), ALL (1), MDS (4), T-PLL (1), HD (2), DLBCL (1) and MCL (1). Only 2 patients had co-existing evidence of disease (morphological relapse of AML, 2% positive by immunophenotyping in T-PLL). The median age was 55 years (range: 22–66). There were 10 males and 7 females. 12 patients had HLA-matched sibling donors and 5 had 10/10 matched unrelated donors (UD). The majority of patients (13) had conditioning with fludarabine, melphalan and alemtuzumab, with cyclosporine post transplant.Each received a single dose of DLI on the trial. The median time to DLI post-transplant was 7.4 months (range: 4–11.7). The median percentage of donor chimerism pre-DLI was 85% (range: 18–93). The median lymphocyte count pre-DLI was 1.3 (range: 1.0–3.8).Of the 17 eligible patients, 11 (65%) responded to a single dose of DLI (CR=9 (>95%), PR=2 (chimerism 94% donor at study end)). Five patients developed GvHD – grade 1 (n=2), grade 2 (n=1), grade III/IV (n=2). GVHD resolved completely in all cases. 3 patients reactivated a virus (CMV, 2 × EBV), although not all required treatment.No pre-DLI clinical factor had a significant impact on DLI responsiveness including: disease type, age, gender, donor type, product source (sibling, UD), pre-DLI chimerism, pre-DLI lymphocyte count, time to DLI or source of DLI (mobilised or non-mobilised cells). Post DLI GvHD did not have a significant association with response.There was a significant decrease in the lymphocyte count, as well as the absolute numbers of CD8 (unpaired t test, p<0.0001) and CD4 (unpaired t test, p<0.005) cells between day D-7 and D0 in all patients. The absolute number of Tregs was not significantly different between D-7 and D0, however, DLI responders had a significantly higher absolute Treg count at all time points compared to non-responders (unpaired t test, p<0.005). In addition, DLI responders had a significantly higher Treg/CD8 ratio of means compared to non-responders at all time points (unpaired t test p<0.05). There were no significant differences in CD4 or CD8 counts between responders and non-responders. CD8 subset analysis showed a significant reduction in the CD8 CM cells in the responders (unpaired t test, p<0.001) between D-7 and D0, while this was not seen in the non-responders (p=0.3).In conclusion, lymphoreduction with fludarabine pre-DLI is associated with high response rates to a single dose of DLI and a low incidence of GvHD. Interestingly, Treg counts do not appear to be reduced by fludarabine and higher Tregs counts at all time points were associated with responsiveness to DLI. Conversely, CD8 subsets were reduced following fludarabine, but relative resistance in CD8 memory cells was associated with poor responses to DLI. These findings suggest future strategies to test novel delivery of DLI as well as potential mechanisms of action. Disclosures:No relevant conflicts of interest to declare.

Low Incidence and severity of Graft-Versus-host disease after outpatient allogeneic Peripheral Blood Stem Cell Transplantation employing a Reduced-Intensity Conditioning

Abstract 4707 Introduction.Graft-versus-host disease (GVHD) is the most common complication of allogeneic hematopoietic cell transplantation (alloHCT) and may affect the transplant outcome. Its incidence is higher when the preparative regimen used is a non-myeloablative and the stem cell's source is peripheral blood after mobilization. Objective.Demonstrate the 10 years incidence of GVHD in 2 Mexican transplant centers using peripheral hematopoietic stem cell transplantation (PHSCT) in related donors after non-myeloablative conditioning. Patients and methods.Three hundred and four patients with hematological and non-hematological malignancies that underwent outpatient PHSCT after non-myeloablative conditioning between October 1998 and July 2008 were included. The age ranged between 1 and 71 years (median of 30.5). One hundred and eighty-five patients were men and 119 women. The median of cells CD34+ infused was 4.9 × 106/kg (0.23-17.70).They received cyclosporine 4mg/kg per day and intramuscular methotrexate 5mg/ m2 in days +1, +3, +5 and +11 for GVHD prophylaxis. Results.Two hundred thirty-nine (80%) patients were successfully engrafted. The conditioning regimen was delivered as an outpatient procedure in all individuals. One hundred and fifty-four patients (64%) developed acute and/or chronic GVHD. Sixty four patients (26.7%) developed acute GVHD, 50 (20.9%) developed chronic GVHD, and 40 (16.7%) with acute GVHD progressed to chronic. Twenty seven (26%) patients who developed acute GVHD were grade III or IV, and 30 (33.2%) of chronic GVHD patients presented in the extensive way. GVHD was the cause of dead in 40 patients; 24 of acute GVHD and 16 of chronic GVHD, even when immunosuppressive therapy (high dose steroids and rituximab) was used. Last death was 24 months ago; subsequently, alemtuzumab was included in GVHD treatment. The cumulative incidence of acute GVHD was 37.6%. Conclusion.The higher incidence of GVHD reported in other studies after PBSC transplant seems to decrease if non-myeloablative conditioning regimens are used. We suggest that the combination of a reduced-intensity conditioning transplant and the outpatient procedure is the responsible for the low incidence of GVHD in our patients. Disclosures:No relevant conflicts of interest to declare.