Donor Lymphocyte Transfusions Research Articles

Commentary on” The FLAMSA Concept Past and Future”

The FLAMSA concept was designed as a conditioning regimen for acute leukemia separating leukemia therapy from reduced intensity conditioning. Leukemia treatment consists of a short and intensive chemotherapy followed after a 3-day rest by conditioning with intensive immune suppression and reduced intensity of myeloablative (stem cell toxic) treatment preferably total body irradiation (TBI). Furthermore, it included T cell depletion “in vivo” with antithymocyte globulin (ATG) for prophylaxis of graft-versus-host disease followed by restitution of immunity by the transfusion of donor lymphocytes

The FLAMSA concept—past and future

The FLAMSA reduced intensity (RIC) concept, also known as "sequential therapy", is a conceptual platform for the treatment of leukemia separated in several parts: induction therapy, a sequence of antileukemic and immunosuppressive conditioning for allogeneic stem cell transplantation, and immune restitution supported by donor lymphocyte transfusions. The antileukemic part consists of fludarabine, cytosine arabinoside, and amsacrine (FLAMSA); non-cross reactive agents like fludarabine and amsacrine have been successfully used in cases of refractoriness and relapse. Immunosuppressive conditioning and transplantation follow after only 3days of rest. This way, the toxicity of allogeneic transplantation could be reduced and the anti-leukemia effects by using allogeneic immune cells could be optimized. This review summarizes available data on efficacy and toxicity of this approach. Further, possible strategies for improvements are discussed in order to provide better chances for elderly and frail patients and patients with advanced and high-risk disease. Among others, several new agents are available that target molecular changes of leukemia for induction of remission and allow for bridging the time after transplantation until adoptive immunotherapy becomes safe and effective.

Allogeneic Hematopoietic Stem Cell Transplantation Offers Similar Outcome after Myeloablative and Sequential Conditioning Regimen in Patients with Primary Refractory or Relapsed Acute Myeloid Leukemia: A Study from the Societe Francaise De Greffe De Moelle Et De Therapie Cellulaire (SFGM-TC)

Introduction: Acute myeloid leukemia (AML) patients failing to achieve complete remission or relapsing after intensive chemotherapy, have a dismal prognosis. In this setting, allogeneic hematopoietic cell transplantation (HCT) is a curative salvage therapy, but myeloablative conditioning (MAC) transplants are usually associated with high non-relapse mortality (NRM). Promising results have been reported using a sequential treatment approach (SEQ) consisting of cytoreductive chemotherapy, followed by reduced-intensity conditioning (RIC) and prophylactic transfusion of donor lymphocytes. Nevertheless, for young patients with refractory or relapsed AML, there is no definitive evidence to prefer SEQ rather than MAC HCT. The aim of this study, analysing the data from the SFGM-TC, was to compare the results of these two approaches in patients transplanted in AML not in complete remission.Methods: Inclusion criteria: a) HCT performed from January 2006 to December 2013 b) Patient age up to 50 years (y), c) Transplant for refractory or relapsed AML, c) SEQ or MAC regimen d) Matched sibling donor, matched or mismatched unrelated donor.PatientsÕ characteristics: 108 patients (median age: 38 y (range 1-50)) were analysed. Eighty-eight percent of patients were diagnosed with de novo AML and 12% with secondary AML. At diagnosis, 4% of patients were classified favorable, 33% Intermediate-1, 25% intermediate-2, and 38% adverse, according to the European LeukemiaNet (ELN) score. Before transplant, patients received a median of 2 lines of chemotherapy (range 0-3). At transplant, 53% of patients were in primary induction failure (PIF), 43% in relapse, and 4% were never treated and the median of bone marrow (BM) and circulating blast percentages were 19% (range 0-96) and 2% (range 0-93) respectively.Transplant modalities: 60 patients received a SEQ approach (cytoreductive chemotherapy with FLAMSA (N=40) or with clofarabine (N=20) + high-dose cytarabine, followed by RIC combining cyclophosphamide (CY) with 4 Gy total body irradiation (TBI) or with busulfan (BU)) while 48 patients received a MAC regimen (CY combined with 10-12 Gy TBI (N=20) or with BU (N=28)). Forty-one percent of patients were transplanted from a sibling and 59% from an unrelated donor. Stem cell source was peripheral blood in 76% and BM in 24%.Results: MAC and SEQ groups had similar ages, ELN scores, numbers of pre-transplant chemotherapy lines and status at transplant. They only differed from the percentage of circulating blasts at transplant (median 1%, range (0-66) in MAC group vs 8%, range (0-93) in SEQ group, p=0.004) and from the time between diagnosis and transplant (median 164 days, range (62-610) and 239 days range (61-3702) respectively, p=0.036). In univariate analysis, type of conditioning regimen (MAC versus SEQ) did not impact post-transplant outcomes: 2-year overall survival (OS) was 37.2% (95% confidence interval (CI): 23.8-50.7%) in MAC patients vs 32.9% (95%CI: 21.4-44.9%) in SEQ patients (p=0.43); 2-year cumulative incidence of relapse was 56.7% (95%CI: 42.2-71.1%) vs 50.1% (95%CI: 37.2-63%) respectively (p=0.89) and 2-year non-relapse mortality was 14.7% (95%CI: 4.5-25%) vs 16.7% (95%CI: 7.1-26.2%) respectively (p=0.46). Grade II-IV acute graft versus host disease (GVHD) occurred more frequently in patients who received MAC: 66.7% vs 36.7% in the SEQ group (p=0.002), there was also a trend for more chronic-GVHD (41.7% vs 25%, p=0.066). Multivariate analysis identified high number of chemotherapy lines (hazard ratio (HR) 1.66, 95%CI 1.11-2, p=0.013) and high percentage of circulating blasts at transplant (HR 1.01, 95%CI 1.003-1, p=0.011), as factors independently associated with poor OS. Heterogeneity analyses performed for OS identified significant interactions between the type of HCT conditioning and BM blast percentage (p=0.01), or CD34+ cell dose (p=0,04), analysed as categorical variables with cut-offs at medians (20% and 6.106/kg, respectively). Though limited by low patients numbers, subgroup analyses suggested that SEQ HCT conferred a higher risk of death in patients with BM blasts > or = to 20% (HR=1.42), but a lower risk (HR=0.59) with BM blasts <20%, compared to MAC.In conclusion, these data confirm that HCT is a valid salvage therapy for relapsed or refractory AML. In patients up to 50 years, both MAC and SEQ conditioning regimens offer similar outcomes, without increased NRM after MAC transplant. [Display omitted] DisclosuresMohty:Janssen: Honoraria; Celgene: Honoraria.

Long-term results of adjuvant donor lymphocyte transfusion in AML after allogeneic stem cell transplantation.

Adjuvant transfusion of donor lymphocytes (aDLT) may reduce the risk of relapse after allogeneic stem cell transplantation in high-risk AML. We performed a retrospective analysis on the safety and efficacy of aDLT in a cohort of 46 patients. To be eligible for aDLT, patients had to be in CR for at least 120 days from transplantation, off immunosuppression for ⩾30 days and free of GvHD. Thirty-four patients with similar disease characteristics and fulfilling the same selection criteria served as controls. Median follow-up among aDLT recipients was 7.2 years. Ten patients (22%) relapsed inspite of aDLT, as compared with 53% in the control group. Induction of GvHD was the main complication. However, non-relapse mortality was low with patients dying from infection (n=2), severe chronic GvHD (n=1) and secondary malignancy (n=2). At the time of analysis, 31/46 aDLT recipients were alive in CR at a median of 5.7 years after first aDLT. Overall survival at 7 years after transplant was 67% as compared with 31% in the control group (P<0.001). In conclusion, aDLT is safe, when given in escalating doses to a well predefined group of patients. Long-term survival can be achieved.

Donor Lymphocyte Infusion After Allogeneic Hematopoietic Stem Cell Transplantation

Donor lymphocyte infusion, a rescue therapy after hematopoietic stem cell transplantation, has been increasingly adopted, as modalities of stem cell transplantation have widened. First described as donor lymphocyte transfusion or cell therapy, it consists of infusion of donor lymphocytes, collected in steady state or after growth factor enhancement. As in literature the most used name is donor lymphocyte infusion, we'll adopt it here. Its most striking efficacy is observed in patients with chronic myelogenous leukemia, who relapsed after allogeneic stem cells transplantation. However, graft-versus-host disease, its main complication, may still hamper its feasibility.

Treatment of leukemia with immunized donor cell infusion after nonmyeloablative haploidentical bone marrow transplantation

This study was purposed to investigate the therapeutic effects of early transfusion of immunized donor lymphocytes after haploidentical transplantation by means of mouse model of nonmyeloablative haploidentical bone marrow transplantation. CB6F1 female mouse was served as recipient and C57BL/6 male mouse was served as donor. Each CB6F1 female mouse was subjected to intravenous transfusion with 1×10(6) erythroleukemia (EL9611) cells at day 4 before transplantation, followed with intraperitoneal injection of Ara-C (0.015 g) respectively at day 2 and day 1, then conditioned for BMT with TBI (450 cGy) at day 1 before transplantation. After conditioning (day 0), each of recipients was transplanted with 6×10(7) mixture of bone marrow and spleen cells from the C57BL/6 mice, and was infused with 6 × 10(7) immunized donor lymphocytes at day 15 after transplantation. All treated animals were evaluated for survival, development of leukemia and aGVHD. The donor CD3(+) cell chimerism and sex determining region Y gene (SRY)in recipients were monitored periodically after transplantation. The results showed tht all mice with only inoculation of 10(6) EL9611 cells survived for 15 ± 1 days (n = 4); all mice of other groups obtained the varying degrees of implantation. SRY could be detected at day 30 and 60 after transplantation. The chimerism of donor CD3(+) cells in mixed bone marrow transplantation (MT) group at day 14, 30 and 60 respectively reached 17.95% ± 12.03%, 37.34% ± 2.78% and 47.06% ± 6.1%. In donor lymphocyte infusion (DLI) group it reached 69.78% ± 12.62%, 75% ± 15.97%, 83.41% ± 16.07% at day 30, 45 and 60 after transplantation. The mice of MT and DLI group survived for 66.66 ± 1.47 days and 78.2 ± 7.82 days. It is concluded that the high tumor burden before transplantation can affect donor cell engraftment and prognosis.Early post-transplanted infusion of immunized lymphocytes from donor can help to improve the therapeutic efficacy and survival.

On the role of HLA antibodies in hematopoietic stem cell transplantation

While the role of donor-specific antibodies (DSA) in solid organ transplantation is well established, their importance in hematopoietic stem cell transplantation (HSCT) is only now becoming clear. A review of the literature reporting on HLA immunization in HSCT provides ample circumstantial evidence that donor-specific HLA antibodies (DSA) are associated with a 2- to 10-fold increase of graft failure of HLA mismatched HSCT, irrespective the type of the graft, or the patient conditioning. Nevertheless, this is not a condition 'sine qua non', and engraftment has been documented despite the presence of DSA. However, prediction of graft failure based on serology is cumbersome. Although sensitivity and specificity of current solid-phase assays (SPAs) for HLA antibody detection are high, correlation with graft failure remains elusive. When lacking an alternative donor, reduction of strong reacting DSA must be attempted. Unfortunately, results of DSA reduction treatments in HSCT are scarcely reported. Case reports show that persisting DSA after plasma-exchange and immunosuppressive treatment can become negative after a 'last rescue' in vivo absorption with antigen-bearing platelets or donor lymphocyte transfusions. The destruction of engrafting hematopoietic cells by antibodies appears to be an immediate event. Blocking antibody mediated effector functions, e.g. with intravenous immunoglobulin (IvIg), may have additional value, despite IvIg often not reducing the antibody titre. An even less explored aspect of HLA-immunization is the presence of non-DSA antibodies in the host or HLA antibodies emerging post-transplantation. Such antibodies, either causally or as confounders, may be associated with negative transplant outcome. We conclude that HLA antibody assessment should be at the forefront in the treatment handbook of HSCT.

Monitoring the Antileukemic Immune Response with an Active Specific Immunization Strategy

AbstractAbstract 4354 Background:Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach. Objective:To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system. Methods:The myeloid blasts derived from five patients with AML relapsed post allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail. The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. Results:Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with the nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay. Conclusion:The active specific immunization strategy was realized to monitor the antileukemic immune response measured with radioactive and nonradioactive assay system. Disclosures:No relevant conflicts of interest to declare.

Flamsa-RIC Sequential Regimen in High Risk CR1 AML Patients: Preliminary Results From a Pilot Study

AbstractAbstract 4549 IntroductionPatients with high risk AML have poor outcomes. However, the only approach with curative potential remains allogeneic HSCT. With the aim to improve the effect of allogeneic HSCT by sequential use of chemotherapy, RIC regimen and pDLI, we are currently conducting a prospective pilot study for high risk AML. High risk AML was defined by unfavorable cytogenetics, adverse molecular abnormality, secondary AML, and AML requiring 2 induction courses to obtain CR1.After identification of a HLA 10/10 donor, patients are received the sequential regimen consisted of fludarabine (30mg/m2/d), Ara-c (2g/m2/d) and amsacrine (100mg/m2/d) (FLAMSA) chemotherapy for 4 days. After 3 days of rest, RIC regimen consisted of 4 Gy TBI, cyclophosphamide for 2 days (40 mg/kg in case of matched related donors, and 60 mg/kg for unrelated or mismatched donors), and ATG (5mg/kg total dose) (German regimen) or Busulfan 3.2mg/kg/d during 4 days followed by ATG (5mg/kg total dose) (French regimen). The modified regimen has been established after our results in refractory AML patients (ASH 2011, poster 1957). Prophylactic donor lymphocyte transfusion was given from day +120 in patients who were not receiving immunosuppression and were free of GvHD.Our objective is to include 20 patients and to compare with a control cohort of patients with the same high risk AML treated according to the conventional strategy during the same period. ResultsBetween August 2010 and November 2011, we have included 12 consecutive patients in first complete response who underwent an allogeneic HSCT after sequential FLAMSA-RIC regimen with a median follow-up of 12 months (range [7–22]). Nine patients were < 55 years old (median age: 54 [28–64]), 7 patients had an unrelated donor and 5 patients had a related donor. The stem cell source was PBSC for 11 patients and two cord blood unit for 1 patient. Before FLAMSA-RIC regimen, 3 patients had received two induction courses to obtain CR1. All patients had adverse cytogenetics or molecular abnormalities and 1 patient had a secondary AML.At the last follow-up, 6 patients (50%) are alive in CR. (Figure 1.) Four patients (33.3%) died in remission. The cause of death was infection for 2 patients, aGvHD for 1 patient and graft failure for 1 patient. Only one patient died from relapse 6 months after transplantation.Five patients (41.6%) experienced aGvHD and 2 patients (16.6%) had an extensive cGvHD including the patient who has been transplanted with 2 cord blood unit. Six patients (75%) in a group of 8 patients aged > 45 years experienced complications (infection (n=3) and GvHD (n=3)). One patient (25%) from a group of 4 patients aged < 45 years had infectious complication after transplantation. Prophylactic donor lymphocyte transfusion was given in 6 patients, the causes of no administration were GvHD for 2 patients, cord blood unit as stem cell source for 1 patient and 3 patients were dead before 120 days after transplantation. From the 6 patients who had received pDLI, 5 patients are alive in CR and 1 patient died from GvHD. ConclusionThe FLAMSA-RIC regimen before allogeneic HSCT is a new approach for high risk AML. Between 2012 January and 2012 July, 8 additionnal patients have been included and the results for the whole study will be communicated later. Our primary results are promising especially for young patients (< 45 years) who seem to better profit from this sequential FLAMSA-RIC regimen. [Display omitted] Disclosures:Nicolini:Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Impact of anti-HLA antibodies on allogeneic hematopoietic stem cell transplantation outcomes after reduced-intensity conditioning regimens

Anti-human leukocyte antigen (HLA) antibodies are associated with several complications in solid organ transplantations, but their impact after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not yet well defined. To evaluate the relevance of anti-HLA antibodies, we have retrospectively analyzed 107 peripheral blood allo-HSCTs after reduced-intensity conditioning regimen between 2005 and 2010. Acute myeloid leukemia and multiple myeloma were the most frequent malignancies in the cohort. The detection of anti-HLA antibodies was systematically performed in all patients before transplantation. Anti-HLA antibodies were present in 24 patients (22%). There was no significant impact of anti-HLA antibodies on engraftment, incidence of relapse, and incidence of acute graft-vs-host disease. The presence of anti-HLA antibodies was associated with significantly worse overall survival (p = 0.006) and event-free survival (p = 0.024) after stratification on sex. The 3-year probability of overall survival was 34% without anti-HLA antibodies and 16% in their presence. Patients with anti-HLA antibodies had a higher transplant-related mortality associated with life-threatening vascular complications. Our study supports that anti-HLA antibodies should be tested and considered as an important impacting factor for transplantation outcomes after reduced-intensity conditioning allo-HSCT. We recommend its consideration before allo-HSCT in the donor-recipient selection parameters.

Lenalidomide plus donor-lymphocytes infusion after allogeneic stem-cell transplantation with reduced-intensity conditioning in patients with high-risk multiple myeloma

Myeloma relapse is the main cause of death after allogeneic stem cell transplantation. The aim of our observational study was to evaluate the anti-myeloma effect of lenalidomide followed by donor-lymphocyte infusion (DLI) as post-transplantation adoptive immunotherapy. Twelve patients with refractory myeloma were analyzed. The median age at transplantation was 56 years (range, 46-64 years). All patients received reduced-intensity conditioning. Patients were included if progressive or residual disease was observed at day +100 and if no signs of graft-vs-host disease were evident. DLIs were administered after two cycles of lenalidomide. Median dose of lenalidomide was 15 mg (range, 10-25 mg). Patients received a median of six cycles (range, 1-10 cycles). Nine patients (60%) received an escalating dose of DLI. The 1 and 2-year probability of progression-free survival was 75% and 50%, and overall survival was 83% and 69%, respectively. Median overall survival was not reached and median progression-free survival was 23 months. Lenalidomide is well tolerated after allogeneic stem cell transplantation; the combination with DLI did not cause a higher risk of graft-vs-host disease; an immunological synergistic effect was probably present with this strategy. This combination should be evaluated further in a larger cohort of patients.

Outcome of High-Risk and Refractory AML/MDS Patients Receiving a FLAMSA Sequential Chemotherapy Regimen Followed by Reduced-Intensity Conditioning (RIC) and Allogeneic Hematopoeitic Stem Cell Transplantation (allo-HSCT)

Abstract 1957▪▪This icon denotes a clinically relevant abstract Introduction:The FLAMSA sequential chemotherapy regimen followed by RIC for allo-HSCT and prophylactic donor lymphocyte transfusion (pDLT) was introduced a few years ago as a salvage therapy for patients with refractory or high risk AML/MDS. This retrospective analysis aimed to assess the outcome of 40 patients with refractory or high risk AML/MDS who received a salvage FLAMSA sequential chemotherapy (including a subgroup of patients who received a modified FLAMSA regimen incorporating Busulfan instead of total-body irradiation (TBI)). Patients and methods:This series included 30 males and 10 females with a median age of 52 years (range, 32–66) treated in 2 transplant centres in France. Diseases characteristics were as follow: progressive or refractory disease after rescue treatment for first relapse (n=21), early relapse without any further salvage therapy (n=4), and primary induction failure (PIF; n=4). The series also included 7 patients with high risk MDS (IPSS ≥ 1.5), and another 4 patients in first CR but having a very poor prognosis based on cytogenetics features (CR group hereinafter). According to cytogenetics and molecular markers, 12 patients were classified in the “standard risk” cytogenetics risk group, while 21 patients had an unfavourable/poor profile. Data were not available for 7 patients.The FLAMSA regimen included Fludarabine (30 mg/m2/d), cytarabine (2 g/m2/d) and amsacrine (100mg/m2/d) from day −12 to day −9. In addition, priming with G-CSF once daily was done in 12 patients. After 3 days of rest, a RIC regimen was administered. In 28 patients, the RIC regimen included 4 Gy. TBI, ATG 5 mg/kg total dose, and cyclophosphamide (40 mg/kg in case of matched related donors, and 60 mg/kg for unrelated or mismatched donors). In the remaining 12 patients, TBI was replaced by I.V. Busulfan 3.2 mg/kg/d for 4 days. Per protocol, pDLT was planned to be given from day +120 in patients who were not receiving immunosuppression and were free of GvHD. Eighteen patients were transplanted using an HLA identical sibling donor, and 22 received transplant from an unrelated donor (14 matched 10/10 and 8 mismatched 9/10). Results:After allo-HSCT, 39 patients (97.5%) engrafted. In the CR group (n=4), after a median follow-up of 5 months (range, 3–31) all patients were still alive in CR at last follow-up. In the remaining 36 patients, 9 patients developed acute GVHD ≥2 with a cumulative incidence at 3 months of 18% (95%CI, 10–26). At day 90 post HSCT, 23 (64%) patients could achieve hematological CR, and 14 of the 23 remained in CR at last follow-up.After a median follow-up of 6 months (range, 1–60), the 2-years probability of OS was 30% (95%CI, 17–52), and the 2-years probability of PFS was 29% (95%CI, 17–50). The cumulative incidence of disease progression at 1 year was 25% (95%CI, 18–33). Interestingly, none of the patients who received Busulfan instead of TBI as part of the RIC, relapsed. The cumulative incidence of TRM at 3 months and 1 year were 14% (95%CI, 8–20) and 22% (95%CI, 15–29), respectively.In the multivariate analysis (taking into account all relevant parameters such as priming with G-CSF, conditioning with or without TBI, related or unrelated donors, HLA matching and number of previous treatment lines), there was a significantly worsened PFS in patients who received transplant from a mismatched donor (HR=3.6; [95%CI, 1.3–10] p=0.01; below Figure). Also, when considering disease relapse, there was a highly significant impact of the type of RIC regimen (in favour of a FLAMSA regimen without TBI (p<0.0001, Figure). Conclusion:Despite its limitations, results from this analysis confirm that a FLAMSA sequential chemotherapy regimen followed by RIC and allo-HSCT is a valid salvage therapy for patients with refractory or high risk AML/MDS. This study shows that HLA mismatched donors should be avoided in FLAMSA regimen. Moreover a modified FLAMSA regimen incorporating I.V. Busulfan instead of TBI is likely to allow better long-term disease control, warranting prospective evaluation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Early allo-SCT for AML with a complex aberrant karyotype—results from a prospective pilot study

In AML, a complex aberrant karyotype is associated with poor response to chemotherapy and dismal prognosis. We prospectively studied the concept of allogeneic haematopoietic SCT (HSCT), performed early and regardless of response to induction treatment in patients with complex karyotype AML (CK-AML). The preparative regimen consisted of fludarabine, Ara-C and amsacrine (FLAMSA) chemotherapy, followed by reduced intensity conditioning (RIC) 3 days later. In vivo T-cell depletion by anti-thymocyte globulin was used to protect from early GvHD, and prophylactic donor lymphocyte transfusion was given from day+120 to augment the GvL effect, once tolerance was established. Eighteen consecutive patients with CK-AML (median age: 53 years) received HSCT from related (n=7) or unrelated (n=11) donors. Before FLAMSA-RIC, nine patients each had received one and two induction courses. Stage at start of FLAMSA-RIC was CR/CRi (n=8) or persistent disease (n=10). Following HSCT, 16 patients achieved CR. After a follow-up of 51 months, 11 patients are alive in CR, whereas seven have died in remission (n=3), or from leukaemia (n=4). Cumulative incidence of relapse, non-relapse mortality, acute GvHD≥II and chronic GvHD were 0.222±0.098, 0.235±0.104, 0.367±0.120 and 0.481±0.123, respectively. Four-year survival from HSCT is 61%. Early HSCT following FLAMSA-RIC may improve the outcome of this unfavourable AML subgroup.

An Improved Bicistronic CD20/tCD34 Vector for Efficient Purification and In Vivo Depletion of Gene-Modified T Cells for Adoptive Immunotherapy

T-cell-based adoptive immunotherapy is widely used to treat graft rejection and relapse after stem cell transplantation (SCT). However, this approach is hampered by a high risk of life-threatening graft-versus-host-disease (GvHD). Clinical trials have demonstrated the value of suicide genes to modify T cells for the effective control of GvHD. Herewith, we show that the combination of a codon-optimized B-cell antigen (CD20op) with a selection marker based on a cytoplasmic truncated version of the human stem cell antigen CD34 (tCD34) allows the generation of highly enriched gene-modified T cells. We demonstrate coordinate co-expression of both transgenes and high expression of CD20op resulting in an increased susceptibility to Rituximab (RTX)-induced cell death. In addition, T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Long-lasting transgene expression was sustained in vivo after adoptive transfer into Rag-1(-/-) mice. Moreover, gene-modified T cells were quickly and efficiently depleted from peripheral blood (PB) and secondary lymphoid organs of transplanted animals after RTX treatment. These results warrant further steps toward a clinical application of CD20op as a suicide gene for adoptive immunotherapy.

Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

A retrospective analysis was performed of 118 patients with hematologic malignancies who received donor lymphocyte infusions (DLIs) after allogeneic stem cell transplantation (ASCT). Treatment was either given because of hematologic relapse (n = 44), molecular/cytogenetic relapse (n = 52), or other causes (n = 22). Molecular relapse was in most cases based on leukemia lineage-specific chimerism analysis. Patients with acute leukemia and myelodysplastic syndrome showed a 3-year survival of 42% if DLI treatment was given because ofmolecular relapse, compared to 16% in hematologic relapse (P < .006). In multivariate analysis, there was a correlation between response to DLI and nonhematologic relapse (risk ratio [RR] 3.36, P = .001), chronic graft-versus-host disease (cGVHD) (RR = 1.51, P = .005), and late relapse (RR = 2.06, P = .017). The overall incidences of acute GVHD (aGVHD) grades I-II and grades III-IV aGVHD were 33% and 8.5%, respectively. Probability of cGVHD was 33%. The development of aGVHD or cGVHD did not significantly influence the response of DLI in patients with molecular/cytogenetic relapse. However, the development of cGVHD was significantly associated with a better response in patients with hematologic relapse because only 4 of 29 patients without cGVHD responded compared to 7 of 12 with cGVHD (P = .007). The development of cGVHD increased significantly if DLI was given >12 months after ASCT (46% versus 27%, P = .04). In contrast, time between ASCT and start of DLI treatment had no significant influence on the risk of developing aGVHD. To conclude, monitoring of leukemia lineage-specific chimerism is of utmost importance for DLI response after ASCT.

Long Term Follow-up of Patients Treated with mTOR Inhibitors for Severe Chronic Gvhd Following Allogeneic Stem Cell Transplantation.

Abstract 2254Poster Board II-231Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality in hematopoietic transplant recipients. The mammalian target of rapamycin inhibitors (mTOR-I) sirolimus and everolimus are immunosuppressants that have activity in the prevention and treatment of acute GVHD. Sirolimus in combination with calcineurininhibitors (CNI) has also been shown to have activity in chronic GVHD, however at the cost of considerable toxicity. Since mTOR-I exert their action by blocking interleukin 2 receptor signaling and thus arresting the cells at G1 phase they are promising candidates for tolerance induction. In addition, they also have antiangiogenic and antiproliferative activity. We hypothesized that treatment of cGVHD with mTOR-I and without CNI would be more effective in inducing tolerance than the combination of both and would have a lower toxicity profile. In this retrospective analysis we report 31 consecutive patients (pts) with severe cGVHD according to the NIH consensus, in whom treatment with mTOR-I (everolimus n=20; sirolimus n=11) was initiated between 2004 and 2006, with a median follow-up after initiation of mTOR-I therapy for surviving pts of 46.5 months. Three pts terminated treatment prematurely (within 3 months after starting therapy) because of adverse events (1xsevere cough; 1xhypertriglyceridemia; 1xmouth ulcers) and they were excluded from further analysis. Of the remaining, 12 pts had de novo or quiescent cGVHD, 5 progressive cGVHD developing from acute GVHD and 11 had cGVHD following donor lymphocyte transfusions. Organ involvement included skin (scleroderma) in 21, lungs in 12, mucous membranes in 9, liver in 6, gut in 2 and eyes in 4 pts. Eighteen pts received mTOR-I in combination with steroids, three received a monotherapy and 7 pts received additional immunomodulatory drugs, no CNI however. To further reduce the risk of adverse events drug trough levels were monitored and the dosing was adjusted to low therapeutical levels (3-8 ng/ml). Eleven pts each had a complete and partial response, respectively, with an overall response rate of 78%. At the time of analysis 6 of the 15 surviving pts were complete off immunosuppression and steroids could be tapered and stopped in 5 additional pts. Best responses were seen in pts with de novo or quiescent cGvHD. Thirteen pts have died. Causes of death were: progressive cGvHD (n=5) relapse of the underlying disease (n=4), infections (n=2), secondary malignancy (n=1), unknown (n=1). No difference was observed in everolimus- and sirolimus-treated pts. With a median treatment time of 24 months (range 3 – 54 mo) the major adverse events possibly related to mTOR-I were hyperlipidaemia and impaired wound healing, especially in pts with ulcerative lesions of skin or mucous membranes. Only one of the pts developed thrombotic microangiopathy (TMA) and infectious complications were rare. In pts treated in combination with steroids frequently a shortened prothrombin time has been observed, suggesting an increased risk for thomboembolic events. Controlled trials comparing this approach with alternative strategies are warranted. Disclosures:Off Label Use:Everolimus and Sirolimus for the treatment of chronic GVHD. Jedlickova:Wyeth: Research Funding.

Long-Term Results of Donor Lymphocyte Transfusions for the Treatment of Relapse After Allogeneic Transplantation for Chronic Myelogenous Leukemia - Is There a Role for Immunotherapy in CML?.

Abstract 2258Poster Board II-235The long-term outcome of donor lymphocyte infusions (DLI) as first line treatment of relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation was studied and compared to other treatment modalities. Forty five patients were treated with DLI and 36 without DLI. Patients given DLI first did not differ significantly from those given other treatments in gender, age, donor, gender and histocompatibility of the donor, source of stem cells (blood vs marrow), depletion of T cells, stage of the disease, time from diagnosis and year at the time of transplant, acute and chronic graft-versus-host disease (GVHD) and remission duration. The survival of the DLI group was 66.4 percent at 10 and 15 years after transplantation, it was 42.2 and 23.8 percent respectively in the non-DLI group (p=0.019, log rank). Excluding patients with early relapse in the first 6 months the survival of the DLI group was 78 percent at 15 and 20 years, 70 and 26 percent respectively of the non-DLI group (p=0.009). Recurrent leukemia was the predominant cause of death in both groups (11 of 14 patients of the DLI-, 21 of 23 patients of the non-DLI group). Three patients in the DLI group died of recurrent infections, bronchiolitis obliterans (BO) and heart failure respectively, and 2 patients of BO in the non-DLI group. The proportion of surviving patients with positive PCR for bcr/abl was not different in both groups (2 of 13 patients of the non-DLI and 7 of 30 patients in the DLI group). The better survival of the DLI group indicates a better control of residual leukemia by a persistent immune effect. The response to DLI was improved by simultaneous treatment with low doses of interferon-a and GM-CSF. The combination of these cytokines without DLI and transplantation remains to be defined in patients that do not tolerate or respond incompletely to Imatinib. The role of immunotherapy for induction and maintenance of remission is well established in transplant patients. Disclosures:No relevant conflicts of interest to declare.

Stem cell transplants for patients with relapsed/refractory leukaemia

Today the indication for allogeneic stem cell transplantation for a high-risk leukaemia in first remission is well defined by most centres. In patients with primary refractory leukaemia the indication is controversially discussed. Similarly patients with relapse and advanced disease have a poor prognosis with chemotherapy, but also with transplantation. Finally more elderly patients with comorbidities seek help from transplantation, most of them in advanced and otherwise refractory disease. The results are reviewed. The role of alloimmunity in the control of leukaemia has been defined and pretransplant conditioning treatment could be reduced to less intensive protocols. Graft-versus-leukaemia reactions have been demonstrated with the transfusion of donor lymphocytes. Using nonmyeloablative regimens allogeneic stem cell transplantation could be offered to elderly patients, the majority of patients with acute myeloid leukaemia and myelodysplastic syndromes. The use of antibodies and radio-immuno-therapy has improved the treatment of lymphoid malignancies. Cord blood transplants have shown improved results with double transplants. The superiority of maternal donors indicates a role of the donor's immune repertoire. Taking advantage of alloimmune reactions and reduced intensity conditioning allogeneic stem cell transplantation has become successful even in elderly and fragile patients. The combination of molecular monitoring, targeted therapy and transplantation as a form of immunotherapy may improve the results of leukaemia treatment further.

Tolerance in DLA-haploidentical canine littermates following CD6-depleted marrow transplantation and donor lymphocyte transfusion

Donor lymphocyte transfusions (DLT) are effective in the treatment of leukemia after allogeneic stem cell transplantation. Graft-vs-host-disease (GVHD) is the major risk factor of DLT. In dog leukocyte antigen (DLA)-identical littermate dogs, DLT given within 3 weeks after transplantation of marrow depleted of T cells using absorbed antithymocyte globulin produced fatal GVHD, whereas at 2 months or later after transplantation, DLT were tolerated without GVHD. Here, we studied tolerance to DLT in DLA-haploidentical recipients of CD6-depleted bone marrow. Bone marrow recipients were DLA-heterozygous and DLA-haploidentical to their DLA-homozygous donors. Marrow transplantation was performed on day 0, one day after total body irradiation with 10Gy. CD6 depletion was achieved by treatment of the marrow with CD6 antibody and rabbit complement. Natural killer cell activity of CD6-depleted cells was studied against canine thyroid adenocarcinoma cells. DLT were given at various time points after transplantation. Seven DLA-heterozygous dogs given undepleted marrow died of GVHD within 28 days. In contrast, three dogs given CD6-depleted marrow had sustained engraftment without occurrence of GVHD. DLT given on either day 3, 7, or 14 produced fatal GVHD. DLT on day 20, however, produced fatal GVHD in only two of four dogs. Mixed chimerism was converted into complete chimerism in all cases. Contrary to T-cell depletion with antithymocyte globulin, CD6 depletion spares canine natural killer cells. We conclude that T-cell depletion with CD6 antibody and complement induces graft-vs-host tolerance without jeopardizing engraftment. DLT on days 3, 7, and 14 after transplantation produced GVHD, but it failed to abrogate tolerance in two of four dogs transfused on day 20.

Graft-versus-leukemia effects of transplantation and donor lymphocytes

AbstractAllogeneic transplantation of hematopoietic cells is an effective treatment of leukemia, even in advanced stages. Allogeneic lymphocytes produce a strong graft-versus-leukemia (GVL) effect, but the beneficial effect is limited by graft-versus-host disease (GVHD). Depletion of T cells abrogates GVHD and GVL effects. Delayed transfusion of donor lymphocytes into chimeras after T cell–depleted stem cell transplantation produces a GVL effect without necessarily producing GVHD. Chimerism and tolerance provide a platform for immunotherapy using donor lymphocytes. The allogeneic GVL effects vary from one disease to another, the stage of the disease, donor histocompatibility, the degree of chimerism, and additional treatment. Immunosuppressive therapy before donor lymphocyte transfusions may augment the effect as well as concomitant cytokine treatment. Possible target antigens are histocompatibility antigens and tumor-associated antigens. Immune escape of tumor cells and changes in the reactivity of T cells are to be considered. Durable responses may be the result of the elimination of leukemia stem cells or the establishment of a durable immune control on their progeny. Recently, we have learned from adoptive immunotherapy of viral diseases and HLA-haploidentical stem cell transplantation that T-cell memory may be essential for the effective treatment of leukemia and other malignancies.