Donor Lymphocyte Infusion Treatment Research Articles

Donor Lymphocyte Infusion Is a Feasible Way to Improve Survival in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndromes Who Relapse after Allogeneic Stem Cell Transplantation

Introduction: Donor lymphocyte infusion (DLI) is used to induce remission in patients who relapse after allogeneic stem cell transplantation (allo-HSCT). During the last decade, the hypomethylating agent Azacitidine has been used together with DLI for a synergistic graft-versus-leukemia (GVL) effect. Here, we report results of DLI/Azacitidine treatment from a retrospective single-center study. Methods: Fifty AML/MDS patients treated for relapse after allo-HSCT between 2001 and 2020 with DLI at the Department of Hematology, at Rigshospitalet, Copenhagen University Hospital were included for analyses. A subgroup of patients who obtained complete remission (CR) after reinduction chemotherapy, received DLI in combination with low-dose (32 mg/m2) Azacitidine. Results: Overall survival in all patients after DLI treatment was 59% at 2 years and 20% at 5 years. Relapse-free survival in patients in CR prior to DLI was 32% after 2 years and 7% after 5 years. In the DLI + low-dose-Azacitidine group, 5-year relapse-free survival was 40%. Conclusion: DLI remains an effective treatment in post-transplant relapse leaving one-fifth of patients’ long-term survivors. Our results support the concomitant use of low-dose Azacitidine in the future use of DLI in order to enhance the GVL effect of donor lymphocytes.

MRD as Biomarker for Response to Donor Lymphocyte Infusion after Allogeneic Hematopoietic Cell Transplantation in Patients with AML.

Donor lymphocyte infusions (DLIs) can directly target leukemic cells through a graft-versus-leukemia effect and play a key role in the prevention and management of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Predictors of response to DLIs are not well established. We evaluated measurable residual disease (MRD) before, 30 and 90 days after DLI treatment as biomarkers of response. MRD was assessed by next-generation sequencing in 76 DLI-treated acute myeloid leukemia patients. MRD status before DLI treatment was independently prognostic for event-free survival (EFS, p < 0.001) and overall survival (OS, p < 0.001). Within 90 days of DLI treatment, 73% of MRD+ patients converted to MRD- and 32% of patients without remission achieved remission. MRD status 90 days after DLI treatment was independently prognostic for the cumulative incidence of relapse (CIR, p = 0.011) and relapse-free survival (RFS, p = 0.001), but not for OS. To evaluate the role of DLI treatment in MRD- patients, 23 MRD- patients who received DLIs were compared with a control cohort of 68 MRD- patients not receiving DLIs. RFS (p = 0.23) and OS (p = 0.48) were similar between the two cohorts. In conclusion, MRD is prognostic before (EFS, OS) and after (CIR, RFS) DLI treatment and may help in the selection of patients who benefit most from DLIs.

Low incidence and morbidity of Epstein-Barr virus reactivation following donor lymphocyte infusions.

Low incidence and morbidity of Epstein-Barr virus reactivation following donor lymphocyte infusions.

Donor Lymphocyte Infusion Is a Feasible Way to Improve Survival in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndromes Who Relapse after Allogeneic Stem Cell Transplantation

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) yet the major cause of death remains relapse after transplantation which occurs in 30-70% of patients for whom the prognosis is dismal. Since the 1990's donor lymphocyte infusion (DLI) has been proven able to induce remission after allo-HSCT and the use of therapeutic DLI at relapse has widely increased. The immunological mechanism in DLI is primarily T-cell-mediated graft-versus-leukemia (GVL) effect driven by genetic differences between donor and recipient in minor and major histocompatibility antigens. DLI treatment at relapse can additionally reverse T-cell exhaustion and increase T-cell receptor diversity, both of which are GVL-enhancing mechanisms. Risks and complications with DLI-treatment are primarily graft-versus-host disease (GVHD). Though dose escalation schedules have been suggested to increase the GVL-effect while minimizing the risk of GVHD, uniform therapeutic algorithms are still lacking, treatment is often individually scheduled, and outcome results are often disappointing with reported 2-year overall survival rates at 14-29% in AML relapse patients (Greiner J, Götz M, Bunjes D, Hofmann S, Wais V. Immunological and Clinical Impact of Manipulated and Unmanipulated DLI after Allogeneic Stem Cell Transplantation of AML Patients. J Clin Med. 2019;9(1):39). During the last decade, treatment with the hypomethylating agent azacitidin (Aza) has become another potential treatment in patients with myeloid malignancies. Immunological mechanisms of GVL in Aza-treatment for relapse include epigenetically reactivation of pro-apoptotic pathways and demasking of tumor-antigens while increased expression of regulatory T-cells protects from GVHD. In recent years DLI and Aza have been used for synergistical effect post-HSCT relapse both in patients who are un-fit to receive high-dose cytoreductive therapy as well as consolidation after reinduction. The aim of this analysis is to report results of retrospective single center-study of patients treated with DLI +/- Aza over a period of twenty years.Methods: Between 2001 and 2020 50 adult patients with relapse after allo-HSCT for AML(n=38) or MDS (n=12) were treated with DLI at the Department of Hematology, Transplant Unit, at Rigshospitalet, Copenhagen University Hospital, table 1. Only patients free from active GVHD were selected as DLI-candidates. Median follow-up time was 57 (1-170) months. Reinduction with high-dose chemotherapy was administered in 35 (70%) of patients prior to DLI and 34 (68%) patients were in complete morphological remission (CR) before DLI. DLI-products were unmanipulated and obtained from leukapheresis of unstimulated peripheral blood in matched related or unrelated donors of the original stem cell graft. Patients received a median of 3 (1-5) doses of DLI with median total doses of 6,1x10 7 (5x10 6- 4,65x10 8) CD3 postive T-cells per kg. Aza was used together with DLI from 2012 and administrered in 28 (56%) patients with a median of 6 (2-20) cycles. Reported outcomes are overall survial (OS) and relapse-free survival (RFS) in patients in CR prior to DLI.Results: At end of follow-up 20 patients were alive, 11 of these in CR and 2 in partial remission. In 7 patients, DLI was discontinued due to the development of GVHD after 1-2 doses, 6/7 of these patients had unrelated donors. Overall, 2 (4%) patients died from GVHD after DLI. Seven patients received a second HSCT after DLI treatment and were censored at this date in survival analyses. Figure 1a+b shows OS in all patients (n=50) and RFS in patients in CR prior to DLI (n=34). 2-year OS was approximately 59% and 5-year OS was 20%. 2-year RFS was approximately 32% and 5-year RFS was 8%. None of the analyzed baseline factors showed significant associations to the probability of OS, table 2, or RFS (data not shown). Reinduktion before first DLI and increasing doses of transplanted CD3 T cell per kg showed trends towards superior survival probability but failed to reach significant levels, possibly due to the limited patient number.Conclusion: Treatment vith DLI +/- Aza is effective and safe as relapse-treatment after allo-HSCT in myeloid diseases. In selected patients, a short-term (2-year) overall survival of 59% is achieved, and 20% of the patients remain long term survivors. [Display omitted] DisclosuresFischer-Nielsen: A.F.N. is employee and shareholder of StemMedical A/S, a biotech company working with cell-enriched fat grafting.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Donor Lymphocyte Infusions After Allogeneic Stem Cell Transplantation in Acute Leukemia: A Survey From the Gruppo Italiano Trapianto Midollo Osseo (GITMO).

We conducted a retrospective multicenter study including pediatric and adult patients with acute leukemia (AL) who received donor lymphocyte infusions (DLIs) after allogeneic hematopoietic stem cell transplantation (HCT) between January 1, 2010 and December 31, 2015, in order to determine the efficacy and toxicity of the immune treatment. Two hundred fifty-two patients, median age 45.1 years (1.6–73.4), were enrolled from 34 Italian transplant centers. The underlying disease was acute myeloid leukemia in 180 cases (71%). Donors were HLA identical or 1 locus mismatched sibling (40%), unrelated (40%), or haploidentical (20%). The first DLI was administered at a median time of 258 days (55–3,784) after HCT. The main indication for DLI was leukemia relapse (73%), followed by mixed chimerism (17%), and pre-emptive/prophylactic use (10%). Ninety-six patients (38%) received one single infusion, whereas 65 (26%), 42 (17%), and 49 patients (19%) received 2, 3, or ≥4 infusions, respectively, with a median of 31 days between two subsequent DLIs. Forty percent of evaluable patients received no treatment before the first DLI, whereas radiotherapy, conventional chemotherapy or targeted treatments were administered in 3, 39, and 18%, respectively. In informative patients, a few severe adverse events were reported: grade III–IV graft versus host disease (GVHD) (3%), grade III–IV hematological toxicity (11%), and DLI-related mortality (9%). Forty-six patients (18%) received a second HCT after a median of 232 days (32–1,390) from the first DLI. With a median follow-up of 461 days (2–3,255) after the first DLI, 1-, 3-, and 5- year overall survival (OS) of the whole group from start of DLI treatment was 55, 39, and 33%, respectively. In multivariate analysis, older recipient age, and transplants from haploidentical donors significantly reduced OS, whereas DLI for mixed chimerism or as pre-emptive/prophylactic treatment compared to DLI for AL relapse and a schedule including more than one DLI significantly prolonged OS. This GITMO survey confirms that DLI administration in absence of overt hematological relapse and multiple infusions are associated with a favorable outcome in AL patients. DLI from haploidentical donors had a poor outcome and may represent an area of further investigation.

T Regulatory Cell Receptor Repertoire Focusing and Clonal Expansion Indicates Control of Acute GvHD after Donor Lymphocyte Infusion

Background: T effector cells (Teff) within the stem cell graft in allogeneic hematopoietic stem cell transplantation (HSCT) can elicit disabling acute graft-versus-host disease (aGvHD) contributing to transplant-related mortality. Teff as donor lymphocyte infusion (DLI) are a therapeutic option to re-induce complete remission (CR) after leukemia relapse. Usually DLIs are given in dose escalating regimens until CR is achieved or first signs of aGvHD develop. To monitor the DLI induced allo-immune response and the efficacy of aGvHD treatment is a clinical challenge, since no established biomarkers are available.T regulatory cells (Tregs) are thought to play an important role in balancing immune responses and studies have shown effects of adoptive Treg transfer as a therapeutic option in GvHD. T cell receptor (TCR) sequencing of T cell subsets, such as Tregs, after DLI might allow the identification of clones inducing control of GvHD. Here we report data of 29 DLI patients with a median follow-up of >1 year allowing us to thoroughly analyze differences of the TCR repertoire in patients with or without aGvHD.Aims: We aimed to analyze Treg TCR diversity and clonality changes over time as a potential biomarker for development and treatment response of aGvHD following DLI.Patients and Methods: The study cohort consisted of 29 leukemia patients after HSCT who received DLI treatment for recurrence of disease, molecular relapse or high risk phenotype. Blood samples were taken before and after DLI. A median of 4 (range 2-6) blood samples per patient were available for TCR-sequencing. The last sample was taken at a median of 123 (27-530) days post DLI. After generation of PBMCs CD4+CD127-CD25+ Tregs were FACS-sorted for cDNA-based CDR3-region amplification of the TCR-β chain. CDR3 amplicons were sequenced on the Illumina MiSeq platform and annotated using the IMGT.org database. Further bioinformatic analyses were based on VDJtools and the tcR R-package.Results: In 18/29 patients we observed clinical symptoms of aGvHD, with blood samples available of the acute onset in 12/18 cases. Treg frequencies and absolute numbers did not differ between aGvHD and noGvHD samples. Treg TCR diversity, assessed via inverse Simpson's diversity index (1/D) increased on average by 322% at the first occurrence of aGvHD compared to the previous sample (Figure 1A, B). Stratifying for aGvHD severity (total grade 1-2 vs. 3-4) did not reveal any group differences. However, stratifying by organ involvement (skin vs. GI/liver) showed a more pronounced increase of 1/D in patients with aGvHD of the GI tract and/or the liver. Moreover, in 11 subjects blood samples were available a median of 7 (3-14) days prior to aGvHD diagnosis. Already at this preclinical time point we detected an increased 1/D of +361% (Figure 1A, B). Again, aGvHD organ involvement significantly affected the result, with GI/liver involvement mainly driving this effect (+567% vs. +1% 1/D). In contrast, patients who did not develop aGvHD at any time after DLI showed on average a slight decrease of -8% 1/D compared to the previous time point. Next, we analyzed all aGvHD patients with at least one sample available after initiation of treatment (local and/or systemic steroids). Control or remission of aGvHD symptoms was accompanied by decreased 1/D of on average -58% compared to the previous samples (Figure 2). In 9/11 patients we saw a focusing of the Treg TCR repertoire; in the other two (Figure 2, red lines) no or only partial clinical response to systemic steroids was reported.Conclusion: Our data describe detailed changes in the Treg compartment on the TCR level following development and treatment of aGvHD. Currently, aGvHD diagnosis following DLI treatment relies solely on clinical symptoms, deciding whether further dose increments of DLI can be administered. As our data suggest, Treg TCR sequencing may support transplant specialists with (1) detection of patients at risk for aGvHD, even prior to clinical symptoms, (2) identification of patients eligible for further dose increments (compare Figure 1B), and (3) assessment of aGvHD treatment with guidance whether higher dosage of steroids and/or alternative immunosuppressive treatment might be required (compare Figure 2, red lines). Future prospective studies are needed to replicate these findings in a large cohort, potentially enabling identification of specific Treg TCR clones controlling the allo-immune response in aGvHD. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:BMS: Consultancy; abbvie: Consultancy; Amgen: Consultancy; Roche: Consultancy.

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.

Final Analysis of a Multicenter Pilot Phase 2 Study of Cytokine Induced Killer (CIK) Cells for Patients with Relapse after Allogeneic Transplantation

Introduction: Patients with disease relapse after allogeneic transplantation (alloHSCT) have a poor prognosis. Donor lymphocyte infusion (DLI) is one of the main clinical options to salvage patients in relapse after transplant. Cytokine Induced Killer (CIK) cells are in vitro activated and expanded T lymphocytes which have acquired NK like cytotoxicity as well as CD56 expression. CIK cells have shown Graft versus Leukemia (GvL) activity with little GvHD and therefore may represent an ideal candidate to treat post-transplant relapse. We report the final results of a phase II multicenter pilot study, whose objective was to evaluate the safety and efficacy of sequential administration of donor derived unmanipulated DLI and CIK cells in patients with recurrent hematologic cancers after alloHSCT.MethodsSeventy-four patients relapsed after alloHSCT, performed using either a matched related (N=42) or unrelated donor (n= 32), were enrolled in the study. This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, and approved by the Agenzia Italiana del Farmaco (AIFA). The trial was registered as (EUDRACT number 2008-003185-26, ClinicalTrial.gov : NCT01186809).ResultsAmong the 74 patients (including 16 children and 58 adults) enrolled into this study (median age 45, range 1-67), 20 had a diagnosis of ALL (27%), 41 of AML (55%), 4 of MM (5%), 3 of HD (4%), 2 of NHL (3%) and 4 of MPN (5%). All patients relapsed after matched allogeneic transplants (32 unrelated and 42 sibling), of whom 44 (59%) suffered from a hematological, 4 (5%) from a cytogenetic and 26 (35%) from a molecular relapse. The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with increasing numbers of CIK cells, in groups of three patients per dose level. Since dose limiting toxicity (DLT) was never observed (acute GVHD of grade III or more), the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to all patients.Ten patients died for disease progression, 1 patient developed aGVHD (grade I, skin only) and 1 withdrawn for medical decision before or during the DLI treatment and could not proceed to the planned subsequent CIK administration. Sixty-two patients received at least one infusion of CIK cells, of whom 43 patients (61%) completed the cell therapy program, while 3 patients are still under treatment. The study flow is outlined in Figure 1.As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 24 (32%) patients (4 during the DLI), no response was observed in 18 (24%) patients, a stable disease in 1 patient (1%), a complete remission in 21 (28%) and a partial remission in 6 (8%), for an overall response rate of 36%. In 4 patients clinical response could not be evaluated (3 patients still in treatment and 1 withdrawn from the protocol). Acute GVHD was observed in a total of 11 patients (15%): grade 1 (n=4), 2 (n=2) and 3-4 (n=5). During follow up, chronic GVHD was observed in 8 patients (11 %) (3 mild, 4 moderate and 1 severe).By univariate analysis, progression free survival (PFS) and overall survival (OS) were significantly associated (p<0.0001) with the type of relapse since at 3 years it was 11% and 23% vs. 54% and 77 % for patients enrolled due to a hematologic vs. a molecular/cytogenetic relapses, respectively (Figure 2A-B). By multivariate analysis, the type of relapse remained the only significant predictor of survival (p=0.0019).ConclusionOur study shows that administration of CIK cells is feasible in patients with recurrent hematologic cancer after alloHSCT with a relatively low toxicity in terms of GvHD. Particularly in the setting of the molecularly relapsed patients, long-term survival can be achieved. In future studies, we are planning to test CIK cells in preventing post-transplantation relapse in high risk AML. Finally, CIK cells may represent an innovative platform to transduce chimeric antigen receptors in allogeneic T cells with a reduced risk to induce GvHD. [Display omitted] [Display omitted] DisclosuresBiondi:Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Rituximab-based treatments followed by adoptive cellular immunotherapy for biopsy-proven EBV-associated post-transplant lymphoproliferative disease in recipients of allogeneic hematopoietic stem cell transplantation

ABSTRACTTo improve prognosis of post-transplant lymphoproliferative disease (PTLD), a sequential therapeutic strategy that rituximab-based treatments followed by donor lymphocyte infusion (DLI) or autologous EBV-specific cytotoxic T lymphocytes (EBV-CTL) for biopsy-proven EBV-associated PTLD in recipients of allogeneic hematopoietic stem cell transplantation was designed. 84 patients with EBV-PTLD were enrolled in this prospective study. After two cycles of the rituximab-based treatments, 68 of 84 patients (81% [95% CI 71–88]) responded and 52 (62% [51–72]) had CRs. This increased to 73 of 77 patients (95% [87–98]) with completion of sequential cell infusions, and 70 of 77 (91% [82–96]) achieved CRs after DLI or autologous EBV-CTL infusion. 22 patients experienced acute GVHD (aGVHD) (grade I in 5 and grade II in 13, grade III in 4) and 13 chronic GVHD (limited cGVHD in 7 and extensive cGVHD in 6) in 62 patients undergoing a median of three doses of DLI. The incidences of GVHD were similar between DLI and EBV-CTL group (aGVHD 35% vs. 33%, p = 0.876; cGVHD 21% vs. 13%; p = 0.503). EBV-CTL activity after the rituximab-based treatments did not change, while increased after cell infusions and reached its maximum in the 3rd or 6th month after EBV-CTL or DLI treatment, respectively. The 5-y cumulative incidence of PTLD relapse was 4.5% ± 3.3%. The 5-y overall survival (OS) and progression-free survival (PFS) after PTLD were 70.7% ± 5.2% and 68.9% ± 5.3%, respectively. Rituximab-based treatments combined with adoptive cellular immunotherapy might elevate CR rates and reduce relapse of PTLD after allo-HSCT.

Expanded umbilical cord blood T cells used as donor lymphocyte infusions after umbilical cord blood transplantation

BackgroundUmbilical cord blood (UCB) is an alternative graft source for hematopoietic stem cell transplantation and has been shown to give results comparable to transplantation with other stem cell sources. Donor lymphocyte infusion (DLI) is an effective treatment for relapsed malignancies after hematopoietic stem cell transplantation. However, DLI is not available after UCB transplantation. MethodsIn this study, in vitro–cultured T cells from the UCB graft were explored as an alternative to conventional DLI. The main aim was to study the safety of the cultured UCB T cells used as DLI because such cell preparations have not been used in this context previously. We also assessed potential benefits of the treatment. ResultsThe cultured UCB T cells (UCB DLI) were given to 4 patients with mixed chimerism (n = 2), minimal residual disease (n = 1) and graft failure (n = 1). No adverse reactions were seen at transfusion. Three of the patients did not show any signs of graft-versus-host disease (GVHD) after UCB DLI, but GVHD could not be excluded in the last patient. In the patient with minimal residual disease treated with UCB DLI, the malignant cell clone was detectable shortly before infusion but undetectable at treatment and for 3 months after infusion. In 1 patient with mixed chimerism, the percentage of recipient cells decreased in temporal association with UCB DLI treatment. ConclusionsWe saw no certain adverse effects of treatment with UCB DLI. Events that could indicate possible benefits were seen but with no certain causal association with the treatment.

Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid–immunoglobulin complexes in humans

Donor lymphocyte infusion (DLI), whereby donor mononuclear cells are infused into patients, is one of the few effective immunotherapeutic strategies that generate long-lasting tumor remissions. We previously demonstrated that chronic myelogenous leukemia (CML) patients treated with DLI develop high-titer plasma antibodies specific for CML-associated antigens, the majority of which have been reported to bind nucleic acids These observations led us to predict that circulating antibody-antigen complexes in DLI-responsive patients carry nucleic acids that can engage innate immune sensors. Consistent with this, we report here that post-DLI plasma from 5 CML patients that responded to DLI treatment induced massive upregulation of MIP-1α, IP-10, and IFN-α in normal blood mononuclear cells. Importantly, this was not observed with plasma obtained before DLI and from DLI nonresponders and imatinib-treated patients. This endogenous immunostimulatory activity required nucleic acid and protein for its adjuvant effect and activated antigen-presenting cells through the RNA and DNA sensors TLR8 and TLR9. Presence of the immunoglobulin Fc receptor CD32 enhanced cellular responses, suggesting that immunoglobulins associate with this activity. Finally, a TLR-induced expression signature was detectable in post-DLI but not pre-DLI blood, consistent with an active circulating TLR8/9-stimulating factor. We have therefore demonstrated that effective tumor immunity correlates with the presence of endogenous nucleic acid-immunoglobulin complexes in patient plasma, thus providing a putative mechanism for the induction of potent antigen-specific immunity against malignant cells.

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.

HLA-Identical Dendritic-Leukemic Cell Hybrids Generate Specific CTLs in Vitro

hile being instrumental in the treatment of leukemic relapse after allogeneic hematopoietic stem cell transplantation, the impact of donor lymphocyte infusion (DLI) and its effectiveness remain debatable. Consequently it is widely accepted that more efforts are needed in order to make DLI more effective. This communication thus deals with the generation of specific CTLs in the clinical setting of HLA matched hematopoietic stem cell transplantation, to be used as an improved DLI treatment for post-transplantation relapsed leukemias. We assessed the potential of fused dendritic cells from donor origin, with leukemic cells from the HLA matched recipient for the generation of donor anti-tumor CTLs. Leukemic cells and donor dendritic cells were fused using polyethylene glycol (PEG). The hybrids were analyzed for double phenotype of both DC and tumor, and used for the education and generation of cytotoxic donor lymphocytes. Results demonstrate that efficient and specific CTLs can be generated and used in vitro for the elimination of the recipient tumor cells. These results form the basis for the establishment of a novel methodology aimed at generating active or passive anti-leukemic vaccine in relapsed patients.

Hematopoietic Chimerism in Patients with Hematological Malignancies Treated with Donor Lymphocyte Infusions Combined with IL-2 for Relapse after Allogeneic Stem Cell Transplantation.

Donor lymphocyte infusions (DLI) are considered to be the treatment of choice in pts with relapses after allogeneic hematopoietic stem cell transplantation (HSCT). This kind of treatment is based on alloreactivity of donor lymphocytes, mediating a graft-versus-leukemia (GVL) effect. However, the relapses of acute leukemia in many cases are not susceptible to DLI. There were few reports that the concominant administration of cytokines (IL-2, IFN-alfa) and DLI is more efficient for conversion to complete donor chimerism (CC) from mixed chimerism (MC) and leads to remission in pts relapsed after HSCT, but the significance of this therapy is still not estimated. The aim of study was to evaluate the efficacy of DLI treatment combined with IL-2 i.v. administration for CC establishment and remission induction in pts with relapses after HSCT for hematological malignancies. 26 pts were treated with DLI for relapses after ASCT from related siblings in 2001–2007. 12 of them were included in the study. Median age was 25 y (16–44), 9 men/3 woman, 4 pts with AML, 1 - APL, 1 - ALL, 1 -ANL, 3 -CML, 2- MDS. 2 pt received reduced intensity HSCT, 10- conventional one. Median time to relapse was 12,5 months (3,5 – 48 months).10 pts demonstrated hematological relapses, 1 pt with APL and 1 pt with CML - molecular relapses. All pts with AL in overt relapse received chemotherapy first and then DLI was performed in aplasia. CML pts were treated with Hydrea and Glivec and then DLI were administrated. Median dose of infused DL was 6,34 x 108/kg (median 6 transfusions per pt) with median of CD3+ cells. Chimerism status was investigated on BM and PB using VNTR/STR analysis at relapse, before every DLI and monthly after DLI completion. All pts demonstrated MC in bone marrow before chemotherapy (median 74% of host DNA). 9 pts were still MC after chemotherapy and before DLI (median 38% of host DNA), 3 pts became CC before DLI. In all pts DLI were combined with IL-2 (2–6 MU with every DLI). In 6 pts with MC after chemotherapy only 1 (with CML) became CC after 1 course of DLI and developed aGVHD II–III and then cGVHD. 5 pts (with AL and MDS) after DLI were still MC remaining in hematological remission. One of them died of CMV infection. 2 pts recieved the 2nd course of DLI + IL-2, became CC and are still well and alive (1 - with cGVHD, 1 - without GVHD) for 30 months. For 2 pts (AML) with MC 2nd DLI course was not possible (donor was not available) and they received the course of IL-2 (5 days for 6 MU daily) in 2 months after DLI. The both are in complete remission with CC and cGVHD. 4 pts showed no effect and soon relapsed despite the 2nd DLI course in 2 pt and 2nd course of IL-2 in 1 pt. Despite the small and heterogenious group we may conclude, that DLI administration concomitantly with IL-2 leads to more prominent GVL effect and quick conversion from MC to CC. The interesting observation is that the additional infusions of IL-2 were effective for conversion to CC and remission induction in 3 of 4 pts resistant for DLI.

Adoptive Immunotherapy with Donor Lymphocyte Infusions after T-Cell Depleted Allogeneic Stem Cell Transplantation for Patients with Acute Myeloid Leukemia.

In this study we analyzed 40 patients with AML who received donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation (alloSCT). Patients with a median age of 42 (range, 11–66) were transplanted with an in vitro T-cell depleted (Campath in the bag) graft from an HLA matched sibling following a myeloablative (n=35) or a nonmyeloablative conditioning regimen (n=5). Patients received DLI for relapsed AML (n=28) or for mixed chimerism (n=12). All patients with relapsed AML were treated with a cytarabine-containing chemotherapy regimen or with gemtuzumab ozogamycin immediately prior to DLI treatment and received daily 3 x 106 U α-IFN sc. The DLI dose administered was depending on the protocol used. Fourteen relapsed patients received high dose DLI with a median of 50 x 106 CD3+ cells/kg and 14 low dose DLI with a median of 5 x 106 CD3+ cells/kg. Ten of 28 patients with relapsed AML obtained a complete remission after the combination of chemotherapy and DLI. One patient obtained a partial remission and fourteen patients showed no response. Three patients were not evaluable. No effect of cell dose was observed on the clinical response after DLI. The median percentage of donor chimerism in patients with relapsed AML before DLI was 74% (range, 5–96%) and increased after DLI to 97% (range, 5–100%). Six patients with relapsed AML became complete chimera after DLI. DLI caused in 12 of 28 relapsed patients grade 3–4 GVHD. cGVHD was observed in 3 patients of whom 2 developed extensive GVHD. With a median follow up of 11 months only 1 pt in the high dose and 4 pts in the low dose group are alive in CR and 23 have died due to progressive disease (16) or GVHD (7).Due to this low rate of long-term survival of relapsed AML patients treated with DLI, we recently developed a new DLI protocol for AML patients. Twelve patients with mixed chimerism at 6 months after T-cell depleted alloSCT were pre-emptively treated with 3 x 106 CD3+ cells/kg. Complete chimeric response was observed in 6 of these 12 patients. Two patients showed increasing mixed chimerism and three patients showed no response. Two of 12 patients treated for mixed chimerism developed aGVHD gr 1–2. No severe aGVHD was observed in this group. Two patients developed chronic GVHD, both after nonmyeloablative conditioning. Incidence of GVHD was significantly lower in this group compared to the relapsed patients. Currently, 7 patients are alive in CR. In conclusion: 1) Long term survival of patients with overt relapse AML after alloSCT treated with DLI remains poor 2) High dose DLI appeared to be less effective than low dose DLI due to increased toxicity 3) DLI for AML may be more successful when it is administered pre-emptively for mixed chimerism after T-cell depleted alloSCT.

Evaluation of immunomodulatory treatment based on conventional and lineage-specific chimerism analysis in patients with myeloid malignancies after myeloablative allogeneic hematopoietic cell transplantation

Both conventional chimerism analysis (CCA) and lineage-specific chimerism analysis (LCA) have potential pitfalls as diagnostic means for the detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). Therefore, the present study examines the results of both methods in order to determine how predictive consecutive evaluations were, with respect to the risk that the patient would relapse during post-transplant follow-up and with respect to responsiveness to immunomodulatory treatment. A total of 168 individuals with acute myeloid leukemia (AML) (n = 137) and myelo dysplastic syndrome (n = 31) were investigated with CCA and LCA at mean intervals of 24 days (range: 11-116). The median follow-up after myeloablative aHCT was 22 months (range: 4-49). Of 168 patients, 65 experienced a clinical relapse after aHCT. CCA and LCA were comparatively sensitive and specific for relapse at the intervals of chimerism testing employed in this study. Of 32 patients, 10 who were offered donor lymphocyte infusions (DLI) treatment for increasing (n = 29) or stable (n = 3) mixed chimerism (MC) achieved at least transitory CC. The observation that all patients with increasing MC relapsed despite DLI treatment (54%) or withdrawal of immune suppression (24%) indicates that novel strategies to deal with rapidly evolving relapse in AML patients, such as shortening of chimerism monitoring intervals, need to be evaluated.

The Application of Sequential and Quantitative Analysis of Donor Chimerism in Adoptive Immunotherapy Following Allogeneic Stem Cell Transplantation.

Objective In order to investigate the value of sequential and quantitative analysis of chimerism to predict relapse or graft rejection after allogeneic stem cell transplantation, to determine the optional time point of adoptive immunotherapy and to estimate the efficacy of adoptive immunotherapy.Methods 70 patients who received HLA compatible allo-HSCT were evaluated. Peripheral blood and bone marrow were collected before and after transplantation in different period. Serial and quantitative analysis of donor chimerism (DC) both prior to and following adoptive immunotherapy were performed by multiplex PCR amplification of STR markers (STR-PCR) and capillary electrophoresis with fluorescence detection.Results 13 of 70 patients relapsed at median time of 6months post transplant (4/13 patients developed hematologic relapse and 9/13 patients appeared cytogenetic or molecular relapse respectively). 2 of 70 patients experienced graft rejection. Before the time of relapse or graft rejection, STR-PCR indicated the decreasing DC in all 15 patients, at levels ranging from 24.8%~86.2%. The declining value of donor chimerism (DC< 90%) was detected in four patients 26 days before relapse or graft rejection diagnosed clinically. Adoptive immunotherapy was used to treat 15 patients(9 CML, 5 AML, 1 MDS). A response to immunotherapy was achieved in 6 patients(6 CML) following withdrawal of CsA and in a further 4 patients(2 CML, 1 M2, 1 MDS) after additional donor lymphocyte infusion(DLI). In these patients, the value of DC increased with convertion to a predominant donor profile (>90%) or converted to stable full donor chimerism(FDC)(6 patients) or stable mixed chimerism(MC)(4 patients) shortly after immunotherapy. All these 10 patients who responsed to immunotherapy developed acute or chronic GVHD. While in the patients without response, the level of DC decreased persistently or declined after transient increasing. Three patients without response received second DLI but remain failed to therapy.Conclution The results demonstrate that the decreasing value of DC can identify the patients who have high risk of relapse or graft failure and can be used to guide adoptive immunotherapy implementation at early stage. The sequential and quantitative monitoring of DC has been shown to be a valuable tool to determine the optional time point of immunotherapy and to early evaluate the efficacy of treatment. Furthermore, it can present a rational basis for treatment intensification in patients who did not respond to first-line DLI treatment

Role of double-negative regulatory T cells in long-term cardiac xenograft survival.

A novel subset of CD3(+)CD4(-)CD8(-) (double negative; DN) regulatory T cells has recently been shown to induce donor-specific skin allograft acceptance following donor lymphocyte infusion (DLI). In this study, we investigated the effect of DLI on rat to mouse cardiac xenotransplant survival and the ability of DN T cells to regulate xenoreactive T cells. B6 mice were given either DLI from Lewis rats, a short course of depleting anti-CD4 mAb, both DLI and anti-CD4 treatment together, or left untreated. DLI alone did not prolong graft survival when compared with untreated controls. Although anti-CD4-depleting mAb alone significantly prolonged graft survival, grafts were eventually rejected by all recipients. However, the combination of DLI and anti-CD4 treatment induced permanent cardiac xenograft survival. We demonstrate that recipients given both DLI and anti-CD4 treatment had a significant increase in the total number of DN T cells in their spleens when compared with all other treatment groups. Furthermore, DN T cells harvested from the spleens of DLI plus anti-CD4-treated mice could dose-dependently inhibit the proliferation of syngeneic antidonor T cells. Suppression mediated by these DN T cells was specific for antidonor T cells as T cells stimulated by third-party Ags were not suppressed. These results demonstrate for the first time that a combination of pretransplant DLI and anti-CD4-depleting mAb can induce permanent survival of rat to mouse cardiac xenografts and that DN T regulatory cells play an important role in preventing long-term concordant xenograft rejection through the specific suppression of antidonor T cells.