HLA-mismatched Stem Cell Transplantation Research Articles

Prevalence and risk factors of antibodies to HLA according to different cut-off values of mean fluorescence intensity in haploidentical allograft candidates: A prospective study of 3805 subjects.

The importance of anti-HLA antibodies in transplantation settings, such as HLA-mismatched or haploidentical hematopoietic stem cell transplantation and platelet refractoriness, is widely recognized. In previous reports, it was mentioned that several cut-off values of donor-specific anti-HLA antibodies mean fluorescence intensity (MFI) were related to graft rejection in the environment of HLA mismatched stem cell transplantation and the aim of this study was to investigate the prevalence and risk factors of anti-HLA antibodies according to those cut-off values of MFI. A total of 3805 patients with hematologic disease were prospectively enrolled and analyzed. When using MFI of anti-HLA antibodies ≥500, ≥1000, ≥1500, ≥2000, ≥5000, and ≥ 10,000 as cut-off values for positivity, the prevalence of class I or II anti-HLA antibodies ranged from 4.6% to 20.2% in all cases. When the MFI cut-off value was ≥500 for positivity, multivariate analysis indicated that platelet transfusion, underlying disease, and pregnancy were the most important risk factors for the presence of anti-HLA antibodies for the total patients. Subgroup analysis according to age, gender, and underlying disease showed that pregnancy was the most important risk factor for the presence of anti-HLA antibodies. For all patients (n=3805), when anti-HLA antibody positivity was defined according to different MFI cut-off values, including ≥1000, ≥1500, ≥2000, ≥5000, and ≥ 10,000, an association of platelet transfusion and pregnancy with anti-HLA antibodies was also demonstrated. Our results suggest that pregnancy and platelet transfusion are the main risk factors for the prevalence of anti-HLA antibodies in haploid allograft candidates, providing evidence for guiding the evaluation of anti-HLA antibodies and helping donor selection for HLA-mismatched transplant candidates.

48 - Cytokine Release Syndrome Post HLA-Mismatched Stem Cell Transplantation Does Not Affect Immune Reconstitution and Is Effectively Treated with Tocilizumab

48 - Cytokine Release Syndrome Post HLA-Mismatched Stem Cell Transplantation Does Not Affect Immune Reconstitution and Is Effectively Treated with Tocilizumab

Rituximab for desensitization during HLA-mismatched stem cell transplantation in patients with a positive donor-specific anti-HLA antibody.

To define the efficacy of a single dose of 375 mg/m2 rituximab for DSA-positive patients with 2000 ≤ MFI < 10,000, we enrolled a prospective clinical cohort including patients with positive DSA treated with rituximab (n = 55, cohort A), a matched-pair cohort including cases with negative DSA (n = 110, cohort B) and a historical cohort including subjects with 2000 ≤ MFI < 10,000 without receiving any treatment for DSA (n = 22, cohort C). The incidences of primary poor graft function (PGF) in cohort A and cohort B were 5% and 1% (P = 0.076), respectively, both of which were lower than that in cohort C (27%, P < 0.001, for all). Rituximab was associated with a reduced incidence of primary PGF (HR 0.200, P = 0.023). The 3-year nonrelapse mortality of patients in cohort A and cohort B were 23% and 24%, respectively, both of which were lower than that in the cohort C (37%), although no statistical significance was observed. These results led to a low 3-year overall survival in patients in the cohort C (58%) compared with those in the cohort A (71%) and the cohort B (73%). We suggest that a single dose of rituximab could be effectively used to prevent the onset of primary PGF. The prospective cohort of this study is registered at http://www.chictr.org.cn/ChiCTR-OPC-15006672.

Rituximab for Desensitization during HLA-Mismatched Stem Cell Transplantation in Patients with a Positive Donor Specific Anti-HLA Antibody: A Prospective Study

A prospective, clinical trial in non-manipulated haploidentical allografts was initiated to define the efficacy of a single dose of 375 mg/m2 rituximab as a desensitization regimen for donor-specific anti-HLA antibody (DSA)-positive patients with 2,000 ≤ mean fluorescence intensity (MFI) &lt; 10,000. In the clinical cohort, compared to pretransplantation [median, 4791, n=28], the median MFI levels at day 7 [0], 14 [0], 21 [0], 30 [0], and 45 [0] following transplantation were significantly decreased (P&lt;0.0001 for all). The incidence of primary poor graft function (PGF) in the clinical cohort (n=55) was comparable to that in the matched-pair cohort (n=110) negative for the DSA (5% vs. 1%, P=0.076), both of which were significantly lower than that in the historical cohort (n=22) with 2,000 ≤ MFI&lt;10,000 without receiving any treatment to cope with DSA (27%, P&lt;0.001, for all). Rituximab desensitization was associated with a reduced incidence of primary PGF (HR 0.200, P=0.023). The 3-year non-relapse mortality of patients in the clinical cohort and the matched-pair cohort were 23% and 24%, respectively, both of which were lower than that of patients in the historical cohort (37%), although no statistical significance was observed. These results led to a low 3-year overall survival in patients in the historical cohort (58%) compared to those in the clinical cohort (71%) and the matched-pair cohort (73%). These results suggest that a single dose of rituximab could be effectively used to desensitize patients and prevent the onset of primary PGF in patients who receive HLA-mismatched allografts. This study is registered at http://www.chictr.org.cn/ChiCTR-OPC-15006672. Disclosures No relevant conflicts of interest to declare.

Prior history of HLA-mismatched stem cell transplantation is a risk factor for graft failure in HLA-haploidentical transplantation.

Prior history of HLA-mismatched stem cell transplantation is a risk factor for graft failure in HLA-haploidentical transplantation

The Outcome of Relapsed Childhood Core Binding Factor Acute Myeloid Leukemia: A Report from the JPLSG AML-05R Study

BACKGROUND: Core binding factor acute myeloid leukemia (CBF-AML), which is characterized by the chromosomal abnormalities of t(8;21) and inv(16), is the most frequent subtype of pediatric AML. Patients with CBF-AML have a better prognosis than patients with a normal karyotype when they receive chemotherapy of appropriate intensity; however, in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study, the excessive treatment reduction was observed to induce a higher incidence of relapse in children with CBF-AML. We investigated the outcomes of patients with relapsed CBF-AML who were registered in the JPLSG AML-05.PATIENTS & METHODS: We conducted a retrospective analysis of the JPLSG AML-05R study using a questionnaire. Furthermore, a mutation analysis was performed to identify NRAS, KRAS, KIT, WT1, NPM1, NUP98-NSD1, FLT3-ITD and MLL-PTD mutations using the available samples. We investigated the probability of overall survival (pOS) after relapse according to the patient's age, time from diagnosis to relapse, site of relapse, FAB classification, t(8;21) versus inv (16), reinduction chemotherapy regimen, obtaining a second complete remission before stem cell transplantation (SCT), the method of SCT and the presence of additional mutations.RESULTS: Forty-one of the 154 children with CBF-AML who were registered in the JPLSG AML-05 study experienced a relapse. The information from 32 of these 41 patients was collected using a questionnaire. Twenty-seven had t(8;21), and 5 had inv (16). It was possible to perform molecular analyses for 28 of the patients. KIT mutations were identified in 16 of 28 patients (57.1%). The follwing mutations were also detected: NRAS (n=4), KRAS (n=1), and WT1 (n=1). There were no cases with FLT3 -ITD, NUP98-NSD1, MLL-PTD or NPM1 mutations. Twenty-five cases achieved a complete remission after the initial reinduction (78.1%). The pOS was 65.6% (21/32) after a mean observation period of 2.7 years. The significant prognostic factors influencing pOS included a failure to obtain complete remission prior to SCT (p=0.03), reinduction chemotherapy regimen [p=0.02, a FLAG-based or ECM (etoposide, cytarabine and mitoxantrone)-based regimen was better than other modalities], donor source (p<0.01, cord blood was better than bone marrow and peripheral blood) and HLA matching between the donor and recipient (p=0.03, a mismatch was better than a full-match). Additional mutations, including the presence of the KIT gene, did not have any effect on pOS (p=0.64).CONCLUSIONS: Relapsed childhood CBF-AML in the JPLSG AML-05 study was still associated with a better prognosis, because almost two thirds of the patients were successfully treated with SCT. Obtaining complete remission prior to SCT was associated with a good prognosis. A FLAG- or ECM-based regimen is therefore recommended for reinduction chemotherapy. Unexpectedly, cord blood transplantation and HLA-mismatched SCT were associated with a better prognosis. Further investigations are necessary to clarify the genes that are associated with a poor prognosis in patients with CBF-AML. DisclosuresNo relevant conflicts of interest to declare.

Extramedullary T-lymphoblastic blast crisis in chronic myelogenous leukemia: A case report of successful diagnosis and treatment.

Extramedullary T-lymphoblastic blast crisis of chronic myelogenous leukemia (CML) is uncommon and the prognosis is poor. It was usually misdiagnosed as the co-existence of T-lymphoblastic lymphoma (T-LBL) and CML. In the present study, we report a patient with CML, who developed extramedullary T-lymphoblastic blast crisis and was successfully treated with human leukocyte antigen (HLA)-mismatched stem cell transplantation. The patient was a 44-year-old man who presented with lymphadenectasis and leucocytosis prior to diagnosis. The bone marrow smear, biopsy and fluorescence in situ hybridization (FISH) of Breakpoint Cluster Region/ Abelson murine leukaemia (BCR/ABL) supported the diagnosis of CML in the chronic phase, while the immunohistochemistry of lymph nodes supported the diagnosis of T-LBL. The FISH test for BCR/ABL in lymph node blast cells was performed and the result was positive; therefore, the patient was diagnosed with extramedullary T-lymphoblastic blast crisis of CML. After several courses of combined chemotherapy, the patient was treated with HLA-mismatched stem cell transplantation and obtained continuous remission for 51 months until the present (September 2013).

Current status of GVHD control in HLA mismatched stem cell transplantation from family donor

Current status of GVHD control in HLA mismatched stem cell transplantation from family donor

Flow Cytometry Based Chimerism Analysis Can Predict Graft Rejection in Pediatric Patients Receiving HLA Mismatched Stem Cell Transplantation

AbstractAbstract 4156Graft failure is a life-threatening complication after allogeneic stem cell transplantation and its probability increases with the degree of HLA disparity between donor and recipient. In HLA-mismatched transplantation, specific T cells against mismatched HLA class I alleles were shown to be associated with rejection. Moreover, HLA mismatches allow to monitor the chimerism status of several cell populations by flow cytometry with specific monoclonal antibodies against HLA alleles with high sensitivity even in very low cell numbers. We report our experience with this method in a cohort of 90 pediatric patients who received T and B cell depleted haploidentical stem cells after myeloablative conditioning regimens. Diagnoses were: leukemias n=43, solid tumors n=28, non malignant diseases n=19. Graft manipulation was carried out by magnetic microbeads and the clinimacsÒ device with 4 to 5 log depletion efficacy. Conditioning regimens were TBI, Bu or Mel based and comprised ATG or OKT3. Immunosuppression with mycophenolate mofetil (MMF) was given in most patients (if donor T cells in the graft exceeded 25.000/kg body weight) Flow cytometry with HLA antibodies for detection of chimerism status of T and NK cell subsets in peripheral blood was started at day 0 and was repeated at least weekly. 14 out of 90 patients (15%) rejected the graft between day 19 and 86 (median day 33). Two different rejection patterns were observed: early rejections (diagnosis of rejection, day 19–40, median day 28) with cytokine release syndrome including high fever, massive elevation of CRP, ferritin, LDH and D-Dimers or late rejections without signs of inflammation (diagnosis of rejection, day 53–86). In all patients, residual T cells of recipient origin were still detectable after the conditioning procedure. The portion of residual recipient T cells within the first week (day 0–7) was predictive: Non-rejectors had a median percentage of 46+-5% recipient T cells detectable (n=42 analyses) whereas rejectors showed a significant higher percentage (85+-7%, n=13 analyses, p<0.001). In more than 90% of the rejectors, the portion of detectable autologous recipient T cells exceeded 60%. Thus, the risk of rejection for patients with >60% detectable recipient T cells was 41% (12 out of 29 rejected), whereas patients with <60% recipient T cells had a risk of 4% (only 1 out of 27 rejected; p=0.001). In non-rejectors, recipient T cells where detectable at a very low level (median 0,7/μl) up to day 60 without proliferation tendency, whereas strongly increasing and significantly higher numbers were observed in rejectors (from 3/μl at day 0–3 up to 617/μl at day 15–18; p< 0,05 for all data pairs). Based on these data, donor lymphocyte infusions (DLI) and cessation of MMF were performed in 12 patients with increasing residual recipient T cells to prevent imminent rejection (1–4 infusions, median number: 1; starting dose: 25.000 CD3+cells/kg bw). Recipient T cells were reduced/eradicated in 7/12 patients (60%) after DLIs and no rejection occurred. In 5 patients, DLIs had no impact on increasing recipient T cells, resulting in graft rejection in 4 out of these 5 patients. Conclusions:Flow cytometry with specific monoclonal antibodies against HLA alleles allows to analyze the origin of lymphocyte subsets and their kinetics after HLA mismatched transplantation with high sensitivity even in very low cell numbers. The percentage of residual recipient T cells in the first week posttransplant was predictive, since patients with >60% detectable recipient T cells had a significantly higher risk to experience graft rejection. In all cases, rejection was associated with increasing recipient T cells. Intervention with DLI and cessation of immune suppression may contribute to prevent rejection. Disclosures:No relevant conflicts of interest to declare.

PRAME-Specific Allo-HLA–Restricted T Cells with Potent Antitumor Reactivity Useful for Therapeutic T-Cell Receptor Gene Transfer

In human leukocyte antigen (HLA)-matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA-reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide-specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells.

Allogeneic stem cell transplantation as treatment for heavily treated, refractory acute graft-versus-host disease after HLA-mismatched stem cell transplantation

No effective treatment has been established for patients with steroid-refractory acute graft-versus-host disease (GVHD). Recently, we demonstrated in a murine tandem bone marrow transplantation model that life-threatening GVHD established by the first bone marrow transplantation was successfully treated by engraftment of a second donor graft after reduced-intensity conditioning. We named the effect by which allografts counteract GVHD "graft-versus-GVHD." To investigate the efficacy of graft-versus-GVHD treatment clinically, 16 patients who developed, after human leukocyte antigen-mismatched stem cell transplantation, severe GVHD, refractory to three to five lines of GVHD-specific treatments, underwent 17 allogeneic stem cell transplantations using reduced-intensity conditioning regimens with grafts from a second donor. Among the 15 transplantations that could be evaluated, rescue donor grafts were engrafted in 11 cases and rejected in 4 cases. For patients who achieved rescue donor engraftment, the response rate was 90.9% (eight complete response, two partial response, and one stable disease). Six of the eight patients with complete response survived without GVHD symptoms, with a median follow-up of 2128 days. No new development of GVHD by the second graft was observed. No patients had recurrence of the original malignant disease. In contrast, no long-term survivors were observed in patients who rejected rescue donor grafts. We propose here a novel graft-versus-GVHD treatment to treat refractory GVHD, and these results strongly suggest that GVHD can be successfully treated by eliminating the harmful lymphocytes responsible for GVHD by a second allogeneic stem cell transplantation.

Costimulatory blockade with monoclonal antibodies to induce alloanergy in donor lymphocytes

Alloreactive donor T cells are central to the pathogenesis of Graft-versus-Host Disease (GvHD). Non-specific T cell depletion of donor grafts reduces GvHD, but increases infection and disease relapse. Several strategies are, therefore, in development to selectively remove alloreactive donor T cells prior to transplant while retaining beneficial donor T cells. An alternative approach is ex vivo alloanergization of donor T cells via allostimulation and blockade of costimulatory signals with fusion proteins or monoclonal antibodies. This strategy, which selectively inactivates alloreactive donor T cells, has been tested with some success in previous clinical trials of HLA-mismatched bone marrow transplantation. To build on the findings of these early trials, the strategy is now being tested in a multi-center clinical study of alloanergized donor lymphocyte infusion after HLA-mismatched stem cell transplantation. Recent advances in the understanding of alloanergization include the recognition of the central role played by CD4(+) regulatory T cells and new applications include the combination of alloanergization with T cell redirection to maximize anti-tumor activity. This mini-review will give an overview of the immunological basis for alloanergization, the previous and current clinical applications, and how new pre-clinical data have helped to create exciting new avenues of translational research in this area.

Full Haplotype Mismatch Stem Cell Transplantation: Myeloablative with T-Cell Depletion Vs. Reduced Intensity Conditioning without T-Cell Depletion

AbstractAbstract 3533Some groups have continuously tried to reveal the possible role of full-haplotype mismatch stem cell transplantation (SCT) in patients who lack a compatible donor. However, the long-term event-free survival (EFS) rate after high-grade mismatch SCT using conventional myeloablative conditioning with T-cell depletion (TCD), even in complete remission (CR), is still only in the range of 10–30%. Most patients who were transplanted had heavily pretreated acute leukemia. The most important obstacle to overcome is the high rate of infection-related mortality associated with delayed immunological recovery in the setting of full-haplotype mismatch SCT, specifically by way of complete depletion of donor T cells in the graft. In contrast, by using unmanipulated donor cells and less aggressive conditioning regimens without TCD, most groups in Asia have reported EFS >40%. We investigated the role of reduced-intensity conditioning without TCD in HLA-mismatched related-donor SCT, specifically in patients with high-risk acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) who received unmanipulated granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs). SCT was performed in 32 consecutive patients. We compared two different protocols: one with more intensive conventional conditioning with TCD (group 1), and one with reduced intensity without TCD (group 2). The protocols differed between the two groups with respect to total body irradiation (1200 cGy in group 1 vs. 800 cGy in group 2) as well as busulfex, fludarabine, and anti-thymocyte globulin (ATG; thymoglobulin, Genzyme) treatments. No patient in group 1 received post-transplant graft versus host disease (GvHD) prophlyaxis or G-CSF. In contrast, all patients in group 2 received 1.25 mg/kg/day ATG for 4 consecutive days, together with our standard GvHD prophylaxis regimen of methotrexate (5 mg/m2 intravenous bolus on days 1, 3, 6, and 11) and tacrolimus starting at day –1. All patients received G-CSF-mobilized PBSCs. The median number of CD34+ cells infused was 14.2 (range, 4.3–20.6) × 106/kg for group 1 and 6.0 (range, 3.6–8.5) × 106/kg for group 2. The median numbers of CD3+ cells infused were 0.03 (range, 0.015–0.057) × 106/kg and 870 (range, 84–1260) × 106/kg, respectively. G-CSF was administered subcutaneously to all patients in group 2 at a dose of 5 mg/kg/day, from day 7 after transplantation until neutrophil recovery. GvHD, relapse, non-relapse mortality (NRM), overall survival, EFS, reconstitution of immunity, and natural killer (NK) cell alloreactivity were compared between the groups. The median patient age was 33 (range, 17–48) years for group 1 and 39 (range, 19–63) years for group 2. The median follow-up for surviving patients was 18 months (range, 8–100). The majority of patients had intermediate or unfavorable cytogenetic features. The transplanted patients were all successfully engrafted. The median times to neutrophil (>0.5 × 109/kg) and platelet (>20 × 109/kg) recovery were 12 and 13 days in group 1, and 11 and 10 days in group 2. The overall rates of acute and chronic GvHD were 50% and 33%, and 80% and 92% in groups 1 and 2, respectively. Of note, NRM differed significantly between the groups: 46.2% in group 1 vs. 9.5% in group 2 (P = 0.0014). The estimated probability of EFS at 2 years was 15.3% for group 1 vs. 59.2% for group 2 (P = 0.05). We did not find NK alloreactivity in any of the patients, based on the NK-killer cell immunoglobulin-like receptor (KIR) disparity between donor and recipient. However, we noted a significant difference in EFS between C1/C1 homozygotes and C1/C2 heterozygotes according to the donor NK-KIR ligands (P = 0.0175). Recovery of CD4+ cell numbers at 3 months after SCT showed a markedly different pattern between the groups, with a median of 4 cells/μl (range, 2–7) in group 1 vs. 311 cells/μl (range, 56–1226) in group 2. Our findings suggest that full-haplotype mismatch SCT using a Korean-adapted protocol is a feasible therapeutic strategy for patients with high-risk AML/MDS, specifically in CR, provided that there is a further defined plan for the investigation of NK/T-cell alloreactivity. Disclosures:No relevant conflicts of interest to declare.

Allo-HLA reactivity of virus-specific memory T cells is common

AbstractGraft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.

High Avidity PRAME Specific T Cells Derived From In Vivo HLA Mismatched Transplantation Setting Potentially Useful for Immunotherapeutic Strategies.

Abstract 4087Poster Board III-1022Adoptive T cell therapy is an attractive strategy to provide cancer patients with antigen specific T cells. For this approach T cells with specificity for non-polymorphic tumor-associated self-antigens that are shared between various tumors are promising candidates. However, the isolation of high avidity T cells specific for nonpolymorphic tumor associated self-antigens is difficult, because of self-tolerance. During thymic selection T cells that exhibit high avidity for self-antigens presented by self-HLA are deleted by positive and negative selection. This explains why most T cells directed against non-polymorphic tumor associated self-antigens characterized to date exhibit low to intermediate avidity. After HLA mismatched stem cell transplantation (SCT) however, T cells educated in the donor have not encountered the allogeneic HLA molecules from the patient during thymic selection. Consequently, these T cells can exhibit high avidity for tumor associated antigens presented by allogeneic patient HLA molecules. In this study we aimed to identify T cells directed against non-polymorphic tumor associated antigens using an in vivo HLA mismatched transplantation setting. Alloreactive T cell clones were isolated and expanded from a patient that experienced graft versus leukemia as well as acute graft versus host disease following HLA-A2 mismatched SCT and donor lymphocyte infusion for the treatment of AML. All isolated T-cell clones were allo-HLA-A2 reactive, and by loading of T2 cells with HPLC fractionations of peptides eluted from HLA-A2 we were able to demonstrate that all alloreactive clones recognized one single fraction, indicative for peptide specific recognition. By two additional peptide HPLC fractionation rounds and mass spectrometry we were able to characterize the peptides of 8 different allo-HLA-A2 reactive T cell clones. One of the T cell clones, was of particular interest since this T cell clone recognized the peptide SLLQHLIGL derived from the preferentially expressed antigen on melanomas (PRAME). Recognition by the clone of HLA-A2 positive COS cells transfected with PRAME, and tetramer staining of the clone, confirmed the specificity against the PRAME derived peptide. Peptide titration demonstrated that the PRAME specific T cell clone exhibited high affinity for the SLL peptide. Since PRAME is overexpressed in a large fraction of tumors, we analyzed whether the PRAME specific clone could recognize HLA-A2 expressing tumor cell lines. The results demonstrated that all 8 tested melanoma cell lines, 2 of 3 RCC cell lines, 1 of 2 mamma carcinoma cell lines and 1 of 2 lung carcinoma cell lines were recognized by the clone. In addition, 5 out of 10 primary acute myeloid leukemia cells, and 2 out of 3 acute lymphoblastic leukemia cell lines were recognized. Since it has been described that also certain normal tissues express low levels of PRAME, the clone was tested against numerous HLA-A2 positive non-malignant cells. No reactivity against fibroblasts, keratinocytes, bronchus epithelial cells, hepatocytes, billiair duct epithelial cells, colon epithelial cells, mesenchymal stem cells, (activated) B-cells, (activated) T cells, monocytes and CD34+ cells was observed. However, the clone demonstrated high reactivity against monocyte derived DC's and a low but significant reactivity against primary tubular epithelial cells. By quantitative PCR we demonstrated that the level of recognition of the different cell types is correlated with the expression levels of PRAME. The results demonstrate that the high avidity PRAME specific T cells clone, derived from an in vivo allo-HLA-A2 immune response, is solely PRAME specific and exerts high reactivity against numerous tumors and limited of target toxicity. Based on these results we conclude that the high affinity TCR from this high avidity PRAME specific T cell may be an effective tool for adoptive T cell therapy using TCR gene modified T cells for the treatment of cancer patients. Disclosures:No relevant conflicts of interest to declare.

The Outcome of Unrelated Hematopoietic Stem Cell Transplants with Total Body Irradiation (800 cGy) and Cyclophosphamide (120 mg/kg) in Adult Patients with Severe Aplastic Anemia.

Abstract 3397Poster Board III-285 Backgrounds:We had reported the results of a pilot prospective study demonstrating the superiority of 800 cGy of total body irradiation (TBI) in combination with a 120 mg/kg of cyclophosphamide (CY) [TBI-800/CY-120] compared to the higher doses of TBI (1000 and 1200 cGy) as a conditioning regimen for unrelated stem cell transplantation (SCT) in adult patients with severe aplastic anemia (SAA) [Biology of Blood and Marrow Transplant 2007;13:836-870]. Patients and methods:To analyze the long-term results of unrelated SCT with TBI-800/CY-120 for adult SAA who failed to respond to immunosuppressive treatment, we prospectively enrolled 50 adult patients with SAA [median age, 28 years (range, 15-53)], including 26 patients of a previous pilot study. All patients received fractionated TBI (400 cGy/day) for 2 days followed by CY (60mg/kg/day, 2 days). Nine patients (18%) were very SAA. Enrolled patients had received multiple transfusions (median 64 units, range, 10-363) and long disease duration (median 48 months, range 2-323). High resolution DNA typing was done at HLA-A, -B, -C, -DR level. Seventeen patients (34%) received HLA-mismatched SCT at allele level. Thirty-eight patients (76%) received bone marrow and 12 (24%) peripheral blood stem cells (PBSC). All patients received tacrolimus and short-course of methotrexate as GVHD prophylaxis. Results:All patients achieved engraftment and the median day of neutrophil and platelet recovery was 13 days (range, 8-30) and 20 days (range, 9-200), respectively. There was one case of delayed platelet recovery, but platelet count rose slowly to the normal level thereafter. At a median follow-up of 38 months (range, 1-84), the estimated 3-year overall survival was 87.8%. The cumulative incidence of acute GVHD (aGVHD) over grade II (6/23, grade III and IV) and chronic GVHD (cGVHD) were 46.0% and 50.3% [limited (39%) vs. extensive (61%)], respectively. Among 7 patients who died, 4 patients and 1 patient died of severe aGVHD (grade III and IV) and cGVHD, respectively, 1 patient multi-organ failure, and 2 patient sepsis. Univariate analysis revealed the following risk factors: very SAA (P=0.018), HLA-mismatched donor at allele level (P=0.012), and older donor age (P=0.080) for aGVHD and previous aGVHD (P=0.030) and PBSC (P=0.024) for cGVHD. Multivariate analysis showed that only a HLA-mismatched donor at allele level (hazard ratio: 2.7, 95% CI: 1.2-6.2, P=0.018) was a significant factor associated with higher cumulative incidence of aGVHD over grade II. Conclusions:TBI-800/CY-120 conditioning resulted in excellent outcome of unrelated transplants in adult patients with SAA who had received multiple transfusions and long disease duration, which was comparable to those of matched sibling transplants. Additional strategies to prevent severe GVHD, for example, adding anti-thymocyte globulin, particularly for HLA-mismatched transplant and in case of PBSC as a stem cell source, will be helpful to improve the outcome in the future. Disclosures:No relevant conflicts of interest to declare.

Leukemic Blasts Acting as Host Antigen Presenting Cells Trigger a Combined CD4 and CD8 Allo-Immune Response Directed against Mismatched HLA Class I

HLA disparity between patient and donor increases the risk and the severity of graft versus host disease (GVHD). In order to safely administer HLA mismatched stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), it is important to understand the mechanisms underlying GVHD in HLA mismatched settings. The degree of patient chimerism, especially of the antigen presenting cells, at the time of DLI has been shown to influence the development of GVHD. This is suggested by the correlation between the time after SCT of DLI administration and the occurrence of GVHD. In mouse models the underlying role of the presence of host APCs in the occurrence of GVHD has been demonstrated in both MHC matched and mismatched models. In this study, the alloimmune response in a patient suffering from GVHD after an HLA class I mismatched DLI was characterized. The patient received a T-cell depleted SCT followed by two DLIs from the same donor. The SCT and first DLI, administered for mixed chimerism, did not result in any signs of GVHD. In contrast, the second DLI led to severe GVHD. Patient chimerism in different cell subsets present prior to the two DLIs showed no significant difference in patient T-cell chimerism and there were no patient B-cell, monocytes or dendritic cells present prior to each DLI. However, a significant difference in the presence of patient leukemic blasts was found at the time of the two DLIs. Prior to the first DLI no leukemic cells could be demonstrated, whereas 9% leukemic blasts were present in bone marrow prior to the second DLI. We showed that the leukemic blasts expressed HLA class II. Correlating with the absence of GVHD, the first DLI did not lead to activation of donor T-cells while the second DLI led to severe GVHD accompanied by a vigorous CD8 and CD4 T-cell response. Activated donor derived CD8 and CD4 T-cells from this response were isolated by single cell sort based on HLA-DR expression. Expanded clones were characterized for alloreactivity, HLA restriction, specificity and clonality. We showed that 50 of the 53 expanded CD8 T-cells were alloreactive and that all alloreactive CD8 T-cells were restricted to the mismatched HLA-A2. Additionally, by sequencing the T-cell receptor (TCR) Vβ CDR3 region, we showed that all alloreactive CD8 clones expressed a different CDR3 region, illustrating polyclonality. Of the 88 expanded CD4 clones, 21 were alloreactive and the majority of the alloreactive CD4 T-cell clones were directed against an HLA-A2 derived peptide presented in HLA-DR1. By Vβ analysis we showed that the CD4 T-cell response was also polyclonal. Finally, to investigate the role of the leukemic blasts in the initiation of the GVHD, the capacity of the different cell subsets present prior to DLI 1 or 2 to stimulate donor CD8 and CD4 T-cells was analyzed. This showed that only the leukemic blasts were able to highly stimulate both the CD8 and the CD4 T-cells. Our data indicate that the leukemic blasts by acting as host APCs triggered a combined CD4 and CD8 allo-immune response directed against the mismatched HLA, and thus initiated not only anti-leukemic reactivity but also lethal GVHD.

Second Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Introduction: In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), relapse after allogeneic stem cell transplantation (SCT) occurs with frequencies of 30–80% in dependence on risk profiles. Selected patients are offered a 2. allo-SCT. As the number of studies is limited, we here performed retrospective analysis of 20 consecutive MDS and AML pts who received at least 2 allografts from related/unrelated donors from 2000–2007. Patients: There were 4 children and 16 adults who received ≥2 allo-SCTs (10 males, 10 females; 2–63 years, median 38 yy). 14 pts had de novo AML, 3 pts s-AML, 2 MDS RAEB-1; one child had juvenile myelomonocytic leukemia (JMML). 19 pts received two allo-SCTs-18 due to another relapse after the 1. allo-SCT, one due to secondary graft failure after 1. SCT. One pt with MDS had 3 allo-SCTs due to relapses. 13/20 pts received treatment before the 2. allo-SCT, but only 6/20 achieved complete remission (CR). 10/20 pts had unfavorable cytogenetics. The median interval between the 1. and 2. allo-SCTs was 20 months (range 2–47 mo). Median disease free survival (DFS) after the 1. SCT was 16 months (range 3–45 mo). Characterization of 2. allo-SCTs: 4/20 second allo-SCTs were performed from related donors (3 HLA-identical, one haploidentical). 16 SCTs were performed from unrelated donors (HLA matched: n= 8, -mismatched: n=8). In 10 cases, the 2. allo-SCTs was performed from different, in 10 cases from the same donor. Reduced intensity conditioning was applied to 16 pts, myeloablative to 4 pts. Antithymocyte globuline was administered to 12/20 pts. One pt received bone marrow, 19 pts peripheral blood stem cells. Immunosuppression was performed in most pts by cyclosporine A plus short-course MTX or mycophenolate mofetil. Outcomes: Leukocyte engraftment was achieved in 16/20 cases with a median interval of 13 days (r. 10–24 d). 3 pts died prior to engraftment. One pt had secondary graft failure being followed by a 2. allo-SCT. CR was achieved by 16/20 pts. At the time of this report, 5/20 pts are still alive after a median follow-up of 15 months (r. 2–36 mo), three of those in CR with follow-ups of 7, 22, and 36 months. 9/20 pts developed relapse (45%), of whom 2 received donor lymphocytes. Median DFS after the 2. SCT was 7 months (range <1–38 mo). One patient received a 3. allo-SCT after another relapse of AML, but died 8 months after 3rd SCT due to treatment related mortality (TRM). The TRM rate was 7/20 pts (35%) either due to sepsis and multiorgan failure, fungal pneumonia, or acute GvHD. AGvHD (II–IV) was described in 11/20 pts (55%). Chronic GvHD developed in 5/20 pts. Two of those 3 pts, who remained in stable CR, had unfavorable karyotypes. They had developed relapse after 3, 28, and 45 months following the 1. SCT. Two achieved CR before the 2. allo-SCT. In two cases, HLA-mismatched SCTs were performed being followed by extensive cGVHD. All had reduced conditioning before SCT. One of those, a male pt aged 16 years, primarily received no immunosuppression. Following severe intestinal aGvHD after 3 weeks tacrolimus and steroids were started. Discussion: Despite a high rate of reduced intensity conditioning, TRM (35%) and relapse rate were high (45%). However, single pts seemed to benefit from a 2. SCT, even in case of unfavourable karyotypes or when no complete remission was achieved before the 2. SCT, and here also in one case with an early relapse after the 1. SCT. Thus, it seems difficult to predict which patients might have a benefit from this approach. Research should focus on the optimization of conditioning regimens especially for recipients of a 2. SCT and try to provide concepts for immunosuppression in this specific situation due to the high relapse risk.

KIR2DS5 is associated with leukemia free survival after HLA identical stem cell transplantation in chronic myeloid leukemia patients

Background Alloreactive NK cells play a role in tumor eradication after allogeneic HLA mismatched stem cell transplantation (SCT). The effect of NK alloreactivity in HLA identical SCT is still under debate and in particular in transplantation for chronic myeloid leukemia (CML) the data are very limited and with conflicting outcome. The aim of our study was to evaluate the effect of KIR genes and KIR ligands on leukemia free survival (LFS) and relapse rate in a well-defined, homogeneous group of CML patients phase upon HLA identical sibling SCT. Methodology We retrospectively analyzed the effect of KIRs and KIR ligands (C1 and C2) on LFS and relapse in 70 CML patients in 1st chronic phase, who had received an HLA identical sibling graft. For KIR typing we used a single PCR based KIR typing protocol that also included primers allowing for the identification of the KIR binding site on HLA-Cw (AA 77 and 80). Principal findings The data show clear differences in transplant outcome between patients having both ligands (C1 and C2) as compared to patients having only one ligand (C1 or C2). In the latter group, the stimulatory KIR2DS5 gene was associated with improved leukemia free survival ( p = 0.007; hazard ratio 4.3; 95% confidence interval 1.3–6.7) and lower relapse rates ( p = 0.028; HR 4.3, 95% CI 1.1–9.1). In contrast, in patients carrying both ligands, KIR2DS5 was associated with reduced LFS ( p = 0.0056; HR 0.3; 95% CI 0.1–0.7) and higher relapse rate ( p = 0.02; HR 0.35, 95% CI 0.1–0.8). Conclusions Our data indicate a role for an NK mediated anti-CML response after HLA identical sibling SCT that is influenced by KIR ligands and, more importantly, by stimulatory KIRs present in the donor.

Allo-HLA Reactive CD8 T-Cells May Recognize Tissue Specific Peptides Explaining Tissue Restricted GVHD after HLA Mismatched SCT.

HLA disparity between patient and donor increases the risk of GVHD after allogeneic stem cell transplantation (SCT). However, similar to fully HLA matched SCT, the clinical manifestation of GVHD after HLA mismatched SCT is frequently restricted to skin, gut and liver. Based on the frequency of allo-HLA reactive T-cells, which is about a 1000-fold higher than the frequency of minor histocompatibility antigen specific T-cells, the immune response after HLA mismatched SCT is expected to be mediated by allo-HLA reactive T-cells. Theoretically allo-HLA reactive T-cells can exert three different types of recognition. They may directly recognize the mismatched HLA molecule, independent of the peptide it presents. Alternatively they could be peptide dependent but promiscuous in their peptide recognition, or peptide specific like in normal T-cell recognition. Previous studies all showed peptide independent recognition by at least part of the studied T-cells. Since HLA class I is expressed by all nucleated cells, these data would imply that after HLA mismatched SCT alloreactive T-cells would be able to equally damage all tissues. Whereas previous reports mainly studied in vitro generated allo-HLA reactive T-cells, we characterized the pattern of peptide recognition of a large number of different T-cells retrieved from a patient suffering from GVHD after HLA-A2 mismatched donor lymphocyte infusion. Activated CD8+ T-cells were single cell sorted based on HLA-DR expression, expanded and tested for alloreactivity and HLA restriction. 46 of the 56 generated CD8 T-cells clones were alloreactive and allo-HLA-A2 restricted. The alloreactive CD8 clones showed usage of different T-cell receptor Vβ chains with different CDR3 sequences, illustrating polyclonality. To evaluate the tissue recognition, the clones were tested against different HLA-A2 positive target cells derived from various hematopoietic and non-hematopoietic tissues. The clones showed major differential recognition of the different tissue target cells, varying from lack of recognition to strong recognition of the same target. To analyze the peptide recognition pattern, the clones were tested against TAP deficient T2 cells loaded with HPLC fractions of peptides eluted from HLA-A2. The different clones recognized different HPLC fractions, but each clone recognized of only one single fraction, indicating peptide specificity. Three clones recognized non-pulsed T2 cells, which was interpreted in previous studies to demonstrate peptide independence. T2 cells, however, do present peptides which are independent on TAP to enter the ER. By testing reactivity against TAP independent peptides, one clone showed recognition of a peptide derived from the signal sequence of TRAPα, which is positioned at the ER membrane, and is therefore TAP independent. This indicates that recognition of T2 cells by the allo-HLA reactive T-cells was also based on peptide specific recognition. Finally, two clones were screened against COS cells expressing HLA-A2 transfected with a c-DNA library constructed from an EBV-LCL. Each clone showed recognition of only one (different) gene out of 40.000 c-DNAs. These results show that all allo-HLA reactive T-cells exerted peptide specific recognition. Differential expression of tissue specific peptides that can be recognized in the context of allo-HLA molecules may explain tissue specific peptides as a cause for tissue restriction in GVHD after HLA mismatched SCT.