HLA-identical Stem Cell Transplantation Research Articles

T Cell-Replete Haploidentical Transplantation with Post-Transplantation Cyclophosphamide for Hodgkin Lymphoma Relapsed after Autologous Transplantation: Reduced Incidence of Relapse and of Chronic Graft-versus-Host Disease Compared with HLA-Identical Related Donors

Allogeneic hematopoietic stem cell transplantation (SCT) represents a potential curative strategy for patients with Hodgkin lymphoma (HL) relapsing after autologous SCT (ASCT), but the incidence of disease relapse is still high. We performed a retrospective study on 64 patients with HL relapsing after ASCT to compare outcomes after HLA-identical SCT (HLAid-SCT; n = 34) and haploidentical SCT with post-transplantation cyclophosphamide (PT-Cy) (Haplo-SCT; n = 30). All patients engrafted, with a significantly shorter median time for neutrophil and platelet engraftment after HLAid compared with Haplo-SCT (14 days versus 19 days and 11 days versus 23 days, respectively; P < .005). With a median follow-up of 47 months, 3-year overall survival (OS), 3 -year progression-free survival (PFS), and 1-year nonrelapse mortality (NRM) were 53%, 44% and 17%, respectively. Recipients of Haplo-SCT were less likely to experience disease relapse (3-year cumulative incidence of relapse, 13% versus 62%; P = .0001) and chronic graft- versus-host disease (GVHD; 3% versus 32%; P = .003), resulting in improved PFS (60% versus 29%; P = .04) and GVHD-free/relapse-free survival (47% versus 17%; P = .06). The 3-year OS did not differ between the 2 groups (56% versus 54%; P not significant), and NRM was higher after Haplo-SCT, but the difference did not reach statistical significance (26% versus 9%; P = .09). On multivariate Cox regression analysis, receipt of Haplo-SCT (hazard ratio [HR], .17; P = .02) and achieving optimal disease control (complete remission before SCT: HR, .6; P < .0001) were the only independent variables associated with a reduced risk of disease relapse. Haplo-SCT is a valid option for patients with HL relapsing after ASCT, with a reduced incidence of relapse compared with HLAid SCT.

ID3012 Evaluation of HLA-identical sibling allogenic peripheral blood stem cell transplantation for acute myelogenous leukemia at BTH, Vietnam

Background: the purposes of this study were to assess the efficacy of HLA-identical sibling allogenic peripheral blood stem cell transplantation in the treatment for acute myelogenous leukemia (AML) at Blood transfusion and hematology hospital at Ho Chi Minh city.&#x0D; &#x0D; Methods: retrospective study, case series.&#x0D; &#x0D; Results: from June 2005 to September 2016, there were 47 AML patients included 24 males and 23 females, with median age was 36 years old. Engraftment was achieved in 47 patients (100%). The median number of transfused CD34+ cells was 7.5 x 106/kg. The median time for neutrophil engraftment and platelet engraftment were 11 ± 2.8 days and 12.5 ± 10.1 days respectively. Oral mucositis occurred in 38 patients (80.9%), febrile neutropenia in 42 patients (89.4%), acute graft-versus-host disease (aGvHD) in 14 patients (29.8%), chronic graft-versus-host disease (cGvHD) in 10 patients (21.3%), and VOD in 6 patients (12.8%). Transplant-related mortality (TRM) was 4 patients (8.5%). Median follow-up time was 52.26 months. Patients remained alive were 27 (57.4%). The 5-year overall survival (OS) and disease-free survival (DFS) were 55% and 54% respectively.&#x0D; &#x0D; Conclusion: HLA-identical sibling allogeneic blood stem cell transplantation can reconstruct hematopoiesis quickly and is a favorable therapeutic method for AML patients. Further development and improvement of hematopoietic stem cell transplantation techniques along with graduated and individualized therapy are expected to provide better outcomes and improved quality of life for AML patients.

移植後シクロホスファミドを用いたHLA半合致移植の現状と課題

Recent studies demonstrated that donor engraftment was achieved without severe graft-versus-host disease (GVHD) after posttransplant cyclophosphamide (PTCy) based human leukocyte antigen (HLA) -haploidentical bone marrow transplantation (PTCy-haploBMT). It has been suggested that PTCy selectively deplete proliferating alloreactive T cells, while preserving regulatory T cells. Although GVHD and non-relaplse mortality appears to be low, relapse remains a major problem after PTCy-haploBMT. Recently, PTCy is increasingly used in the setting of peripheral blood stem cell transplantation or myeloablative stem cell transplantation in order to reduce rejection and disease relapse. Furthermore, this strategy can be applied to patients with nonmalignant disorders. This strategy also represents a promising platform to establish calcineurin-inhibitor free GVHD prophylaxis in HLA-identical stem cell transplantation.

Differences in Natural Killer(NK) Reconstitution Between Unmanipulated Haploidentical and HLA Identical Stem Cell Transplantation and Relationship with Citomegalovirus and Graft Versus Host Disease (GVHD). Experience in One Centre in 22 Patients.

Introduction: It´s well known that NK cells exert an important role in postransplant immune reconstitution. Their maturation process through the contact with recipient´s bone marrow stromal cells is crucial for the development of their functional capacities. Previous studies showed the relevance of NK alloreactivity in relapse prevention and the influence of NK subpopulations and expression of activating receptors on CMV infection control, antitumor effects, and graft versus host disease (GVHD). Unmanipulated Haploidentical (Haplo) transplantation with postrasplant cyclophosphamide (Cy) is an emerging alternative for patients (pts) with no identical sibling donor with encouraging results. We previously reported preliminary data of NK cell reconstitution in 7 pts in Haplo setting. Our aim is to contrast our previous data in 22 pts comparing with conventional HLA-identical (HLAid) Stem Cell Transplantation (SCT) and to analyse the relationship between NK reconstitution and CMV reactivation and GVHD.Methods: 22 pts received an Haplo SCT and 7 pts an HLAid SCT in Gregorio Marañon Hospital between January 2013 and April 2014. Peripheral blood was used in all cases. Conditioning regimen comprised fludarabine (Flu), Cy and busulfan (Bux) for Haplo and Flu/Bux or Flu/Melphalan for HLAid SCT. Prophylaxis for GVHD consisted of high dose Cy on +3, +4, cyclosporine (CsA) A and mycophenolatemofetil for Haplo and CsA/Methotrexate for HLAid. Table1 show patients characteristics. For analysis of NK reconstitution and receptor expression multi-colour flow cytometry on FC500 and Navios Beckman Coulter® was used. Total NK cells, CD56 intensity and expression of NKG2A, NKp30, Nkp46 and NKG2D was studied at +15, +30, +60, and +90. For comparison between the two groups Mann–Whitney U-test was used.Results: Median NK cells/mm3 on +15 and +30 were significantly lower on Haplo than HLAid group ( 0 (0-5.5) and 28.5 (12.2-96.5) vs 69.5 (55-134) and 276 (151-422); p<0.05), being similar on + 60 and +90 (86 (49.5-158) and 147 (85-204.5) for Haplo vs 105 (39.2-162.5) and 117 (82-197) for HLAid; p ns). In Haplo pts NK cells reached normal levels on +60, while only 6/22 (27%) on +30. As previously reported NK high percentages of immature CD56bright (CD56b) cells and high expression of immature receptor NKG2A appears on +30, decreasing on +60 and +90 (CD56b 80% (72.5-88.5), NKG2A 88% (80-94) on +30, 39% (25-49) and 73% (62.2-86.7) on +60, 34% (23-43) and 69% (54.7-77.5) on +90. Compared with HLAid patients, the percentage of NKb cells was significantly higher (p<0.05) on Haplo group on +30, +60 and +90 and expression of NKG2A was also higher on +15, +30 and +60 (p<0.05). Interestingly, expression of activating receptors NKP46 and NKG2D linked to NK maturation are only significantly decreased in Haplo group comparing with HLAid on +15 (p<0.05), with appropriate expression and no differences on +30, +60 and +90. In fact, expression of NKP46 tended to be higher in Haplo on +30 (81% (62.5-90) vs 61% (42-71)), with no statistical significance (p 0.06). NKP30 had a low expression, similar in the 2 groups. In Haplo pts, no differences were found in total NK cell between pts with and without CMV reactivation at any time. Patients with acute GVHD had lower NK cells/mm3 at +30 (21.5 (10.7-35) vs 122 (21.7-260.7) and +60 (75(46-137) vs 156 (51-258); p 0.1), but statistical significance was not reached. Proportion of CD56b and NKG2A expression did not show any difference in pts regarding presence of CMV reactivation or GVHD.Conclusions: After Haplo SCT with postransplant Cy NK cell reconstitution seem to be delayed comparing with HLAid sibling SCT, reaching normal levels at +60. As previously reported, we confirm in our series that NK maturation also show a different pattern. In Haplo setting NK cells remains in an immature phenotype with high proportion of NK CD56b and expression of NKG2A much longer than in HLAid pts. Interestingly activating receptors expression, normally linked to maturation is only lower in Haplo pts in +15, but it is similar in both SCT types by day +30 and later, what should translate in appropriate cytotoxic capacity. In Haplo group, those patients with and without CMV reactivation had similar NK cell reconstitution. Patients with GVHD seem to have an impaired early NK reconstitution, but this fact needs to be contrasted.Acknowledgments: Fundación Mutua Madrileña. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preimplantation Genetic Diagnosis for Congenital Immunodeficiencies

Preimplantation genetic diagnosis [PGD] is presently a realistic option for at risk couples to avoid affected pregnancies and have a genetic disease free offspring of their own. PGD has been also performed together with HLA typing for couples with children affected by congenital disorders, requiring the HLA identical stem cell transplantation therapy. As the HLA matched stem cell transplantation is known to significantly improve or completely replenish the immune system, PGD was applied as an alternative treatment strategy for congenital immunodeficiencies, so that the couples could have unaffected children, who are also HLA compatible to serve as stem cell donor progeny for the affected siblings. We present here our experience of PGD cycles for inherited immunodeficiencies, which is presently the world’s largest series of PGD for this group of diseases, for which there is still no available treatment other than stem cell transplantation.

Donor Genotypes For Interleukin-17A Gene Single Nucleotide Polymorphisms (SNPs) Allow Anticipation Of Complications After HLA-Identical Allogeneic Stem Cell Transplantation (allo-SCT)

Introduction Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in in the promoter region of cytokine genes have shown to alter their expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 17 (IL-17) is secreted by CD4+ T-cells and has been implicated in the pathogenesis of various autoimmune diseases but its importance in SCT is not well-known. Objective To analyse the influence of IL-17A SNP genotypes on the risk and severity of GvHD and other complications after HLA-identical allo-SCT. Patients and Methods Genomic DNA obtained from peripheral blood samples belonging to 546 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). Genotyping of the polymorphisms of interest, rs8193036 (-737C&gt;T), rs2275913 (-197G&gt;A), rs3819024 (-444A&gt;G), rs4711998 (-877A&gt;G), were performed by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Results Genotype frequencies are shown in Table 2 and the association between IL-17A genotypes and complications after allo-SCT are shown in Table 3. Patients transplanted from donors harboring genotype CC for the SNP rs8193036 show increased risk of grade III-IV acute GvHD (7/26 vs 47/397, p=0.035) and of grade II-IV acute GvHD (13/26 vs 133/409, p=0.048). Patients transplanted from donors harboring allele A in the SNP rs4711998 show increased risk of extensive chronic GvHD (53/161 vs 43/177, p=0.045). Relapse rate was not related with IL-17A SNP genotypes. Finally a higher risk of toxicity-related mortality (TRM) was observed in patients transplanted from donors harboring allele A for SNP rs2275913 (78/293 vs 46/227, p=0.048), donors harboring allele G for SNP rs3819024 (78/279 vs 46/242, p=0.011) and donors harboring allele A for SNP rs4711998 (68/250 vs 55/229, p=0.044). Conclusions IL-17A SNP genotyping might be useful to anticipate complications after sibling HLA-identical allo-SCT and, therefore, to improve the clinical management of transplanted patients. This results further support the idea of a genetic predisposition to certain complications after allo-SCT. Paper presented on behalf of the GvHD/Immunotherapy committee of the Spanish Group for Hematopoietic Transplantation (GETH). Disclosures: No relevant conflicts of interest to declare.

Preimplantation Hla Typing- Practical Tool for Stem Cell Transplantation Treatment of Congenital and Acquired Disorders

It is well known that to achieve an acceptable engraftment and survival in stem cell therapy, an HLA identical stem cell transplant is strongly required. However, the availability of the HLA matched donors even among family members is extremely limited, so pre-implantation HLA typing provides an attractive practical tool of stem cell therapy for children requiring HLA matched stem cell transplantation. The present experience of PGD for HLA typing of over one thousand cases shows that PGD provides the atrisk couples with important prospect not only to avoid an inherited risk of producing the offspring with congenital disease, but also to establish an unaffected pregnancy, which may benefit the affected member of the family with hemoglobinopathies, immunodeficiencies and other congenital or acquired bone marrow failures. Despite ethical issues involved in pre-implantation HLA typing, the data presented below show the increasing attractiveness of this option for the couples with affected children requiring HLA compatible stem cell transplantation. So the couples at risk of having children with congenital bone marrow disorders have to be informed about presently available option not only for avoiding the birth of affected child, but also for selecting a suitable stem cell donor for their affected siblings.

Effective Prevention of Acute and Chronic Graft-Versus-Host Disease with Anti-Lymphocyte Globulin (ATG) without Increase of Relapse in HLA-Identical Sibling Peripheral Blood Stem Cell Transplantation.

Abstract 3055Acute and chronic graft versus host disease (GvHD) are severe complications after allogeneic stem cell transplantation but occurrence of GvHD is associated with reduced risk of relapse. Therefore, strategies to prevent GvHD without increasing the risk of relapse are urgently needed.We investigate retrospectively whether anti-lymphocyte globulin (ATG Fresenius®, Fresenius Biotech, Graefelfing, Germany) as part of the conditioning regimen may reduce the risk of GvHD without increasing the risk of relapse after peripheral blood stem cell transplantation from HLA-identical siblings or HLA-compatible relatives. Out of 462 patients who received HLA-identical stem cell transplantation between 1990 and 2011 in our institution, we selected 238 consecutive patients who received allogeneic peripheral blood stem cell grafts after the year 2000. The median age of the patients was 48 years (r.,18–73y) and diagnosis were: AML (n=93), ALL (n=24), CML (n=25), MDS (n=23) or lympho-proliferative disorders (n=73). Patients were classified as good risk (n=95) or bad risk (n=143). 79 patients did receive ATG within the conditioning regimen with a median dose of 30mg/kg (r., 20–90mg/kg) and 159 patients did not receive ATG. In the ATG group there were more HLA mismatch donors (6% vs. 1%, p=0.02), more bad risk patients (70% vs. 50%, p=0.04), more reduced intensity conditioning regimens (65% vs. 34%, p< 0.001) and older patients (median age 50 vs. 46 years, p=0.03). Acute GvHD grade I to IV was less observed in the ATG group (27% vs. 40%, p= 0.04), but the difference in severe GvHD grade III and IV did not show statistical significance (10% vs. 18%, p=0.1). Chronic GvHD was less observed in the ATG group (30% vs. 52%, p=0.002), which was most obvious for extensive chronic GvHD (14% vs. 40%, p<0.001). After a median follow up of 57 months in the non-ATG and 63 months in the ATG group the cumulative incidence of non-relapse mortality at 5 years was 20% for the ATG and 34% for the non-ATG group (p=0.06) and the cumulative incidence of relapse at 5 years was 34% for the ATG group and 29% for the non –ATG group (p=0.2), resulting in a comparable 5 year progression-free (ATG: 46% and non-ATG 37%, p=0.5) and overall survival (ATG: 54% and non-ATG 46%, p=0.4).This retrospective study shows that ATG can prevent severe chronic GvHD without obvious risk of relapse and similar PFS and OS in HLA identical sibling transplantation. A randomized trial including QoL measurement comparing ATG vs. no ATG in HLA identical sibling transplantation is ongoing (registered NCT 00678275). Disclosures:Kröger:Fresenius Biotech: Honoraria, Research Funding. Off Label Use: ATG in HLA identical sibling transplantation.

Rapid Increase of CD8+ T Cell Count in Peripheral Blood of Pediatric Patients After HLA-Identical Stem Cell Transplantation for African Sickle Cell Anemia

Abstract 4769 Background.Sickle Cell anemia (SCA) remain a disease with high risk of morbidity and early death, especially in African patients. Allogeneic haematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCA. To analyze immunohematological reconstitution after transplant, we report our experience concerning 12 geno-identical HSCT for SCA-patients prepared with the same myeloablative conditioning regiment consisting of Busulfan and Cyclophosphamide. Patients and Methods.Twelve patients with a median age of 12 years (range, 2–16), affected by sickle cell anemia (SCA), received hematopoietic stem cell transplantations from HLA-identical, related donors following a myeloablative conditioning regimen. To analyze the mechanisms involved in immunological reconstitution post transplant, we analyzed T cell subsets by flow cytometry at + 60 post transplant. Results.All patients had sustained engraftment and remained free of any SCA-related events after transplantation. Sixty days after the transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (15.6 ± 5.9 % vs. 47.5 ± 6% respectively), whereas CD8+ T cells were the first lymphocytes to repopulate the peripheral blood with up to 45% of these cells being CD8+ T cells (in mean 48.5 ± 14.3 % vs. 20 ± 7%). All patients displayed reduced numbers of B cells versus normal value, and 10/12 patients had only 0% to 1% of control levels of CD19+ cells. CD3-CD56+bright NK cells were 18.6 ± 12.2 %, whereas CD3-CD16+ (with cytotoxic functions) were 16.7 ± 11.8 %. Conclusion.Our primary finding include the following: 1) rapid increase of lymphocytes in peripheral blood after transplant; 2) rapid expansion of CD8+ T cell but not CD4 T cell counts. Probably reactivation of cytomegalovirus (CMV) infection, observed in 11/12 patients on the early stages of T-lymphocyte recovery after transplant, might induce a dramatic increase in CD8 but not in CD4 T-cell counts. Disclosures:No relevant conflicts of interest to declare.

Decreased Apoptosis in Bone Marrow of Pediatric Patients After HLA-Identical Stem Cell Transplantation for African Sickle Cell Anemia

Abstract 4751 Background.In thalassemia patients ineffective erythropoiesis appears to be caused by accelerated apoptosis, in turn caused primarily by deposition of a-globin chains in erythroid precursors. The bone marrow is the main site of the erythroid cell death. The occurrence of apoptosis at the polychromatophilic stage coincides with the stage where intensive hemoglobinization occurs and could be explained by the increase in a-globin production and precipitation. The mechanism of BM apoptosis is also observed in sickle cell anemia (SCA), but is reduced. We report our experience concerning 12 geno-identical hematopoietic stem cell transplantation (HSCT) for SCA-patients, to analyse the effect of transplant on “normalization” of intramedullary apoptosis. Patients and Methods.Twelve patients with a median age of 12 years (range, 2–16), affected by sickle cell anemia (SCA), received hematopoietic stem cell transplantations from HLA-identical, related donors following a myeloablative conditioning regimen. We studied Fas (CD95+) and caspases-3 expression on erythroblast subpopulation. Using both CD71 and FSC parameters, we obtain 3 principal populations, which we labelled A, B, C erythroblasts (Ery. A, Ery. B, Ery. C). Ery A (CD71high FSChigh) are basophilic, Ery. B (CD71high FSClow) are late basophilic and polychromatic, whereas Ery. C (CD71low FSClow) are orthochromatic erythroblasts and reticulocytes. Results.We observed an expansion of Ery A, Ery B and Ery C population at baseline, probably as an essential process needed to maintain a constant red cell production in SCA patients (42.4% Ery A; 24.7% Ery B; 35.6% Ery C). After BM transplant decreased levels was observed especially on Ery A and Ery B populations (12.9% Ery A; 7% Ery B; 35% Ery C). After BM transplant Fas expression was reduced in all 3 erythroid population, but significantly on more immature Ery A (17% vs 3.5% CD95+ on Ery A; 32.2 vs 24.4 on Ery B; 5% vs 3.3% on Ery C). This observation may suggest that Fas, expressed by early erythroblasts in vivo, might contribute to the cell death of erythroid precursors in bone marrow, but this was corrected after HSCT. In addition, only after transplant we observed an initial increase of caspases 3 on more mature Ery C population (2.3% at baseline vs. 15.3% after transplant), as observed during later steps of “normal” cell maturation. Conclusion.After HSCT in SCA we observed a “normalization” of erythroid populations, in parallel with decreased intramedullary apoptosis rate, suggesting normal erythroid maturation in ex-SCA patients. Disclosures:No relevant conflicts of interest to declare.

Overview of T-cell depletion in haploidentical stem cell transplantation.

Overview of T-cell depletion in haploidentical stem cell transplantation.

Unmanipulated HLA-Haploidentical (2-3 antigen-mismatched) Stem Cell Transplantation Using Myeloablative or Reduced-Intensity Preconditioning Regimen,

Abstract 4117Related haploidentical donors, as cord blood, can be alternative donor sources in stem cell transplantation (SCT). Severe GVHD, however, has interfered the progress of haploidentical SCT (haploSCT). To deal with this strong GVHD, T cell depletion has usually been used in US and European countries. In order to pursue the controllable GVL effect by T cells, we have performed unmanipulated haploSCT using myeloablative or reduced intensity preconditioning regimen accompanied with intensified GVHD prophylaxis, including steroids. In this meeting, we will summarize our experience of haploSCT for more than ten years. From August 1998 to September 2010, we have performed 351 cases of haploSCT (all cases were HLA 2–3 antigen mismatched in GVH direction). Patients’ characteristics are sex: male 186, female 168, age: 16–65 years old (median 39), disease: AML/MDS 149, ALL 81, ML 67, others 54. Eighty-three percent of cases underwent SCT in non-complete remission (non-CR) status. Patients under 45 years old underwent myeloablative preconditioning regimen consisting of FLU/CA/CY/TBI 8Gy (haplo-full, n=100), and patients over 45 years old or with comorbidities or repetitive SCT (including second to fifth SCT) underwent reduced intensity preconditioning regimen consisting of FLU/(CA)/BU/ATG or FLU/(CA)/MEL/ATG (haplo-mini, n=251). High dose Ara-C (CA) was optional to reduce tumor burden. As ATG, ATG (Fresenius) 8mg/kg, or thymoglubulin (genzyme) 2–4mg/kg were integrated into conditioning treatments mainly for reduced-intensity transplantation. GVHD prophylaxis consisted of taclolimus (TAC), methylprednisolone (mPSL) 2mg/kg/day, short term MTX, and mycophenolate mofetil (MMF) 15mg/kg/day in haplo-full, and TAC, and mPSL 1mg/kg/day in haplo-mini, respectively. For elderly patients over 50 years old in haplo-mini, MMF was added. Hematopoietic engraftment in haploSCT was as rapid as that in HLA-identical SCT, except 10 cases of graft rejection. The median time to reach a neutrophil account of >0.5 × 109/l was 10 days for haplo-mini and 13 days for haplo-full. Platelet recovery was achieved in 66 % and 60% of patients undergoing haplo-mini and haplo-full, respectively. The median time to reach a nontransfused platelet count of 3 20 × 109/l was 22 days for haplo-mini and 33 days for haplo-full. Sixty percent of haplo-mini patients and 54 % of haplo-full patients did not develop acute GVHD. Acute GVHD (grade II-IV) was observed in 20% for haplo-mini and 36 % for haplo-full. Overall survival at five years was 30% for haplo-full and 40% for haplo-mini, respectively. If limited to CR cases, overall survival reached over 60% in haplo-mini. There is no difference in survival rate among patients’ diseases. In multivariate analysis on survival using variables, including disease status before transplantation, haplo-full vs haplo-mini, mismatches in GVH direction, mismatches in HVG direction, patients’ age, and the number of transplantation times, the disease status (CR) was found to be only a significantly favorable factor (P= 0.0026). Unmanipulated haploSCT is feasible and effective for refractory diseases. ATG dose used in haplo-mini is critical, and rather low compared with that of European cases reported so far. Although it should be too early to refer long term outcome, unmanipulated haploSCT could be considered as an option to control refractory diseases. Disclosures:No relevant conflicts of interest to declare.

Cardiovascular Risk: Analysis of Known Factors in Patients After Treosulfan-Based Conditioning for Haploidentical and HLA-Identical Stem Cell Transplantation

Abstract 4471Allogeneic stem cell transplantation (HSCT) is an emerging risk factor for cardiovascular disease. Long-term survivors seem to be at higher risk for premature arterial vascular disease compared with sex- and age-matched population. In HSCT, pre-transplant treatments and the conditioning regimen play a key role in endothelial and organ damage. The use of immunosuppressant drugs to prevent graft-versus-host disease (GvHD) influences as well the development of cardiac disease. To better understand the impact of treosulfan-based conditioning regimens in leading to the emergence of cardiovascular disease, we retrospectively evaluated 94 consecutive patients who had survived longer than 1 year, transplanted at Our Institution between 2002 and 2010 (62 males – median age 46, range 14–69). For 48 patients an HLA-identical sibling was available, 6 patients found an unrelated donor while 40 patients underwent haploidentical HSCT from family donors. Forty-six patients received in vivo T-cell depletion and 72 B-cell depletion. GvHD prophylaxis was based upon cyclosporine + methotrexate for the HLA-identical transplants (48 – median EBMT risk score 3) whereas rapamycin + MMF were used for patients undergoing transplantation from HLA-haploidentical donors (35 – median EBMT risk score 4); 9 did not receive any immunosuppression because of application of suicide gene strategy as tool to prevent GvHD (median EBMT risk score 3). We compared clinical variables (i.e. GvHD, OS, TRM, major cardiac events and comorbidities) with biochemical and functional tests (i.e. cardiac function, lipid profile and ferritin levels) both before and 1 year after transplantation. After a median follow-up of 29 months, only 3 patients experienced a major cardiovascular event; no one experienced a late congestive heart failure. In all series, we found that ferritin levels significantly decrease after 1 year of follow-up, compared to pre-transplant values (P <0.0001); viceversa, the lipid profile (total cholesterol, HDL and LDL fractions) remarkably increased (p <0.05). Interestingly, the linear regression analysis for the overall survival showed no difference for both ferritin level and cholesterol level. Moreover, no significant variations were detected comparing cyclosporine versus rapamycin-treated patients, nor comparing patients with or without GvHD. Increased rates of cardiovascular disease are commonly associated to HSCT. In our experience, no differences were found in the incidence of cardiovascular events comparing different donor source or immunosuppressants. This suggests that haploidentical transplants are feasible and have similar cardiac toxicity than standard HLA-identical ones. Our analysis was surely limited by the short time of observation. In future, the evaluation of dynamic biomarkers on a greater series and on a longer follow-up will help us to identify a tailored schedule of cardiac monitoring after HSCT to prevent major lethal events in our patients. Disclosures:No relevant conflicts of interest to declare.

Gene Therapy with IgG-HY Fusion Proteins to Reduce Male-Specific T-Cell ReactivityIn Vitro

Graft-versus-host disease is still a major complication in stem-cell transplantation. In HLA-identical stem-cell transplantation, mismatches in minor histocompatibility antigens contribute to the development of graft-versus-host disease. As treatment of graft-versus-host disease with immunosuppressive drugs potentially also reduces the graft-versus-leukemia responses, antigen-specific tolerance induction may be used to reduce graft-versus-host disease while leaving the graft-versus-leukemia responses intact. In mouse models, induction of antigen-specific tolerance using IgG-antigen-transduced B cells can prevent and ameliorate autoimmunity. In this study, the principle of the in vivo animal model has been applied to a human in vitro model wherein induction of tolerance against the HLA class II-restricted male-specific minor histocompatibility antigen RPS4Y1 has been analyzed. T cells precultured in the presence of B cells expressing the IgG-coupled HY antigen showed reduced responsiveness selectively against the minor HY antigen RPS4Y1 in different in vitro protocols. These first observations serve as a basis for further studying and understanding the tolerizing potential of IgG constructs with human T cells.

Low usage rate of banked sibling cord blood units in hematopoietic stem cell transplantation for children with hematological malignancies: Implications for directed cord blood banking policies

Directed sibling cord blood banking is indicated in women delivering healthy babies who already have a sibling with a disease that is potentially treatable with an allogeneic cord blood transplant. We evaluated the effectiveness of a national directed cord blood banking program in sibling HLA-identical stem cell transplantation for hematological malignancies and the factors influencing the usage rate of the stored cord blood units. Fifty families were enrolled from which, 48 cord blood units were successfully collected and 2 collections failed due to damaged cord/placenta at delivery. Among enrolled families 4 children needed transplantation; however, only one was successfully transplanted using the collected cord blood unit containing 2 × 10 7 nucleated cells/kg in conjunction with a small volume of bone marrow from the same HLA-identical donor. Two children received grafts from matched unrelated donors because their sibling cord blood was HLA-haploidentical, while the fourth one received bone marrow from his HLA-identical brother, since cord blood could not be collected due to damaged cord/placenta at delivery. With a median follow-up of 6 years (range, 2–12) for the 9 remaining HLA-matched cord blood units, none from the prospective recipients needed transplantation. The low utilization rate of sibling cord blood in the setting of hematopoietic stem cell transplantation for pediatric hematological malignant diseases necessitates the development of directed cord blood banking programs that limit long-term storage for banked cord blood units with low probability of usage such as non-HLA-identical or identical to patients who are in long-term complete remission.

Near-Tetraploidy in Childhood Acute Lymphoblastic Leukemias Does Not Adversely Affect Prognosis: An EGIL Retrospective Study On 36 Cases Petr Lemez, Andishe Attarbaschi, Marie-Christine Bene, Yves Bertrand, Gian-Luigi Castoldi, Erik Forestier, Richard Garand, Oskar A. Haas, Emmanuelle Homolle, Sandrine Kagialis-Girard, Wolf-Dieter Ludwig, Estella Matutes, Marie-Pierre Pages, Winfried Pickl, Anna Porwit, Alberto Orfao, Jan Stary, Herbert Strobl, Pascaline Talmant, Mars B. Van't

Abstract 1585Poster Board I-611The first larger study on near-tetraploid (metaphases with 80∼104 chromosomes) acute lymphoblastic leukemias (NT-ALL) described 20 children: 9 of the T-lineage (T-ALL) and 11 of the B-cell precursors (BCP-ALL), and concluded that: “NT-ALL represented a unique subgroup of ALL, characterized by preponderance of T-cell immunophenotype, specific morphologic features, and possibly a poorer prognosis” (Pui et al, Blood 1990;76:590). The aim of our study was to enlarge the limited knowledge on these rarely occurring NT-ALL. The EGIL collected retrospectively laboratory and clinical data of 36 children (under the age of 18 years) with NT-ALL from 6 European countries diagnosed and treated in international studies between 1992 and 2008. Bone marrow blasts of all 36 children were negative for peroxidase. Immunophenotyping of leukemic blasts showed that 10 cases were diagnosed as T-ALL and 26 cases as BCP-ALL. In 4 cases aberrant near-diploid metaphases were simultaneously present. At least a single normal metaphase was found in 27 cases. Leukemic blasts of all patients were BCR/ABL1 negative. T-ALL was diagnosed in 7 boys and three girls of a median age 11.9 (3.6 – 17.5) years. CNS infiltration was found in one child. Heterogeneity of NT-T-ALL cases was apparent with immunophenotyping because they belonged to all 4 EGIL categories: TI or TII were each diagnosed in two cases; TIII or TIV each in three cases. Their blasts were negative for HLA-DR in all 10 cases and for CD34 in all 6 examined cases. Cytogenetic examinations revealed different structural abnormalities in 6 cases. All 10 cases achieved complete remission (CR) after induction chemotherapy and 8 are remaining in the 1st CR, exhibiting event-free survival (EFS) 80 % with a median follow-up of 37+ months. One case died of septic shock in pancytopenia after his 3rd consolidation therapy and survived only 7 months. Another case relapsed in the bone marrow and maxilla after 25 months. He reached the 2nd CR with chemotherapy and is alive after unrelated HLA-identical stem cell transplantation (SCT) for 14 months. Survival of NT-T-ALL cases has been favorable - 90 % with a median of 41 (7-120) months. NT-BCP-ALL was further divided according to the cytogenetic and molecular results into 4 categories. ETV6/RUNX1 positive BCP-ALL was diagnosed in 7 boys and 5 girls with a median age of 7.0 (3.3-15.5) years. Immunophenotyping of bone marrow blasts lead to the diagnosis of EGIL categories BII in 10 cases and BIII in two. Blasts from all cases were positive for HLA-DR and for CD34 in 8 of 9 cases. All these 12 cases have achieved CR after induction chemotherapy and are alive in the 1st CR for 18-147 (median 65) months. Twelve cases of ETV6/RUNX1 negative BCP-ALL fall to at least two categories: two cases exhibited simultaneously hypodiploid metaphases with NT ones, 10 cases had only NT metaphases. These 10 cases were 6 boys and 4 girls with a median age of 9.5 (4.4-14.5) years and WBC 0.9-13.0 × 109/l. Immunophenotyping of blasts established the diagnosis of BII in 9 cases and BIII in one. All tested cases were positive for HLA-DR and CD34, three expressed CD2 antigen. All these cases reached CR after induction chemotherapy and are remaining in the 1st CR for 12-155 (median 73) months. Hypodiploid ETV6/RUNX1 negative BII BCP-ALL was diagnosed in two girls. One with a karyotype of 44 chromosomes relapsed after 37 months and died of infection in the 2nd CR after unrelated allogeneic SCT. The other with 45 chromosomes reached CR and is remaining in CR for 7 months. Leukemic blasts of two boys, 9.5 year-old, with BII NT-BCP-ALL were not examined for ETV6/RUNX1. One is in the 1st CR for 82 months. Another relapsed twice after 6 and 11 years. He underwent cord blood SCT in the 3rd CR and died 2.5 months later of cerebral toxoplasmosis. In summary, our data show immunophenotypic, cytogenetic, and molecular heterogeneity of childhood NT-ALL. In contrast to previous reports, we observed favorable prognosis of NT-ALL patients after current more intensive induction and consolidation chemotherapy. Near-tetraploidy did not deteriorate prognosis of childhood NT-ALL cases across different immunophenotypic, cytogenetic and molecular ALL types.Supported by grants MSM0021620813, MZOVFN2005. DisclosuresNo relevant conflicts of interest to declare.

Primary Exploration of Relationship of HLA-Cw typing with graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Objective To determine the effects of HLA-Cw typing on graft-versus-host disease and re-lapse after allogeneic hematopoietic stem cell transplantation. Methods HLA - Cw typing was performed in HLA identical allogeneic hematopoietic stem cell transplantation,including 9 patients with allogeneic peripheral blood stem cell transplantation(allo-PBSCT) and 10 patients with umbilical cord blood(UCBT) respectively. A-cute GVHD data were compared between the groups with different HLA-Cw typing results (matched and un-matched). Results In 16 HLA-Cw matched patients,15 cases got engrafted(94%) ,and 11 patients had Ⅰ～Ⅱ acute GVHD and 2 patients had Ⅲ acute GVHD. Fifteen patients had chronic GVHD but no patients had re-lapse. In 3 HLA-Cw unmatched patients,2 patients had Ⅲ acute GVHD and died in early stage and 1 patient had early relapse. When cases were classified into two groups accoring to HLA-Cw typing results, there is statisti-cally significant difference in both Ⅰ～Ⅱ and Ⅲ acute GVHD between these two groups (P < 0. 05). Conclu-sion In hematopoietic stem cell transplantation cases, HLA-Cw matching may be helpful to reduce GVHD. The results suggest that HLA typing is important for the prognosis of hematopoietic stem cell transplantation in addi-tion to HLA-A, B, DR/DQ matching. Key words: HLA-Cw typing; Allogeneic ; Hematopoietic stem cell transplantation; Graft-versus-host disease

HLA-Identical Allogeneic Stem Cell Transplantation (alloSCT) for Patients With Primary Cutaneous T-Cell Lymphoma (CTCL): A Retrospective Analysis of the Lymphoma Working Party of the European Blood and Marrow Transplantation Group (EBMT-LWP)

HLA-Identical Allogeneic Stem Cell Transplantation (alloSCT) for Patients With Primary Cutaneous T-Cell Lymphoma (CTCL): A Retrospective Analysis of the Lymphoma Working Party of the European Blood and Marrow Transplantation Group (EBMT-LWP)

Lower Relapse Incidence and Relapse Mortality in Patients Transplanted from a Younger Sibling in HLA-Identical Stem Cell Transplantation: Does Microchimerism Matter?

Background: There is some evidence that microchimerism plays a role in outcome of HLA-identical stem cell transplantation (SCT). Evaluation of primacy of birth revealed a superior outcome for firstborn patients (Bucher et al. 2007). The underlying mechanism may include pre-existing microchimerism due to fetomaternal and transmaternal sibling cell trafficking. In addition, persistent fetal microchimerism in women may be associated with specific HLA-alleles.Aims: To analyze the impact of relative donor/recipient birth order on outcome of HLA-identical sibling SCT.Methods: We retrospectively analyzed HLA-identical sibling SCT for all consecutive patients with hematological malignancies from 1995 to 2007 at our center. 215 patients were assigned to one of two groups: 102 (47%) and 113 (53%) recipients had an older (D>R) and younger (R>D) donor, respectively. Transplantation related data for age, sex, disease, conditioning regimen, stem cell source, T-cell depletion (TCD), incidence and severity of acute GvHD, relapse incidence (RI), relapse (RM) and non-relapse mortality (NRM) were analyzed.Results: Hematologic malignancies at time of SCT in the D>R and R>D group were acute lymphoblastic leukemia (ALL; 19% and 9%), acute myeloid leukemia (AML; 49% and 51%), myelodysplastic syndrome (MDS; both 3%), chronic myeloid leukemia (CML; 13% and 14%), other myeloproliferative syndrome (MPS; 2% and 3%), and Non-Hodgkinlymphoma (NHL; 14% and 20%), respectively. For all evaluable patients (n=215), RI was 37% in the D>R group and 27% in the R>D group (p=0.09). In unmanipulated SCT without TCD (n=157) RI was significantly reduced with 38% as compared to 23% in the D>R and R>D group (p=0.04). Of 91 patients transplanted with unmanipulated grafts after myeloablative conditioning 42 (46%) and 49 (54%) patients belong to R>D and D>R. In this subgroup RI was 33% and 12%, respectively (p=0.02). In addition, RM also compared favorably for the R>D group: 9% versus 24% (p=0.05) with no difference in NRM in the two groups (37% in D>R versus 33% for the R>D (p=0.73). Patients in the R>D group seemed to experience less aGvHD ≥II°: 21% versus 35%, (p=0.16). RI in patients receiving a SCT with myeloablative conditioning without TCD for myeloid malignancies (AML, MDS, CML, n=65) corresponded to that of the whole cohort with 13% for R>D and 33% for D>R, respectively (p=0.04). Similarly, RM and incidence of aGvHD in the R>D and the D>R group were similar for myeloid malignancies as for the whole cohort of patients transplanted with unmanipulated grafts after myeloablative conditioning.Conclusions: In this retrospective analysis patients with hematological malignancies transplanted from a younger HLA-identical sibling donor have a superior outcome in HLA-identical SCT in terms of relapse incidence and relapse mortality. Multi-center studies for specific diseases are required to establish the impact of donor and recipients birth order on outcome of HLA-identical sibling transplantation.

Identification of New Minor Histocompatibility Antigens (mHags) in Patients with Chronic Graft Versus Host Disease (GvH) after Allogeneic Stem Cell Transplantation by SEREX.

Background: Minor histocompatibility antigens are antigenic peptides derived from normal cellular molecules which are presented in the context of major histocompatibility antigens (MHC class I and MHC class II). After an allogeneic hematopoietic stem cell transplantation (aHSCT) recipient-derived mHags can be recognized by T-cells of the transplanted immune system an mediate both a graft-versus-host disease and graft-versus- leukemia reaction. All mHags known to date have been identified by allo-reactive T-cells. Due to complex logistics needed for the establishment of such T-cell clones only a few mHags have been molecularly defined to date. Since patients with GvH develop antibodies against mHags and the presence of mHag antibodies has been shown to correlate with GvH and maintenance of remission [Miklos et al. (2005) Blood 105:2973-9] we set out to identify new mHags using SEREX the serological identification of antigens using recombinant expression cloning [Sahin et al. (1995) PNAS 92:11810-3].Methods: Fibroblasts obtained by skin biopsies from patients with chronic GvH were propagated in vivo and used as a source to establish a cDNA library. cDNA was expressed by lambda phage in E.coli and expressed clones were screened for reactivity with antibodies in the serum of patients with chronic GvH. Positive clones were monoclonized and sequenced. The sequences were compared with known sequences using BLAST data bank. Reactive sera were compared with patient's pre transplant serum and the serum from the donor to exclude pre-existing antibodies against (auto-)antigens expressed by the fibroblasts. Only neo-antigens recognized by the transplanted immune system were analyzed further.Results: cDNA libraries obtained from 7 patients with chronic GvH after HLA-identical stem cell transplantation were screened with the sera of these 7 patients by SEREX. Antibodies from one patient with chronic GvH were absent in the donor and the patients pre-transplant serum, reacted with c1ORF107. This constellation proves that the c1ORF107 antibodies in the patient's serum developed after transplant and recognized c1ORF as a neo-antigen. The immunogenic c1ORF107 of the patient differed at previously described polymorphic position 275 (rs585627) with G in the recipient leading to glutamate and C in the donor leading to glutamine. The definition of the c1ORF107 epitopes eliciting a T-cell response by “reverse T-cell immunology” is currently underway.Conclusion: This study proves the principle that the analysis of the humoral immune response in patients with chronic GvH by SEREX allows for the definition of new mHags. SEREX in combination and followed by reverse T-cell immunology is a straight-forward approach which will expand considerably the number of molecularly defined mHags causing GvH in patients after allogeneic stem cell transplantation. Supported by Jose Carreras Foundation Germany