Post-transplant Graft-versus-host Disease Prophylaxis Research Articles

CD34+Selected Hematopoietic Stem Cell Transplants Conditioned with a Myeloablative Regimen Is Well Tolerated and Results in Good Outcomes in Patients with Myelofibrosis

Background: Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) remains the only curative treatment for myelofibrosis (MF). Due to high incidence of non-relapse mortality (NRM) in patients with MF who underwent allo-HCT with myeloablative conditioning (MAC), transplants in these patients are mostly done by utilizing a reduced intensity conditioning regimen with calcineurin inhibitors for graft versus host disease (GVHD) prophylaxis. We previously published very good outcomes in patients with acute leukemia (AML and ALL) and myelodysplastic syndrome (MDS) who underwent Ex- vivo CD34+-selected T-cell depleted (TCD) allo-HSCT following MAC regimens (Barba P et al. BBMT 2017; Tamari R et al. BBMT 2018).Aim: To study retrospectively the outcomes of patients with primary or secondary myelofibrosis (PMF or SMF) who underwent a CD34+ cell-selected Allo-HSCT.Methods: Twenty-one MF patients who underwent a CD34+-selected Allo-HSCT at a single center between October 2010 and November 2017 were included in this retrospective analysis. All patients received MAC regimen including busulfan, melphalan and fludarabine and antithymocyte globulin to prevent graft rejection. None of the patients received post-transplant GVHD prophylaxis. G-CSF mobilized peripheral blood stem cell grafts were depleted of T-cells using immunomagnetic CD34+ selection by CliniMACS device. Overall survival (OS), relapse free survival (RFS), relapse, NRM and the composite endpoint of GVHD-free/relapse-free survival (GFRS: defined as grade 3-4 acute GVHD, chronic GVHD requiring systemic treatment, relapse, or death) were estimated using the Kaplan-Meier and cumulative incidence method, with death considered a competing risk for relapse. Log-rank and Gray's tests were used to assess differences in patient and treatment characteristics.Results: Patient's and donor's characteristics are summarized in table 1. Neutrophils engraftment occurred in all patients at a median of 11 days (range: 8 - 14) and 90% (N = 19) achieved platelet engraftment at a median of 24 days (range, 14 - 77). Another patient achieved platelet engraftment only after splenectomy which was performed on post-transplant day 54. With a median follow-up of 54.06 months, the estimated 3-year OS and RFS were 84.4 % (95% CI, 69.6% to >99.9%) and 74.7% (95% CI, 57.6 to 96.9%), respectively (figure 1). The cumulative incidence of grade II-IV acute GVHD at day 100 was 33.3% (95% CI 11.2-54.1%); majority (N=5) had grade II and 2 patients had grade III (N=1) and grade IV (N=1) acute GVHD. Chronic GVHD developed in 2 patients including only 1 case requiring systemic treatment. The 3-year cumulative incidence rate of relapse was 9.5% (95% CI, <0.1 to 22.4%); three relapse cases include 2 patients with molecular/cytogenetic relapse and 1 patient with clinical relapse. Patients who relapsed were treated with donor lymphocyte infusion (DLI) (N=1), with azacytidine plus DLI (N=1) and both are alive with minimal molecular disease. The 3rd patient is alive without evidence of disease recurrence 43 months after second unmodified Allo-HSCT from the original donor. NRM at 3 years was 15.6% (95% CI, <0.1% to 32.4%) and the cause of all deaths (n=3) was primarily attributed to acute GVHD. The estimated 3-year GRFS was 66.7% (95% CI, 49.3 to 90.2%) (figure 2). TCD boost and unmodified boost were conducted successfully for patients with poor graft function (N=1) and late graft failure (N=1). Six patients received 8 DLIs for the following indications: mixed chimerism (N=3), relapse (N=3) and poor immune reconstitution (N=2).Conclusions: In this analysis we demonstrate that CD34+ selected Allo-HSCT following a chemotherapy only based MAC regimen is well-tolerated and an effective treatment for patients with myelofibrosis. We noted higher incidence of acute GVHD when compared to our reported outcomes in patients with acute leukemia and MDS undergoing a CD34+ selected allo-HSCT and all cases of mortality in this analysis were secondary to GVHD. This may suggest differences in the biology of the diseases and a more inflammatory milieu in pts with MF. Relapse incidence was notably low and all patients who relapsed were salvaged with further cellular therapy suggesting a strong graft-versus-leukemia effect in this disease. DisclosuresSauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. Perales:Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Novartis: Other: Personal fees; Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees.

Long-Term Results on Outcome of Children with Hematological Malignancies Transplanted from an Unrelated Donor Enrolled in an Open Label, Randomized Trial Comparing Two Different Dosages of Rabbit Anti-Thymocyte Globulin

▪Introduction. Although rabbit anti-thymocyte globulin (ATG) is largely used for preventing immune-mediated complications in patients given allogeneic hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD), the optimal dosage of this drug in children is still undefined. We conducted an academic open-label, multicenter, randomized trial comparing two different dosages of rATG in children with hematological malignancies transplanted with either bone marrow (BM) or peripheral blood stem cell (PBSC) from a MUD selected using high-resolution typing for HLA class I/II (i.e. A, B, C, DRB1) loci. The study aimed at testing whether a higher dose of rATG (30 mg/Kg over 3 days, from day -4 to day -2) was superior to a lower dose (15 mg/Kg over 3 days, from day -4 to day -2) in terms of grade II-IV acute graft-versus-host disease (GvHD) prevention. Secondary end-points included cumulative incidence (CI) of chronic GvHD, non-relapse mortality (NRM), disease recurrence and event-free survival (EFS). The drug was kindly provided by Fresenius/Neovii.Patients and methods. Inclusion criteria were: diagnosis of acute or chronic leukemia in remission/chronic phase, non-Hodgkin lymphoma or myelodysplasia; age at HSCT 0-19 years; availability of an unrelated donor selected using high-resolution molecular typing of HLA-A, B, C and DRB1 loci, completely matched or with a single allelic disparity at one of the HLA loci or with 2 allelic disparities, or with one antigenic disparity; use of G-CSF-mobilized PBSC or BM-derived hematopoietic stem cells.From January 2008 to September 2012, 180 patients were enrolled in the study. Eight patients, 5 randomized to the rATG 30 mg/kg group and 3 to the 15 mg/kg group, did not proceed to transplant due to further relapse before HSCT. The remaining 172 patients, 84 belonging to the 30 mg/kg group and 88 belonging to the 15 mg/kg group, were transplanted and included in this analysis; 94 children had acute lymphoblastic and 42 acute myeloid leukemia. The 2 randomization groups were comparable for all demographic and transplant-related variables evaluated (see Table 1). Patients were stratified according to the stem cell source (BM vs. PBSC), degree of HLA compatibility with the donor (unrelated donor perfectly matched or with 1 allelic disparity vs. unrelated donor with 2 allelic disparities or with 1 antigenic disparity) and disease risk group (standard- vs. high-risk, SR and HR). All patients received a fully myeloablative preparative regimen. All patients received cyclosporine-A and short-term methotrexate as post-transplant GvHD prophylaxis. The trial was registered at ClinicalTrials.gov (NCT00934557). Data were analyzed as of January 31st, 2016.Results. With a median follow-up of 4.5 years (range 3.3-7.6 years), the 100-day CI of grade II-IV acute GvHD in the high and low rATG group was 29% and 36%, respectively (P=NS, Figure 1A); the CI of extensive chronic GvHD in the two groups was 6% and 10%, respectively (P=NS, Figure 1B). The CI of NRM was 19% and 9% in the high and low rATG group, respectively (P=0.09). Children receiving the higher rATG dosage had a greater incidence of viral reactivations as compared to those who received the lower dosage. The difference was statistically significant for EBV reactivation (37% vs. 23%; P = 0.038) and for Adenovirus reactivation (12% vs. 1%; P = 0.004). The overall CI of disease recurrence was 17% and did not differ between high and low dose rATG.The 5-year EFS for the whole cohort of patients was 69%; it was 61% and 77% for children given high and low dose rATG, respectively (P=0.028, Figure 1C). EFS was 78% and 55% in the SR and HR groups, respectively (P=0.001, Figure 1D). EFS of the 136 children with acute leukemia given either high or low dose rATG was 60% and 77%, respectively (P=0.049). In multivariate analysis on EFS, the following variables were associated with an unfavorable outcome: rATG dose of 30 mg/kg (relative risk 1.90; P=0.026), HLA mismatch > 1 allele (relative risk 2.08; P=0.01) and HR disease (relative risk 2.46; P=0.0015).Conclusions. Our data indicate that, in children with hematological malignancies transplanted from a MUD selected through high-resolution HLA typing, the use of low dose rATG results into decreased incidence of NRM and better EFS. Low dose rATG is able to spare life-threatening viral infections, without significantly increasing the incidence of acute and chronic GvHD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chemotherapy-Based HLA Haploidentical Transplantation with Treg/Tcon Immunotherapy in Unfit/Elderly Leukemia Patients: Powerful Gvl Effect and Insights from Animal Models

In patients with acute leukemia (AL) at high risk of relapse, because of unfavorable cytogenetics, molecular markers and disease status, the most powerful therapy is hematopoietic stem cell transplantation. In HLA-haploidentical stem cell transplantation we showed adoptive immunotherapy with naturally occurring T regulatory cells (nTregs) followed by conventional T cells (Tcons) prevented graft-versus-host-disease (GvHD) (Di Ianni et al., Blood 2011) and was associated with low transplant-related mortality (TRM) and relapse rate (Martelli et al., Blood 2014) in patients with AL conditioned with single-dose TBI.However, patients that are heavily pretreated (e.g., because of multiple induction chemotherapy cycles, previous autologous or allogeneic transplant, previous irradiation, infections, etc.) and elderly patients are not expected to withstand a high-intensity TBI-based conditioning regimen. For these patients with an indication to transplant we, therefore, designed a chemotherapy only-based conditioning regimen. We enrolled 24 patients with high risk AL (19 AML and 5 ALL, median age: 46, range 21-64). Twenty-two patients were transplanted in CR and 2 in relapse. For 5 patients this was the second transplant. Patients received a conditioning regimen that included thiotepa (10 mg/Kg on days -10 and -9), fludarabine (200 mg/sqm from day -10 to -7) and melphalan (80-120 mg/sqm on day -5). Fiftheen patients also received ATG (6 mg/kg on days -24 and -23). Seven patients also received cyclophosphamide (20 mg/Kg on days -8 and -7) (and no ATG). Two patients received only Treosulfan (36 gr/sqm from day -8 to -6), fludarabine and thiotepa. On day -4 patients were received an infusion of 2x10e6/kg nTregs (93% ± 8 SD FoxP3+ cells). On day 0 they received CD34+ cells (mean 9.5x10e6/kg ±3.1 SD) and Tcons (1x10e6/kg). No post-transplant pharmacologic immune suppressive GvHD prophylaxis was given. Twenty-two of the 24 patients engrafted. CD4+ and CD8+ cell counts reached 200/µL on days 56 (range 30-100) and 35 (range 20-120) respectively with a wide T-cell repertoire as analyzed by Spectratyping. Eight of the 22 engrafted patients (36%) developed ≥ grade II acute GvHD. Three of the 8 patients died, 3 are alive with cGvHD and 2 are currently under immunosuppressive therapy. The incidence of TRM was 32% (7/22). Only one patient (in relapse at the time of transplant) relapsed. At a median follow-up of 24 months (range 5-44), 62% of the patients are alive and leukemia-free. The mechanisms underlying Treg suppression of GVHD with no loss of GVL activity were clarified by our murine transplant models. NSG mice received human myeloid or lymphoblastic leukemia and HLA mismatched Tregs/Tcons. Mice that received leukemia and Tcons (without Tregs) cleared leukemia but died of GvHD. Human T cells harvested from their bone marrow, liver and gut displayed a CD8+ phenotype and mediated alloreactivity against human leukemia. Mice treated with Tregs alone died of leukemia. No human T cells were found in their bone morrow. Human CD4+ T cells were instead recovered in the liver and gut. They had retained their regulatory function as they inhibited mixed lymphocyte reaction. Mice that received human leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. Human T cells harvested from liver and gut were composed of CD4+/FOXP3- T cells (60%) and CD8+ T cells (40%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against human leukemia. In contrast, human T cells harvested from bone marrow were still predominantly CD8+ and still displayed potent alloreactivity against human leukemia. In conclusion, peripheral blood Tregs used for adoptive immunotherapy were predominantly CD45RO+ (and negative for the bone marrow homing receptor CXCR4) and, when infused in mice, migrated to GvHD target organs (i.e., liver and gut) where they blocked Tcons and prevented GvHD. However, they were unable to migrate to the bone marrow, thereby allowing Tcons to exert unopposed alloantigen recognition and leukemia killing.These results demonstrate haploidentical transplantation with a chemotherapy-based conditioning regimen and Treg/Tcon immunotherapy is not only feasible in fragile patients but also exerts a powerful anti-leukemic effect. Apparently, the Treg/Tcon immunotherapy plays a key role in the GvL effect. DisclosuresPierini:Stanford University: Patents & Royalties.

Transplantation from Matched Unrelated and Haploidentical Donor in Pediatric Severe Aplastic Anemia: Experience with TCR Alpha/Beta and CD19 Depletion As Graft Processing Method

Introduction Hematopoietic stem cell transplantation from non-sibling donors remains the only curative option for severe aplastic anemia patients refractory to ATG/CsA immunosuppression. Although the results of MUD and haploidentical transplantation in SAA have improved significantly, graft-versus host disease (GVHD) remains a serious problem, associated with significant morbidity and mortality. We investigated the role of new method of graft processing - TCR alpha/beta depletion as a way to improve the results of MUD and haploidentical transplants in SAA.Patients and methods Forty two patients with SAA were treated since November 2012 till February 2016. Median age at HSCT was 11(3-22) years, 27 male/15 female. All pts. were refractory/relapsed (36/6) after at least two courses of ATG/CsA, 3 pts. had concurrent severe hemolytic PNH. Time from diagnosis to transplant was 17(143-8,6)/15(99-5,5) months. Donors were unrelated volunteers in 32 cases, haploidentical parents in 10 cases. Preparative regimen included cyclophosphamide 100-150 mg/kg, fludarabine 150mg/kg, ATG and 2-6Gy thoraco-abdominal irradiation, in haplo transplants patients additionaly received thiophosphamide at 10mg/kg. Two patients recieved alemtuzumab instead of ATG because of anaphylaxis. Post-transplant GVHD prophylaxis included Tacro and Mtx on days +1, +3, +6. PBSC grafts were depleted of TCR alpha/beta cells and CD19 cells with CliniMACS device as recommended by the manufacturer. Patients received a median of 10(6,0-23) x106 CD34 per kg, 8(1-39) x104 TCR alpha/beta per kg.Results All patients engrafted with a median of 15 days for WBC and 13,5 days for platelets. In 4 patients after MUD transplantation secondary graft failure (rejection) developed, two of them were successfully retransplanted. Cumulative incidence of aGVHD 2-3 was 9% (95% CI: 3-27%) and 40%(95% CI: 18-85%) in MUD and haplo respectively, 90% patients with aGVHD had only skin involvement. No grade 4 aGVHD detected. Cumulative incidence of grade 3 aGVHD was 3%(95% CI: 0,5-21%) and 10%(95% CI: 2-64%) in MUD and haplo, respectively. Cumulative incidence of cGVHD was 12%(95% CI: 0,5-58%) and 30%(95% CI: 11-77%) in MUD and haplo respectively. A median follow up is 2 years. Seven patients died of viral infection - CMV (2 pts.), viral infection - CMV plus GVHD (2 pts.), microangiopathy (1 pt), 2 pts. died after second transplantation (1 - disseminated toxoplasmosis, 1 - ADV and CMV and GVHD). Event and GVHD-free survival is 73%(95% CI: 57-89%) and 60% (95% CI: 30-90%) in MUD and haplo respectively (pic.1)Overall survival is 86%(95% CI: 74-99%) and 78%(95% CI: 50-100%) respectively for MUD and haploidentical (pic.2). Prolonged stable mixed chimerism in T-cells was detected in recipients of MUD grafts in contrast to haploidentical grafts (pic/3).Conclusion TCR alpha/beta depletion is a robust platform for allogeneic transplantation from MUD and haploidentical donors in severe aplastic anemia. Results should be further improved by additional measures to control viral infections and prevent rejection in MUD transplants. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Donor Lymphocytes Depleted of Alloreactive T-Cells (ATIR101) Reduce Transplant Related Mortality and Improve Overall Survival in Haploidentical HSCT for Patients with AML and ALL, Using an Immunosuppressant-Free Transplant Regimen

IntroductionHaploidentical family donors are becoming an effective alternative for patients in need of an hematopoietic stem cell transplantation (HSCT) but lacking an HLA-matched donor. However, to prevent graft-versus-host disease (GVHD), intensive in-vivo or ex-vivo T-cell depletion is often utilised, resulting in slow immune reconstitution, frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival.To overcome these limitations, we have developed a strategy that allows additional donor lymphocytes to be infused post-HSCT without the risk of inducing severe GVHD and maintaining the ability to react against infections and leukemic cells.Patients and MethodsIn this open-label, multi-centre phase 2 clinical trial (CR-AIR-007; NCT01794299), 23 patients with high-risk AML or ALL were enrolled. Patients underwent myeloablative conditioning and were given a CD34+ selected stem cell graft from a haploidentical family donor. In addition, donor lymphocytes from the same donor were processed using a selective photodepletion technology, creating a donor lymphocyte product depleted of alloreactive T-cells (ATIR101). ATIR101 was to be given to patients at 28-32 days post-HSCT at a fixed dose of 2x106 CD3+ cells/kg. No post-transplant GVHD prophylaxis was administered.The primary endpoint of the study is transplant-related mortality at 6 months post-HSCT. Patients are followed to determine disease-free and overall survival.Patients undergoing a similar T-cell depleted haploidentical HSCT without ATIR infusion (CR-AIR-006; NCT02188290) were used as controls. Most centers providing the historic control data also participate in this phase 2 trial with ATIR101.ResultsTwenty-three patients, median age of 41 years (range 21 - 64), have been enrolled into this study. Seventeen patients, 7 male and 10 female, had AML, 12 in CR1 and 5 in CR2 at the time of HSCT. Six patients, 5 male and 1 female, had ALL, 4 in CR1 and 2 in CR2 at the time of HSCT.Conditioning regimen consisted of a] TBI (1200 cGy; n=10) or b] melphalan (120 mg/m2; n=12), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x 5d) and ATG (2.5mg/kg x 4d). No patient experienced graft rejection, with neutrophil and platelet engraftment achieved at a median of 11 and 12 days, respectively (range 8-18, range 9-35). 21 patients to date received ATIR101, at a fixed dose of 2x106 CD3+ cells/kg at a median of 28 days (range: 28-73 days) post-transplant. Median follow-up is 270 Days post-HSCT.No patient developed grade III-IV acute GVHD. Two cases of grade II acute GVHD were reported thus far. The onset of these two acute GVHD cases was delayed, at day 173 and day 247 post-HSCT, 145 and 219 post ATIR respectively.No patient died within the first 100 days post-HSCT. There are 3 deaths as a result of transplant related mortality within 6 months post-HSCT (primary endpoint).When compared to the matched historic control group (N=35), TRM was significantly lower in patients given ATIR101 after a T-cell depleted haplotransplant with a 6-month TRM for HSCT + ATIR101 of 17% versus 34% for HSCT only (Figure 1a).Figure 1a: Kaplan Meier of Transplant related mortality: HSCT + ATIR101 vs HSCT alone (p=0.0075), data as per 3 August 2015.Thus far, in CR-AIR-007 study, only one patient experienced a relapse, which occurred at 90 days post-HSCT and resulted in death of the patient on day 122.The overall survival of patients given ATIR101 is significantly improved, compared to the control group, with a 1-year survival of 67% in the HSCT + ATIR101 group and 20% in the control group (Figure 1b).Figure 1b: Kaplan Meier of Overall Survival: HSCT + ATIR101 vs HSCT alone (p=0.0032), data as per 3 August 2015.ConclusionsAddition of ATIR101 to a T-cell depleted haploidentical HSCT protocol significantly improves transplantation outcome, with reduced TRM and improved OS. Specifically, no patient died during the first 100 days post-HSCT. Administration of ATIR101 at the dose of 2x106 cells/kg does not induce severe GVHD, confirming the efficacy of alloreactive T-cell elimination from the donor lymphocyte infusion. We attribute the reduced TRM (mainly infections in the control group) to the additive immunological protection afforded by ATIR101. Moreover, the low number of relapses observed thus far is most encouraging and supports the preservation within ATIR101 of T-cells able to recognize leukemic antigens. [Display omitted] [Display omitted] DisclosuresRoy:Kiadis Pharma: Consultancy, Research Funding; Novartis: Honoraria. Rudiger:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Rovers:Kiadis Pharma: Employment.

Selective Photodepletion of Recipient-Alloreactive T-Cells Enables Safe and Efficacious Haploidentical HSCT: Initial Results from a Phase 2 Trial in Patients with AML, ALL, and MDS

▪IntroductionFor patients in need of a hematopoietic stem cell transplant (HSCT) but lacking an HLA matched donor, a haploidentical family donor is a particularly appealing alternative. However, to prevent graft-versus-host disease (GVHD), haploidentical HSCT necessitates intensive in vivo or ex vivo T-cell depletion that results in frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival. To overcome this limitation, we have developed a strategy that photodepletes host-reactive cells from the donor T cell graft, while preserving anti-infection and anti-leukemia reactivity.Patients and MethodsIn an open-label, multi-center phase 2 clinical trial (CR-AIR-007; NCT01794299), 12 of a planned 23 patients with high-risk hematologic malignancies were treated to date with this immunotherapy approach consisting of donor lymphocytes selectively allodepleted of host-reactive T-cells using photodynamic therapy (ATIR). ATIR was infused 28-32 days after haploidentical CD34-selected HSCT. No post-transplant GVHD prophylaxis was used. These patients were compared to a control group of 28 patients treated in a previous Phase 2 study with an investigational product manufactured using a process different from the Phase 1 trial and resulting mainly in dead and inactive cells instead of ATIR (CR-AIR-004).ResultsTwelve patients, mean age of 45 (range 21-64), 6 females/6 males with AML (n=9) and ALL (n=3) were treated with ATIR so far. ATIR consisted mainly of T-cells (>90%), with residual B and NK cells (≤10%). Selective depletion of recipient-reactivity in each ATIR cell graft was assessed using a CFSE-based proliferation assay. Cell division numbers upon stimulation were analyzed using Modfit LT software (Fig 1A), which generated a proliferation index representing viable/reactive T-cells in donor cells (blue) and final ATIR product (green)(Fig 1B). Selective depletion of recipient-reactive T-cells with preservation of reactivity towards 3rd party antigens and anti-CD3/CD28 was observed in all ATIR cell grafts and used as a release criteria in the 007 study. [Display omitted] Preparative regimen consisted of A) FTBI (1200 cGy; n=5) or B) melphalan (120 mg/m2; n=7), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x5 d) and ATG (2.5 mg/kg x4 d). Neutrophil and platelet engraftment was achieved in all patients at a median of 12 days (range: 9-35). No patient experienced graft rejection.Patients (n=28) in the 004 control group, mean age of 42 (range 18-61), 13 females/15 males had AML (n=19), ALL (n=6) or MDS (n=3). CFSE proliferation in T-cell grafts could not be assessed a posteriori due to low cell viability. These 004 patients received the same A) FTBI- (n=14), B) melphalan- (n= 10) based preparative regimen as 007 patients, except for 4 patients receiving single fraction (800 cGy) TBI. Neutrophil and platelet engraftment was achieved at a median of 16 days (range: 7-54). Three patients showed secondary graft rejection.Two patients in study CR-AIR-007 developed acute GVHD grade I (skin only) approximately 130 days post HSCT, which was of short duration, (18 and 41 days). Two patients died of infection and no patient relapsed at a mean follow-up of 8 months post HSCT (range 1-14 months). In the CR-AIR-004 control group, 2 patients developed grade I, 1 patient grade II and 3 patients grade III GvHD, none of these cases were lethal. Seventeen patients died of transplant related complications and 2 patients of relapse/disease progression. TRM is 20% in 007 group vs 63% in the 004 control group and OS is 80% in 007 group vs 35% in the 004 control group at 9 months post-transplant (Figures 2A and 2B). [Display omitted] [Display omitted] ConclusionsThese data confirm that a novel immunotherapy strategy consisting of donor lymphocytes selectively photodepleted of alloreactive cells (ATIR) can be manufactured consistently and reproducibly. Results to date show that ATIR is safe and does not cause any grade III/IV GvHD. Moreover, haploidentical HSCT patients treated with ATIR demonstrate very promising TRM and OS rates when compared to the control group. DisclosuresRoy:Kiadis Pharma: Consultancy, Research Funding. Foley:Hoffman-LaRoche: Advisory Board/Lectures Other, Honoraria; Lundbeck: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Sanofi: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Celgene: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Pfizer: Advisory Board/Lectures Other, Honoraria; Novartis: Advisory Board/Lectures Other, Honoraria; Jansen: Advisory Board/Lectures Other, Honoraria; Alexion: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Roche Canada: Honoraria, Research Funding, Unrestricted educational grant, Unrestricted educational grant Other. De Jong:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Wagena:Kiadis Pharma: Employment.

Allo- HSCT for Advanced Myelodysplastic Syndrome:MSKCC Vs MDACC

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is the only curative treatment for myelodysplastic syndrome (MDS). Relapse, infections and graft-versus-host disease (GvHD) are the main causes of treatment failure. We compared the outcomes of patients receiving T cell depleted (TCD) grafts at Memorial Sloan-Kettering Cancer Center (MSKCC) with patients receiving unmodified grafts at MD Anderson Cancer Center (MDACC) for advanced MDS (RAEB-1 and 2).Adult patients transplanted between 2001 -2012 were included in this retrospective analysis. All recipients of TCD grafts (N=60) received myeloablative conditioning (MAC) and antithymocyte globulin (ATG) to prevent graft rejection. None of them received post-transplant GvHD prophylaxis. Of the 129 recipients of unmodified grafts, 87 received MAC and 42 reduced intensity conditioning (RIC); GvHD prophylaxis consisted of tacrolimus and mini-dose methotrexate in the majority of patients (N=113). ATG was given to all matched unrelated donor (MUD) recipients. Patients in the unmodified group had more therapy-related MDS (MDS-t), very poor risk cytogenetics by IPSS-R at diagnosis and bone marrow (BM) blast count >5% at transplant. Only the TCD group had mismatched donors (Table 1).Univariate analysis identified a lower incidence of grade II-IV acute GvHD in the TCD group with 100-day cumulative incidence (CI) of 13.3% vs. 34.1% in the unmodified group (p=0.031). There was no difference in grade III-IV acute GvHD with a 10% CI in both groups at day-100 (p=0.546). The incidence of chronic GvHD was lower in the TCD group with a CI at 3-yrs of 3.4% vs. 44.3% in the unmodified group (p < 0.001). The non-relapse mortality (NRM) in both groups was similar. CIs at day 100, 1yr, and 3 yrs in the TCD group were 8.3%, 20.2% and 32.7% vs. 12.4%, 22.5% and 28.1% in the unmodified group (p=0.628). Relapse was lower in the TCD group, with CI at 1 and 3 yrs of 8.5% and 15.5%, vs. 31.0% and 39.4% in the unmodified group (p=0.002). Since the unmodified recipients had worse disease characteristics, further analyses in patients with good/intermediate risk cytogenetic showed that the relapse incidence was similar between these subgroups, with 3-yr CIs of 7.9% in TCD vs. 18% in unmodified group (p=0.185). The most common causes of death in the TCD group were infections (32%) and relapse (28%), while in the unmodified group it was relapse (55%), GVHD (20%) and infections (13%). Considering the differences in disease characteristics between the groups, multivariate regression models were performed for relapse-free survival (RFS) and overall survival (OS) adjusting for MDS-t, high-risk cytogenetics at diagnosis and high blast count at HSCT. No significant differences were observed between the groups for RFS (HR=1.44, p=0.128) and OS (HR= 1.35, p=0.236) (Table 2). High-risk cytogenetics at diagnosis (very poor risk) was the only significant prognostic factor for RFS (HR=5.32, p<0.001) and OS (HR=4.81, p<0.001).Allo-HSCT is an effective treatment for patients with MDS with similar long term survival with either unmodified or TCD allografts. TCD is associated with a lower incidence of acute and chronic GVHD and without an increased risk of relapse.Table 1CharacteristicsTCD (N=60)Unmodified (N= 129)p-valueMedian follow-up, months (range)43.4 (3.8-119.5)49.4 (12.2-136.3)Age, years (range)57.1 (21.9-72.0)57.0 (19.0-72.0)0.769Female gender34 (56.7%)45 (34.9%)0.008MDS-t7 (11.7%)43 (33.3%)0.003Cytogenetic risk at diagnosis ( IPSS-R)0.009Good25 (42.4%)48 (37.2%)Intermediate15 (25.4%)19 (14.7%)Poor11 (18.6%)15 (11.6%)Very poor8 (13.6%)47 (36.4%)Missing1Blasts at transplant< 0.001< 5%48 (81.4%)57 (46.3%)5-9%11 (18.6%)35 (28.5%)10-19%031 (25.2%)Missing16Donor type< 0.001MRD21 (35.0%)65 (50.4%)MUD25 (41.7%)64 (49.6%)MMD14 (23.3%)0Stem cell source0.002BM5 (8.3%)46 (35.7%)PB55 (91.7%)83 (64.3%)Table 2:Multivariate analysisVariablesOS HR (95% CI)RFS HR (95% CI)Disease etiology0.5180.511De novo11MDS-t0.87 (0.56-1.34)0.87 (0.56-1.33)Cytogenetic risk at diagnosis IPSS-R)< 0.001 < 0.001 Good11Intermediate1.41 (0.74-2.68)1.39 (0.73-2.64)Poor2.06 (1.13-3.73)2.49 (1.40-4.44)Very poor4.81 (2.88-8.02)5.32 (3.22-8.80)Blasts at transplant0.2860.502< 5%115-9%1.11 (0.70-1.77)1.03 (0.65-1.62)10-19%1.57 (0.91-2.71)1.37 (0.80-2.34)Type of transplant0.2360.128TCD11Unmodified1.35 (0.82-2.19)1.44 (0.90-2.31) DisclosuresNo relevant conflicts of interest to declare.

HLA-Haploidentical Stem Cell Transplantation with Treg and Tcon Adoptive Immunotherapy promotes a Strong Graft-Versus-Leukemia Effect

In HLA-haploidentical stem cell transplantation we showed adoptive immunotherapy with naturally occurring T regulatory cells (nTregs) followed by conventional T cells (Tcons) prevented acute and chronic graft-versus-host-disease (GvHD), favoured lymphoid reconstitution and immunity against pathogens (Di Ianni et al., Blood 2011). One major concern is whether a graft-versus-leukemia (GvL) effect is maintained since FoxP3+Tregs can also suppress immune response against tumour. However, we showed in immunodeficient NSG mice engrafted with human myeloid and lymphoblastic acute leukemia cells (7x106 iv), Treg (3x106)+Tcon(3x106) infusion rescued mice from leukemia death in the absence of GvHD. In contrast, controls died of leukemia (when not treated or infused with Tregs only) or of GvHD (when infused with Tcons only).Since September 2008, 45 consecutive patients (21 male; 24 female; median age 40, range 20-59) with high-risk acute leukemia ( 33 AML, 12 ALL) were transplanted. The conditioning regimen included TBI, thiotepa, fludarabine and cyclophosphamide (25 patients in the 1st clinical trial) or alemtuzumab/ATG (20 patients in the 2nd clinical trial). On day -4, patients received an infusion of freshly isolated donor Tregs (mean 2.5/kg ±1; Foxp3 81.01%±16.47; Helios/FoxP3 54%±8.4; CD127 11.72%±7.653) followed on day 0 by a megadose of purified CD34+ cells (9.7x106/kg ± 3.1) and Tcons (mean 1.1 /kg ±0.6; CD3 90.72%± 9.6; CD4 57.77% ± 8.85; CD8 31.21% ± 8.59). No post-transplant GvHD prophylaxis was given. 43 of the 45 patients achieved primary, sustained full-donor type engraftment. Only 6/43 evaluable patients developed ≥ grade II acute GvHD (3 alive, treated and now off-therapy). At a median follow-up of 28 months (range 4-58) no patient has developed chronic GvHD. CD4 and CD8 counts reached 100/µL medianly on days 40 (range 25-150) and 45 (range 18-100) and 200/µL on days 55 (range 45-160) and 60 (range 50-140). In KIR-ligand mismatched transplants (Ruggeri et al., Science 2002; Ruggeri et al., Blood 2007), donor versus recipient alloreactive NK cells were also preserved. At a median follow-up of 46 months (range 7-58) 23/43 patients are alive and disease free with a probability of DFS of 0.56. The cumulative incidence of TRM is 0.37. The cumulative incidence of relapse was 0.06 as only 2 AML patients transplanted from non-NK alloreactive donors have relapsed to date. This incidence is extremely low considering the high-risk patient population (9 with FLT-3/ITD; 8 with complex karyotypes; 4 with t(9:22); 2 PIF; 1 secondary AML; 1CR after second-line induction; 1 with CNS and skin localization at diagnosis; 19 ≥ 2CR). In conclusion, adoptive immunotherapy with Tregs and Tcons exerts a powerful GVT activity in both AML and ALL patients in the absence of GvHD. Disclosures:No relevant conflicts of interest to declare.

Transplantation of TcRαβ/CD19 Depleted Stem Cells From Haploidentical Donors: Robust Engraftment and Rapid Immune Reconstitution In Children with High Risk Leukemia

Abstract 1005In haploidentical transplantation with mobilized peripheral blood stem cells (PBSC’s), in vitro T-cell depletion of the graft is an effective method to prevent or completely avoid Graft-versus-Host Disease (GvHD). In order to increase the T-cell depletion efficacy of PBSC’s while maintaining the anti-tumor and anti-infectious properties of the graft, we have investigated a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, Natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of posttransplant EBV-associated lymphoproliferative disease. The αβ+ T-cells and CD19+ B cells were then removed using the CliniMACS system. So far, 23 patients have been treated in two centers, namely Tübingen and Rome. Graft manipulation resulted in a consistent efficiency of αβ+ T-lymphocyte removal at the two centers. The overall depletion of αβ+ T-cells was 4.5 log (range 3.8–5.0) and 4.3 log (range 3.7–5.0) in Tübingen and Rome, respectively, with a median number of transplanted αβ+ T-cells of 14 × 103/kg. The recovery of CD34+ stem cells was 72% and 89% in the two centers, and the median number of infused CD34+ stem cells was 11.9 ×106/kg (range 7.5 –30 × 106/kg) and 13.3 ×106/kg (range 8.3 –19.8 × 106/kg), respectively. Patients were given 107 × 106/kg (range 35 –186 ×106/kg) and 123 × 106/kg (range 51–202 ×106/kg) CD56+ NK cells in Tübingen and Rome, respectively. The median number of infused γδ+ T-lymphocytes was 11.9 ×106/kg (range 7.5–30.2 × 106/kg) and 10.3 ×106/kg (range 6.5–25.1 × 106/kg) respectively.The 10 patients transplanted in Tübingen had advanced/refractory leukemias (ALL, n=5; AML, n=5; active disease, n=6; 2nd transplantation, n=2; CR2, n=2). For this poor-prognosis patients, a reduced-intensity conditioning regimen (melphalan, thiotepa, fludarabin or clofarabin and OKT-3 or ATG) was used. No further post-transplant GvHD prophylaxis was given. All 10 patients engrafted. The median time to reach neutrophil (PMN) and platelet (PLT) recovery was 9 (range 8– 12) and 15 days (range 6 –28) respectively. All patients reached complete donor chimerism and showed a very rapid immune reconstitution with 350 (range 21–824) CD3+ T cells/μl, 66 (12–177) CD3+4+/μl and 599 (227–1390) CD56+ NK cells/μl at day +28 posttransplant. Three patients had no signs of acute GvHD, 5 patients had GvHD grade I and 2 patients had skin GvHD grade II. Only 1 patient experienced a transient grade 3 GvHD of the skin which required only topical treatment. No patient experienced chronic GvHD. Three patients relapsed after transplantation, 7 patients are in remission for 5 months (range 3 –12). There was no transplant-related death sofar.The second cohort given transplantation in Rome comprised 13 patients with ALL (9), AML (3) and NHL (1). All children but 1 had relapsed/refractory disease. In particular, 11 patients were transplanted in CR and 2 with active disease. Conditioning regimen was myeloablative and consisted of fractionated TBI, Thiotepa, fludarabine and ATG (8 mg/Kg). No further post-transplant GvHD prophylaxis was given. All patients engrafted, the median time to reach PMN and PLT recovery being 11 (range 7–13) and 12 (range 10–16) days, respectively. Only 2 patients had skin grade I acute GvHD. No patient experienced chronic GvHD. With a median follow-up of 4 months (range 1–9) 10 patients are alive and disease-free; 2 patients relapsed (1 died) and 1 had fatal lung aspergillosis. In both cohorts, γδ+ T cells started to expand faster than αβ+ T cells in the early post-transplant period, whereas at day +100, αβ+ T-cells were predominant. In addition to a rapid reconstitution of αβ+ T-lymphocytes, Vbeta spectratyping revealed a broad T-cell receptor repertoire early after transplantation.Altogether, these data indicate that transplantation of TcR αβ+/CD19 depleted cells from a haploidentical donor results in sustained engraftment, rapid immune reconstitution and low incidence of both acute and chronic GvHD. The anti-leukemic efficacy of this approach in comparison to other methods of T-cell depletion needs to be evaluated with a longer patient follow-up. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Therapy In Eight Patients with Metachromatic Leukodystrophy – Relevance of Post-Transplant Medication.

AbstractAbstract 3727The lysosomal storage disease metachromatic leukodystrophy (MLD) is caused by mutations in the arylsulfatase A (ASA) gene leading to demyelination in the central nervous system (CNS). Children often present impaired motor skills and progress to an inability to walk, paresis and cognitive deterioration. Therapeutic options are limited and currently focus on enzyme replacement, gene therapy and hematopoietic stem cell transplantation (HSCT). Both, gene therapy and HSCT aim to establish a continuous endogenous supply of ASA, which can be taken up by somatic cells of the recipient to correct their own lysosomal metabolism.We analyzed 8 patients with MLD, who underwent HSCT between 7 months and 15 years of age, regarding toxicity of a reduced intensity condition regimen and GvHD prophylaxis. The mean follow up was 2.8 years (range 8 months to 9.5 years). All patients received conditioning with treosulfan (3 × 14 g/m2), fludarabin (4 × 40 mg/m2) and thiotepa (10 mg/kg). Six patients received bone marrow from a 10/10 matched unrelated donor, one patient from her brother and one patient with late infantile MLD was transplanted from her haploidentical mother. Patients received a mean dose of 7.74 × 106 CD34+ cells/kg BW and engrafted at day 12 (range d11 to d22) with permanent full donor chimerism. Organ toxicity reached mucositis grade III and skin grade I. Three patients developed transient acute graft-versus-host disease (GvHD) of the skin grade II, which responded well to steroids. There was no case of transplant-related mortality and no chronic GvHD. The relative ASA activity in peripheral blood mononuclear cells (PBMCs) pre-transplantation was between 0 and 0.11 A514nm/106 cells and after transplantation between 0.52 and 2.24 A514nm/106 cells. Patients who were asymptomatic prior to transplantation stayed asymptomatic, but those who presented with neurological symptoms showed various degrees of progression. As the conditioning regimen showed no immediate neurotoxicity, we asked if other drugs may account for the disease progression early after transplantation. Cyclosporine A (CsA) is commonly used as post-transplant GvHD prophylaxis and is known for neurotoxic side effects. Thus, we analyzed effects of CsA on the activity of ASA in comparison to tacrolimus or mycophenolic acid (MPA). PBMCs were cultured with and without 75 or 150 ng/ml CsA (pharmacological level) for 8 days. During this time period, the ASA activity decreased to 70% of the activity of PBMC without CsA treatment. By contrast, the ASA activity of PBMCs did not decrease under treatment with pharmacological levels of tacrolimus (7,5 or 15 ng/ml) and MPA (3 or 7,5 μg/ml). Cell viability and metabolic activity were comparable in the presence of CsA and tacrolismus as assessed by MTS assays. This implies that the decrease of the ASA activity was not due to direct cell toxicity of CsA.Taken together, the conditioning regimen was well tolerated with low toxicity and good engraftment. HSCT is an option for treatment of asymptomatic MLD patients, whereas further studies are needed to identify symptomatic patients who may still benefit from the procedure. Here, the choice of GvHD prophylaxis may be an important factor. In vitro data suggested that CsA should be reconsidered with regimens including tacrolimus or mycophenolate mofetil as an alternative. Disclosures:No relevant conflicts of interest to declare.

HLA-Identical Sibling-Matched, CD34+ Selected, T Cell Depleted Peripheral Blood Stem Cells Following Myeloablative Conditioning for First or Second Remission Acute Myeloid Leukemia (AML): Results of Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 0303.

Abstract 655Allogeneic hematopoietic cell transplantation (HCT) is the most effective means to prevent relapse in patients (pts) with AML in complete remission (CR). However, quality of life and overall survival (OS) are often affected by both acute and chronic graft versus host disease (GVHD). GVHD is most effectively prevented by ex vivo T cell depletion (TCD) of the allograft, but has been limited in its use by logistical difficulties, lack of an FDA-approved method, and concerns regarding potential risk of graft rejection, post transplant infections, and leukemic relapse. Most reported TCD studies represent single centers, multiple disease types and processing methods with varying degrees of TCD, all of which affect outcome. Therefore we designed a trial using a single processing method providing extensive TCD that did not require post transplant GVHD prophylaxis involving adult pts with AML in first or second CR. We hypothesized that the undesired side effects of TCD HCT would be reduced if combined with a conditioning regimen that was highly immunosuppressive and anti-leukemic. The primary objective was to achieve a disease-free survival (DFS) rate at 6 months (mos) post transplant that exceeded 75%. Secondary objectives included assessments of engraftment, transplant related mortality (TRM), GVHD, relapse, and performance of a single TCD method (CD34+ cell selection using the Miltenyi CliniMACS device) at participating centers. From 10/2005 to 12/2008, 47 pts were enrolled and 44 transplanted at 8 different centers. Median age was 48.5 years (range 21-59) with 28 female and 16 male pts. Of 37 AML CR1 pts, 49% had an unfavorable cytogenetic or molecular risk profile. The conditioning regimen consisted of hyperfractionated total body irradiation (1375cGy in 11 fractions) with partial lung shielding, thiotepa (10mg/kg), cyclophosphamide (120mg/kg), and rabbit antithymocyte globulin (2.5mg/kg). The donors, all HLA-identical siblings, were given G-CSF for mobilization and scheduled to undergo at least 2 leukapheresis procedures to ensure a graft with a high CD34+ cell content. All allografts were CD34-enriched and were targeted to contain ≥ 5×10e6 CD34+ cells/kg and < 1.0×10e5 CD3+ cells/kg. The median CD34+ and CD3+ doses achieved were 8.1 × 10e6/kg (range 2.4-46.2) and 0.07 × 10e5/kg (range 0.01-0.85), respectively. The majority (81%) of pts received the targeted CD34+ cell dose and no pt received > 1.0×10e5 CD3+ cells/kg. No pharmacological GVHD prophylaxis was given post transplant. There were no significant toxicities related to infusion of the CD34 enriched allografts. The most common grade 3-5 regimen-related toxicities included grades 3 or 4 mucositis (39%) and grades 3-5 pulmonary abnormalities (11%). Only 1 pt experienced hepatic veno-occlusive disease. All pts engrafted rapidly with a median time to neutrophil recovery (ANC > 500/ul) of 11 days (range 9-19). There was 1 secondary graft failure. The assessed outcomes are shown below.Estimate (95% Confidence Interval)Outcome100 Days6 Months12 MonthsAcute GVHD II-IV20.5% (8.7 – 23.3%)Acute GVHD III-IV4.5% (0 – 10.6%)Chronic GVHD17.7% (5.8-29.6%)Extensive Chronic GVHD7.6% (0-15.7%)TRM17.8% (5.8-29.8%)Overall Relapse18.2% (5.9-30.5%)Relapse 1st CR9.6% (0- 19.8%%)Relapse 2nd CR64.3% (27.5-100%)DFS81.3% (66.1-90.2%)64.0% (46.5-77.1%)DFS 1st CR89.2% (73.7-95.8%)72.1% (53.0-84.6%)OS74.3% (57.3-85.4%)The absolute peripheral CD4+ cell count remained on average below 200/ul until day +365. Donor cell chimerism increased in the CD3+ cell compartment through day +365. There were 14 deaths. The most common causes of death were relapse (N=5) and pulmonary toxicity (N=4). The median follow-up of survivors is 489 days (range 96-776). There was no difference in OS or DFS for pts above or below the median age of 48.5 years. We conclude that TCD HCT following myeloablative chemoradiotherapy can be performed in a multi-center setting using a single TCD method without additional post transplant prophylaxis with excellent DFS and OS, consistent engraftment, low TRM, and low incidence of relapse even in pts with unfavorable risk AML in CR1. The low incidences of acute and chronic GVHD in the absence of post transplant prophylaxis were particularly encouraging. A follow-up study of TCD HCT in AML recipients of unrelated donor allografts is being planned by the BMT CTN Disclosures:No relevant conflicts of interest to declare.

The Effect of Natural Killer Cell Killer Ig-Like Receptor Alloreactivity on the Outcome of Bone Marrow Stem Cell Transplantation for Severe Combined Immunodeficiency (SCID)

Natural killer (NK) cell alloreactions against recipient cells in the setting of bone marrow transplantation have been associated with decreased rates of graft-versus-host disease (GVHD) and improved survival in transplant recipients with myeloid leukemia. These alloreactions are predicted by the absence of recipient HLA class I ligands for donor inhibitory killer Ig-like receptors (KIR). We hypothesized that donor NK cell alloreactions against recipient cells may affect the development of T and B-cell functions and incidence of GVHD in infants with severe combined immunodeficiency (SCID). Of the 156 patients with SCID who had received related bone marrow transplants without pretransplant chemotherapy or posttransplant GVHD prophylaxis, 137 patient-donor pairs were evaluated for the absence of recipient HLA class I ligands for donor inhibitory KIR. Analysis showed that the absence of a KIR ligand had no effect on the incidence or severity of GVHD (RR [corrected] = 0.95, p = 0.84), development of T-cell function (RR [corrected] = 1.05, p = 0.69), production of IgA (p = 0.46) or IgM (p = 0.33), or on 5-year survival (RR [corrected] = 1.21, p = 0.10). Further, in patients possessing native NK cells, the absence of KIR ligands in donors for recipient-inhibitory KIR did not alter transplantation outcomes. This study suggests that inhibitory KIR/HLA interactions do not play a significant role in bone marrow transplantation for SCID.

Allogeneic Stem Cell Transplantation (ASCT) in CML with Partial T-Cell Depletion, No Prophylaxis for Graft-Versus-Host Disease (GvHD) and Preemptive DLI for Patients with Post-Transplant Minimal Residual Disease (MRD): An Alternative Approach in CML Transplantation with Low Morbidity and Long Term Disease Free Survival.

The introduction of imatinib mesylate has revolutionized the treatment of CML, but despite significant cytogenetic responses primary therapy failure and emergence of resistance pose a major obstacle to successful treatment. ASCT still has a pivotal role in the treatment of CML and reduction of morbidity and mortality remains an important focus of ongoing studies in CML. We conducted a prospective study with ablative conditioning and partial T cell depleted ASCT from a matched sibling donor with no post-transplant GvHD prophylaxis. This was followed by an escalated donor lymphocyte infusion (DLI) administered if there was evidence for the presence of MRD, as determined by FISH and PCR/RQ-PCR for bcr-abl. Patients and methods: 40 consecutive patients underwent ASCT for CML between 1999–2005. The transplant regimen contained busulfan (12mg/kg), cyclophosphamide (120 mg/kg), anti-thymocyte globulin (ATG-Fresenius) (25mg/kg) and fludarabine (200mg/kg) followed by an infusion of ≥5×106/kg of G-CSF-mobilized and positively selected CD34 cells and 1×105/kg T cells. No post-transplant GvHD prophylaxis was given. The presence of MRD was determined every 3 months. DLI was administered in escalated doses starting with 3×106/kg and escalating in increments up to 1×108/kg. Results: 40 patients, median age 38 (19–63), 35 transplanted in first chronic phase (CP), 1 in second CP and 4 in accelerated phase. Patients were transplanted at a median of 4 months from diagnosis (1–66). The median of positively selected CD34+ cells was 9×106 cells/kg (5–24). 39 patients engrafted neutrophils at a median of 11 days (9–25) and platelets at a median of 15 days (9–134). Primary non-engraftment occurred in one patient and late graft failure in two, all salvageable by back-up marrow in two patients or donor stem cell boost in a single patient. The median follow-up of the entire cohort is 44 months (6–84).Overall Survival80%Transplant Related Mortality (TRM)day 1005%day > 10010%Acute GvHDPost transplant (n = 38)Post DLI (n= 24)grade I-II21%21%grade II-III08%grade IV00Chronic GvHDPost transplant (n = 38)Post DLI (n= 24)limited2.6%16%extensive00Need for DLIOne dose63%Two doses31%≥ Three doses16%Disease-Free SurvivalMajor cytogenetic response6%Major molecular response6%Complete cytogenetic and molecular response88%Disease Progression10%None of the patients developed significant VOD. Post transplant acute GvHD developed in only 21% of patients and was confined to grade I–II, all responding to a short course of immunosuppression. Additional 7 out of the 24 patients receiving DLI developed acute GvHD. There was no grade IV or mortality from GvHD. At the time of this analysis all apart from 1 patient were able to stop immunosuppressive treatment for GvHD. There were no significant infectious complications in this group. All late TRM were secondary to autoimmune complications, unrelated to GvHD, and occurred in 5 patients (13% of all) at 6–14 months post transplant. These complications included: ITP, AIHA, autoimmune pneumonitis, nephritic syndrome, TTP and Kaposi sarcoma. The mortality from these complications was 80% (4 out of 5 patients). 4 patients developed blast crisis post transplant; 2 patients have died; and the others are currently in CR post salvage treatment, DLI and imatinib. Twenty five percent of patients attained complete molecular remission without ever needing DLI, while 75 % responded after DLI, mostly following a single dose. Two patients are in major cytogenetic response, one of whom received back-up marrow and for the other a DLI donor is not available. The two patients in major molecular response are awaiting DLI. Conclusions: Matched ASCT for CML can be performed with significantly reduced TRM and morbidity using partial T cell depletion, no GvHD prophylaxis and preemptive DLI. This regimen offers > 80% long-term curative potential. Programmed DLI is safe and associated with a low risk of GvHD. The late immunological complications pose a major risk. This appears to be related to an imbalance between B and T cell reconstitution and the precise mechanisms of this need to be elucidated. In the imatinib era ASCT using this approach may be reserved for patients not responding or resistant to the imatinib mesylate.

Megadose CD34-Selected Haplocompatible Donor Stem Cell Transplantation in Children.

We report a retrospective review of 18 children receiving haplocompatible related donor hematopoietic peripheral blood SCT and consecutively enrolled at four U.S. transplant centers. The median age was 8 yrs (range 1–20). Patients with malignancy (n=13) included: AML-CR1 (primary induction failure, failed cord blood transplant) [1], CR2 [3]; MDS-RA/RARS [2], RAEB [2]; AML and Fanconi anemia [1]; CML-CP2 [1]; ALL-CR3 [2]; NHL-CR2 [1]. Patients with non-malignant diseases included severe aplastic anemia [n=4] and Wiskott-Aldrich syndrome [n=1]. Thirteen donors were a 3/6 HLA match and 5 were a 4/6 match. CD34 positive selection was used to select stem cells and deplete T lymphocytes. Conditioning for 13 of the patients consisted of TBI 12–14 Gy in 6 fractions, thiotepa, fludarabine and ATG. Fractionated TBI was replaced by single fraction TBI (n=2) or melphalan (n=3). Cyclophosphamide replaced thiotepa for 1 patient with FA. No post-transplant graft-versus-host disease (GVHD) prophylaxis was given. Patients received a median of 18 × 106 CD34+ cells/kg (range 6–28) and 3 × 104 CD3+ cells/kg (range 0.3–11). Sustained primary engraftment occurred in 15/18 (83%) patients. Primary graft failure occurred in one patient. Two patients had immunological rejection following HHV-6 reactivation. Both engrafted after a second transplant; therefore the overall engraftment success was 94%. The median time to an ANC >0.5 × 109/L was 12 days (range 9–21). Platelet recovery occurred in 16/18 at a median of 17 days (range 9–22). Primary (occurring after SCT but prior to DLI) grade II acute GVHD was seen in 4/17 patients (24%). Grade III-IV GVHD was seen in 1 patient (6%) manifest as overlap syndrome in association with HHV-6 reactivation. One pt had primary extensive chronic GVHD. Of nine patients who received DLI and/or stem cell boosts, 4 had grade II GVHD (3/4 had prior acute GVHD), none had grade III-IV GVHD, 2 developed chronic GVHD and 1 developed overlap syndrome. Infections were common but manageable. All patients were at risk for CMV reactivation based on CMV serology: recipient/donor +/+ (9), +/− (3), −/+ (6). Seven patients (39%) reactivated CMV. All cases were responsive to anti-viral therapy and/or DLI. No CMV disease was seen. Seven patients had adenovirus reactivation and 6 had HHV-6 reactivation. EBV reactivation occurred in 5/18 (28%) patients. Rituximab (5) and DLI (2) yielded rapid resolution of EBV in all patients. Four of 13 (31%) at risk patients have relapsed: 1 pt with cytogenetic relapse remains in CR2 > 6 mo later and another who recently relapsed is undergoing salvage therapy. The 100 day and 1 year transplant-related mortality was 11% and 19%, respectively. The overall survival is 72% with a median follow-up of 31 months (range 7–89). Among patients transplanted for malignant diseases (13) and non-malignant diseases (5), overall survival is 69% and 80%, respectively. The K-M 2 year survival was 70% +/− 22%. All survivors were complete donor chimeras by DNA methods. The use of megadose CD34-selected PBSC without post-transplant GVHD prophylaxis yielded rapid engraftment, low 100-day mortality and incidence of severe GVHD, and excellent survival. The overall survival compares favorably with MSD and MUD HSCT.

Immune reconstitution after haematopoietic transplantation with two different doses of pre-graft antithymocyte globulin

Antithymocyte globulin is widely used before haematopoietic transplantation with HLA-matched unrelated donors or mismatched relatives to prevent rejection and graft-versus-host disease (GVHD). However, optimal dosage is still under debate. Thirty-one consecutive children, mainly with haematological malignancies, were transplanted in a single institution with such donors, selected by HLA-A -B compatibility by serology and DRB1* by DNA typing. Antithymocyte globulin (Thymoglobuline; Sangstat) was infused at days -3, -2, -1. Total dosage varied: 16 patients received a median of 7.5 mg/kg (2.5 to 10.5: low-dose group), and 15 a median of 15.5 mg/kg (14.4 to 19.4: high-dose group). Post-transplant GVHD prophylaxis consisted of cyclosporine, short-course methotrexate and steroids. CD3(+), CD4(+) and CD19(+) cell reconstitution was slower in the high-dose group. Median time to reach 100 CD4(+) cells was 8 months vs 4 months (P = 0.03). Median time to normal CD19(+) cells was 16 months vs 8 months (P = 0.01). CD16(+)CD56(+) and CD8(+) cell reconstitution was similar. Nine patients in the high-dose group and two in the low-dose group experienced life-threatening opportunistic infections (P = 0.009). Although obtained from a limited number of patients, our data suggest that a higher pre-graft dose of antithymocyte globulin may negatively influence immune reconstitution.