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Patients with sickle cell anemia (SCA) have increased susceptibility to infections partially explained by functional splenic and alternative complement pathway defects. Cord blood transplants (CBT) and high doses anti-thymoglobulin (ATG) are suspected to be responsible for viral complications and EBV lymphoma but most of the reports concerned unrelated SCT. The aim of the present study was to compare the immune reconstitution after CBT vs BMT from HLA-identical sibling, in patients prepared with the same myeloablative conditioning regimen (MAC). This retrospective analysis concerns SCA-children all followed in the same CHI-Créteil referral center and transplanted from a HLA-identical sibling with MAC consisting of busulfan, cyclophosphamide and rabbit ATG (Genzyme at 20mg/kg). Pre- and post-transplant clinical and biological data were prospectively recorded in the local database. Lymphocyte subpopulations (CD3+, CD4+, CD8+), IgG, IgA, IgM were recorded each month during the first year post-transplant. Jolly bodies (classified as 0=absent, 1=rare, 2=a few, 3=numerous) and HBs, DTPolio vaccinal serologies were assessed at transplant time (T0), 1 (T1) and 2 years (T2) post-transplant. Revaccination with DTPolio was performed at 1y post-transplant One-hundred-seven SCA-patients (41F,56M) with severe disease were transplanted (1992-2012) with BM (n=83), CB (n=21), CB+BM (=3) at median (range) age: 9.7y (3.4-22.2) for BMT and 6.1y (3.2-12.9) for CBT (p=0.002). Four patients had splenectomy and 5 others partial splenectomy. Rate of rejection was higher after CBT (p=0.002) with 2 non-engraftments and no late rejection whatever the source. TRM was not different despite the occurrence of 3 deaths only after BMT (obliterant bronchiolitis at 1.1year, hemorrhagic stroke at day36, adenoviral encephalitis at month5). Acute GVHD ≥II was observed in 18 patients (16 BMT, 2 CBT) and mild and extensive chronic-GVHD in 5 and 2 patients respectively after BMT and 1 mild after CB+BMT. At 5-year DFS was 95.3% (CI:91.3-99.3%). No significant difference in GVHD and DFS rates was observed according to the source. Neutrophils reached 500/mm3 at mean day32 vs day21 (p Paired analysis comparing results at T0 vs T1 and T2 showed a significant decrease of the mean (SD) Jolly bodies score from 1.38 (0.85) at T0 to 0.50 (0.81) at T1 and 0.28 at T2 (p We confirm the improvement of splenic function after SCT and conclude that contrary to unrelated CBT and SCT using high dose ATG, CBT from HLA-identical sibling do not expose significantly to more frequent viral infections or reactivations and have satisfactory vaccinal response Figure Disclosures Bernaudin: BlueBirdBio: Consultancy; AddMedica: Honoraria, Other: Help for travel; GBT: Membership on an entity's Board of Directors or advisory committees. Socie:Alexion: Consultancy.

Hematopoietic stem cell transplant (HSCT) from an HLA identical sibling is a well-established curative therapy for sickle cell disease (SCD). HSCT from an unrelated donor is a treatment option, but the likelihood of finding a donor varies according to ethnicity and results are still limited. HLA haploidentical relatives can be alternatively used but, to date, only small series of patients have been described. We report outcomes of patients (pts) transplanted with related haploidentical (Haplo) or unrelated (UD) donors grafts and reported to EBMT/EUROCORD databases. Sixty four pts transplanted in 22 EBMT centers between 1991 and 2017 were retrospectively analyzed. Pts were described according to the donor type: haploidentical (n=40) and unrelated (n=24) [adult UD n=19; cord blood (CB) n=5]. The objective of the study was to describe alternative donor transplants for SCD in Europe without performing comparison analyses due to the size and heterogeneity of the groups. Primary endpoint was 3-year overall survival (OS). Median follow-up (FU) was 28 months (range: 1.6-156) [29.5 months (range: 2.1 - 133.5) for Haplo and 24.6 (range: 1.6 - 156) for UD]. Median age at HSCT was 14.2 years (range: 3-31.7) in Haplo and 11.8 (range: 2.1-42.8) in UD, with a predominance of children ( In both groups, vaso-occlusive crisis and cerebral vasculopathy were the most frequent SCD complications and the main indications for HSCT. Other complications were acute chest syndrome (44%), liver disease (31%) and infection (23%). In Haplo, median year of transplant was 2014 (range: 1991-2017) and in UD 2011 (range: 2004-2015). In Haplo, two major protocols were used: (1) post -transplant cyclophosphamide (PTCY) with G-CSF primed bone marrow (BM) and a fludarabine+ cyclophosphamide+thiotepa+2Gy TBI conditioning regimen [16 pts and 2 centers performing most (n=13) of the transplants]; (2) a protocol (performed in 2 centers) consisting in the use of G-CSF mobilized peripheral blood stem cells (PBSC) with ex-vivo B and T cell depletion (BT depleted) (15 pts) and a fludarabine+thiotepa+ treosulfan conditioning regimen (14/15 pts). Haplo donors were most frequently the parents [mother (50%), father (29%), brother (14%) and cousin (7%)]. ATG was used in 95% of transplants and the most frequent combination for graft versus host disease (GvHD) prophylaxis was mycophenolate mofetil (MMF)+sirolimus in PTCY and MMF+ cyclosporine A (CSA) in BT depleted. In UD, graft source distribution was 14 BM, 5 PBSC and 5 CB. Conditioning regimens were mainly myeloablative (83%) with fludarabine+thiotepa+ treosulfan in 54% of HSCT. ATG was used in 87% and campath in 9% of transplants; GvHD prophylaxis was CSA and methotrexate in 50%. Neutrophil engraftment at 60 days was 95±4% in Haplo and 84±8% in adult UD, after a median engraftment time of 18 and 22 days, respectively. In Haplo, 7 pts experienced graft failure (3 primary and 4 late), of those 3 had a second allogeneic transplant and were alive at last FU, at 16, 16 and 63 months respectively; 1 patient died after rescue with autologous transplant and 3 were alive after autologous reconstitution. In adult UD, 3 pts had a primary and 1 a late graft failure, none of them had a second transplant and were all alive at last FU, at 2, 13, 28, 118 months respectively. Grade II-IV acute GvHD at 100 days was 25±7% in Haplo and 21±9% in adult UD; acute GvHD grade III-IV was observed in 3 pts in Haplo (none in BT depleted) and 2 pts in adult UD. Chronic GvHD was observed in 10 pts in Haplo (5 extensive, 3 of these in PTCY) and 3 pts in adult UD (2 extensive). OS at 3 years was 88±4%; being 89±5% in Haplo (88±8% for PTCY, 92±8% for BT depleted) and 94±5% in adult UD. 3-year event free survival was 58±7%; in detail, 60±9% in Haplo (56±12% for PTCY, 68±13% for BT depleted) and 60±12% in adult UD. Overall, 8 pts died (5 Haplo and 3 UD) due to infections or GVHD. Among the 5 pts receiving CB transplant 3 are alive (1 of which after graft failure and a second allogeneic transplant). Conclusion: This preliminary analysis shows that, despite an acceptable OS, rejection and chronic GvHD are still of concern; therefore alternative donor transplants for SCD should be performed in experienced centers with prospective clinical trials. Disclosures Pondarré: Blue Bird Bio: Honoraria; Novartis: Honoraria; Addmedica: Membership on an entity's Board of Directors or advisory committees. Zecca:Chimerix: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy. Bader:Medac: Patents & Royalties, Research Funding; Riemser: Research Funding; Neovii: Research Funding; Cellgene: Consultancy; Novartis: Consultancy, Speakers Bureau. Bernaudin:AddMedica: Honoraria; Pierre fabre: Research Funding; BlueBirdBio: Consultancy; Cordons de Vie: Research Funding.

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

Natural killer (NK) cell alloreactivity is reported to mediate strong GvL (graft versus leukemia) effect in patients after haploidentical stem-cell transplantation (SCT) for acute myeloid leukemia (AML). Because subsequent immune reconstitution remains a major concern, we studied NK-cell recovery in 10 patients with AML who received haplomismatched SC transplants, among whom no GvL effect was observed, despite the mismatched immunoglobulin-like receptor (KIR) ligand in the GvH direction for 8 of 10 patients. NK cells generated after SCT exhibited an immature phenotype: the cytotoxic CD3-CD56dim subset was small, expression of KIRs and NKp30 was reduced, while CD94/NKG2A expression was increased. This phenotype was associated to in vitro lower levels of cytotoxicity against a K562 cell line and against primary mismatched AML blasts than donor samples. This impaired lysis was correlated with CD94/NKG2A expression in NK cells. Blockading CD94/NKG2A restored lysis against the AML blasts, which all expressed HLA-E, the ligand for CD94/NKG2A. Our present study allows a better understanding of the NK-cell differentiation after SCT. These results revealed that the NK cells generated after haplomismatched SCT are blocked at an immature state characterized by specific phenotypic features and impaired functioning, having potential impact for immune responsiveness and transplantation outcome.

Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 × 1011/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (< 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 × 1011 platelets, the high dose between 6 and 8 × 1011, and the very high dose > 8 × 1011; for the children, the three doses corresponded to 2 to 4, 4 to 6, and > 6 × 1011 platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 × 109/L, respectively) than the medium dose (33 × 109/L, P < .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P< .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 × 1011 per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets.The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure. © 1998 by The American Society of Hematology.

Introduction: Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for sickle cell disease (SCD), though rarely used due to inadequate donor availability, associated regimen-related toxicities, and high mortality rate in adults. A reduced intensity, haploidentical bone marrow transplantation (Haplo-BMT) regimen with post-transplant cyclophosphamide (PTCy), reported by the Johns Hopkins Group (JHG) in adults with SCD, resulted in successful engraftment, minimal toxicity, abrogation of sickle cell related symptoms, but was associated with a high graft failure rate of 43% (Blood. 2012;120(22):4285-4291). A multi-institution learning collaborative was developed in 2013 with main objective of reducing the graft failure rate in individuals with SCD who undergo a haplo-BMT with PTCy from first-degree relatives. Material and methods : The collaborative included 3 sites, similar eligibility criteria, monthly phone conferences for sharing of group learning experiences, and independent IRB approvals at each site, allowing for aggregate data sharing. A common conditioning regimen was used based on the JHG haplo-HSCT protocol - fludarabine 150 mg/m2, Cy 29 mg/kg, Thymoglobulin 4.5 mg/kg, and TBI 2Gy), and graft versus host disease (GVHD) prevention with PTCy 100 mg/kg, mycophenolate mofetil and sirolimus. However two additional modifications were systematically added which were believed to improve engraftment rates with two different strategies. In strategy 1, a Data Safety Monitoring Committee (DSMC) planned an interim analysis after enrollment of the first 5 participants using the JHG haplo-HSCT protocol, with modifications for > 20% mortality rate, graft rejection, or severe GVHD. In strategy 2, the participants in this group had preconditioning with 2 drugs for 3 months (azathioprine 3mg/kg/d, hydroxyurea 30 mg/kg/d) and hypertransfusion, plus thiotepa (10 mg/kg/day) on D-7. No DSMC was established. All donors were mobilized with filgrastim (5-10 μg/kg/d x5 days). Primary graft failure was defined as day +42 in patients with prior documentation of > 5% donor cells by day +42. Results: All potential patients that sought haplo-BMT had a HLA haplodonor (n=34). Median total nucleated and CD34+ cell doses were 8.40 x 108 /kg and 3.63 x 106/kg respectively; median follow-up was 20.3 months (1.4-37.5). In strategy 1 (n=5), median age was 26.4yr (12-50), and 3 out of 5 had graft failure, (2 primary; 1 secondary), triggering the stopping rule. Subsequently, thiotepa alone (10 mg/kg/day; D-7) was added; for 7 patients transplanted in this cohort, median age was 18.1yr (7-26), 100% engrafted, median time to neutrophil engraftment was 25 days; 33.1 and 36.9 days for platelets >20 x 109/L and >50 x 109/L, respectively, with an EFS and OS of 100%, respectively. Main complications were 2 independent viral reactivation events (CMV and EBV), 2 patients had > grade 2 acute GVHD (gut) but no significant chronic GVHD developed in any participant. One patient with graft failure using the non-thiotepa approach, was re-transplanted >1 yr after initial haplo-BMT with the thiotepa alone containing regimen with successful engraftment. In strategy 2 (n=22), median age was 10yrs (3-18), 100% engrafted, median time to neutrophil engraftment was 17days; 36.5 and 38.5 days for platelets >20 x 109/L and >50 x 109/L, respectively. Mortality was 13.6% (3/22) mainly from infectious complications and macrophage activation syndrome; 2 patients had secondary graft failure (9.1%). The cumulative incidence of acute and chronic GVHD was 18.2%, respectively, all resolved; EFS and OS was 81.8% and 86.4% respectively. All patients were off immunosuppressive therapy > I year posttransplant. Conclusion: We have provided preliminary evidence that haplo-BMT with PTCy plus thiotepa alone when compared to no thiotepa, improves donor engraftment without increasing morbidity or mortality. Preliminary evidence also indicates that there is no benefit for the 2 drug preconditioning regimen with azathioprine, hydroxyurea plus thiotepa when compared to addition of thiotepa alone. An NIH sponsored BMTCTN phase II trial, of haplo-BMT with PTCy plus thiotepa is underway to determine if this regimen maximizes donor engraftment, with reduced morbidity and mortality. Disclosures Brodsky: Achillion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

Allogeneic marrow transplantation can cure sickle cell disease; however, HLA-matched donors are difficult to find, and the toxicities of myeloablative conditioning are prohibitive for most adults with this disease. We developed a nonmyeloablative bone marrow transplantation platform using related, including HLA-haploidentical, donors for patients with sickle cell disease. The regimen consisted of antithymocyte globulin, fludarabine, cyclophosphamide, and total body irradiation, and graft-versus-host disease prophylaxis with posttransplantation high-dose cyclophosphamide, mycophenolate mofetil, and tacrolimus or sirolimus. After screening 19 patients, we transplanted 17, 14 from HLA-haploidentical and 3 from HLA-matched related donors. Eleven patients engrafted durably. With a median follow-up of 711 days (minimal follow up 224 days), 10 patients are asymptomatic, and 6 patients are off immunosupression. Only 1 patient developed skin-only acute graft-versus-host disease that resolved without any therapy; no mortality was seen. Nonmyeloablative conditioning with posttransplantation high-dose cyclophosphamide expands the donor pool, making marrow transplantation feasible for most patients with sickle cell disease, and is associated with a low risk of complications, even with haploidentical related donors. Graft failure, 43% in haploidentical pairs, remains a major obstacle but may be acceptable in a fraction of patients if the majority can be cured without serious toxicities.

In 1992, we reported the results of the first randomized trial comparing the combination of cyclophosphamide (CY) (120 mg/kg) and total body irradiation (TBI) (CYTBI) versus CY and busulfan (BU) (BUCY) as preparation for an allogeneic bone marrow transplantation (BMT) for adult patients with acute

Transcranial Doppler (TCD) is used to detect children with sickle cell anemia (SCA) who are at risk for stroke, and transfusion programs significantly reduce stroke risk in patients with abnormal TCD. We describe the predictive factors and outcomes of cerebral vasculopathy in the Créteil newborn SCA cohort (n = 217 SS/Sβ0), who were early and yearly screened with TCD since 1992. Magnetic resonance imaging/magnetic resonance angiography was performed every 2 years after age 5 (or earlier in case of abnormal TCD). A transfusion program was recommended to patients with abnormal TCD and/or stenoses, hydroxyurea to symptomatic patients in absence of macrovasculopathy, and stem cell transplantation to those with human leukocyte antigen-genoidentical donor. Mean follow-up was 7.7 years (1609 patient-years). The cumulative risks by age 18 years were 1.9% (95% confidence interval [95% CI] 0.6%-5.9%) for overt stroke, 29.6% (95% CI 22.8%-38%) for abnormal TCD, which reached a plateau at age 9, whereas they were 22.6% (95% CI 15.0%-33.2%) for stenosis and 37.1% (95% CI 26.3%-50.7%) for silent stroke by age 14. Cumulating all events (stroke, abnormal TCD, stenoses, silent strokes), the cerebral risk by age 14 was 49.9% (95% CI 40.5%-59.3%); the independent predictive factors for cerebral risk were baseline reticulocytes count (hazard ratio 1.003/L × 109/L increase, 95% CI 1.000-1.006; P = .04) and lactate dehydrogenase level (hazard ratio 2.78/1 IU/mL increase, 95% CI1.33-5.81; P = .007). Thus, early TCD screening and intensification therapy allowed the reduction of stroke-risk by age 18 from the previously reported 11% to 1.9%. In contrast, the 50% cumulative cerebral risk suggests the need for more preventive intervention.

The toxicity of autologous bone marrow transplantation (ABMT) is correlated to neutropenia. Although recombinant human granulocyte- macrophage colony-stimulating factor (rhu GM-CSF) seems to hold promise in accelerating neutrophil recovery, few analyses from randomized studies are presently available. Ninety-one patients with non-Hodgkin's lymphoma receiving high-dose ablative chemotherapy followed by ABMT with unpurged or purged marrow were included in a randomized, double- blind, placebo-controlled trial. Forty-four patients received 250 micrograms rhu GM-CSF (Escherichia coli)/m2 and 47 patients received placebo. Treatment was administered daily as continuous infusion from day of ABMT until the absolute neutrophil count (ANC) reached 0.5 x 10(9)/L for 7 days or until day 30, whichever was first. With rhu GM- CSF, 50% of the patients reached an ANC count greater than 0.5 x 10(9)/L at day 14 as opposed to day 21 with placebo (P less than .0001). Patients transplanted with marrow purged by mafosfamide also recovered earlier when treated with rhu GM-CSF (16 v 20.5 days, P = .013). The hospitalization duration was shorter in the rhu GM-CSF group (median, 23 v 28 days, P less than .05). No difference was observed in fever, number of infections, and antibiotic administration between the two groups. The major adverse event ascribed to rhu GM-CSF was a capillary leak syndrome in three patients graded as severe in two patients, moderate in one, and reversible in all three patients. In addition, one patient in the rhu GM-CSF group died suddenly with no explanation. In long term follow-up, the relapse rate was identical in both groups and there was no significant difference in the number of deaths at 1 year (12 with rhu GM-CSF v 9 with placebo), although deaths seemed to occur slightly earlier in the rhu GM-CSF group. We conclude that after ABMT with purged or unpurged marrow, rhu GM-CSF (E coli) significantly reduces neutropenia duration and hospitalization stay. A positive causative relation between the study drug and/or its mode of application with an increased toxicity as compared with GM-CSF from other sources and/or other modes of application cannot be deduced from the experiences in this study. Additional randomized trials would be necessary for an appropriate answer.

Allogeneic hematopoietic stem-cell transplantation (HSCT) is the only curative treatment for sickle cell disease (SCD); nevertheless, its use has been limited by the risk of transplantation-related mortality (TRM). Between November 1988 and December 2004, 87 consecutive patients with severe SCD ranging from 2 to 22 years of age received transplants in France. Cerebral vasculopathy was the principal indication for transplantation (55 patients). All the patients received grafts from a sibling donor after a myeloablative conditioning regimen (CR). The only change in the CR during the study period was the introduction of antithymocyte globulin (ATG) in March 1992. The rejection rate was 22.6% before the use of ATG but 3% thereafter. With a median follow-up of 6 years (range, 2.0 to 17.9 years), the overall and event-free survival (EFS) rates were 93.1% and 86.1%, respectively. Graft versus host disease (GVHD) was the main cause of TRM. Importantly, cord blood transplant recipients did not develop GVHD. No new ischemic lesions were detected after engraftment, and cerebral velocities were significantly reduced. The outcome improved significantly with time: the EFS rate among the 44 patients receiving transplants after January 2000 was 95.3%. These results indicate that HLA-identical sibling HSCT after myeloablative conditioning with ATG should be considered as a standard of care for SCD children who are at high risk for stroke.

To the editor: We have read with great interest the comprehensive and detailed review on hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD) published by Hsieh et al in a recent issue of the journal.[1][1] As emphasized by the authors, allogeneic HSCT is currently the only

Background Sickle Cell Anemia (SCA) remains a disease with high risk of morbidity and early death. HSCT is currently the only curative treatment for SCA, but its use has been limited by the risks of transplant-related mortality (TRM) and GVHD. We have previously demonstrated that antithymocyte globulin (ATG) efficiently decreases the rejection rate in HSCT for SCA; however, its optimal dose has not been defined, prompting us to retrospectively analyse outcomes in patients from the SFGM-TC cohort receiving different ATG doses. Patients and Methods The cohort includes 236 SCA-patients (109F, 127M), (229 SS, 5 Sb0, 2 SDPunjab) transplanted in France (1988-2012) with geno-identical donors after homogeneous myeloablative conditioning regimen (CR; BU-CY), and rabbit ATG (n=215) at 5-15 mg/kg (n=35), 20 mg/kg (n=160) or no ATG (n=20). Cell sources were bone marrow (BM, n=197), cord blood (CB, n=30), CB+BM (n=8), and peripheral blood cells (PBC, n=1). Chimerism was assessed by quantitative real-time PCR from PBC at Day-30, 60, 90, Month-6, 12 and every year post-transplant, and defined as total donor chimerism (TDC): >95% donor (D); low (5-50%D) or high (50-95%D) mixed chimerism (MC),; or rejection (< 5%D). Disease-free survival (DFS) was defined as the absence of HSCT-related death and rejection. GVHD prophylaxis consisted of CSA-short MTX for BMT and CSA alone for CBT. Results Mean age at transplant was 9.7yr (range: 2.2-28.9), with 33 patients older than 15. With a mean follow-up of 5.6yr (range 0.5-22.5), TRM was not different with or without ATG (3.4%; 0.8-6%) whereas DFS was higher (p=0.004) with ATG (95.4%; 92-4-98.4) than without (72.7%; 51.7-93.7), due to differences in rejection rates (p15 (OR=7.9, 95%CI: 2.5-25.6,p Conclusion This study confirms that myeloablative CR with ATG offers 95% DFS to SCA-patients and that stable MC in absence of SCA-symptoms does not require intervention such as DLI. It reports for the first time in HSCT for SCA that donor’s age significantly increases GVHD risk, suggesting to choose the youngest sibling when possible, and that high ATG dose (20 mg/kg), which significantly reduces cGVHD risk without enhancing viral risk, should be recommended. Disclosures: Bernaudin: Novartis: Research Funding. Bertrand:ERYTECH Pharma: Principal Investigator Other. Gluckman:Cord use: Honoraria; gamida: Honoraria.

We report on 71 consecutive patients with de novo myelodysplastic syndromes referred to physicians belonging to the Societe francaise de greffe de moelle from 1982 through 1991 and transplanted with marrow from HLA-identical siblings. There were 16 cases of refractory anemia, 27 of refractory anemia with excess of blast cells, and 28 of refractory anemia with excess of blast cells in transformation. Seventeen patients had received cytoreductive chemotherapy before the graft. The disease progressed in 17 patients between diagnosis and grafting. Twenty-three patients are alive with a median follow-up of 6 years, whereas 24 died from relapse and 24 from transplant-related complications. Kaplan-Meier estimates of event-free survival, relapse and transplant-related mortality at 7 years were 32%, 48%, and 39%, respectively. The log-rank test and Cox's model revealed better outcome among young patients, patients in an early stage of the French-American- British (FAB) classification or with a low percentage of marrow blasts before transplantation, patients who did not undergo cytoreductive chemotherapy before transplantation, and patients conditioned with total body irradiation and cyclophosphamide. The high rate of relapse in advanced FAB stages has led us to graft patients earlier in the course of the disease, and we are currently conducting a multicenter, randomized study to determine the value of intensive chemotherapy before grafting in patients with an excess of marrow blasts.

We report a case of mild, clinically asymptomatic, immune thrombocytopenia after allogenic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML) caused by the presence of a recipient- origin Br(a) antibody that recognized the donor platelets. Although the antibody titer decreased, it remained detectable more than 3 years after BMT. Chimerism studies were performed combining cytogenetics, blood cell phenotype studies, and genomic amplification of hypervariable sequences. Cytogenetic studies and molecular analysis of peripheral blood cells, purified B- and T-lymphocyte subpopulations, and bone marrow colonies showed the hematopoiesis to be of donor origin, but absorption-elution experiments with peripheral RBCs showed a small amount of recipient RBCs. The CML chimeric transcript was also detected by means of polymerase chain reaction on samples collected until day +867 post-BMT. This case shows that recipient-origin platelet alloantibodies can cause thrombocytopenia after BMT and that the persistence of small numbers of recipient cells (even leukemic) is not necessarily associated with hematologic relapse.

Abstract 3516 Allogeneic Bone Marrow Transplantation (ABMT) is currently the only curative treatment for sickle cell disease (SCD). However, in adult patients, myeloablative conditioning is usually considered as excessively toxic and non-myeloablative procedures have resulted in high rejection rates or frequent mixed chimerism. In these mixed chimeras, erythropoiesis is usually of donor origin, but the long-term stability of this chimerism remains unknown. In this context, we report the French experience of myeloablative ABMT in young adults, using a preparative regimen previously successful in children (Bernaudin et al. Blood 2007). Patients and Methods: Twelve SCD patients,8 males and 4 females, 16 to 28 years old (mean 20,6) were transplanted with an HLA identical sibling between 1999 and 2009 in five french centers. These patients were listed in the French BMT registry (SFGM-TC). All patients received the same BU-CY +ATG (varying dosage) preparative regimen and Cyclosporine and Methotrexate for GVHD prophylaxis. Reason for transplant included a stroke history in 4 patients, asymptomatic pulmonary hypertension (TRV: 2.9m/s) in 1 patient, and frequent vasoocclusive crisis and/or acute chest syndrome in all the others. Before transplant, 10 patients had received numerous transfusions (>20 units) and none had received Hydroxyurea. Erythrocyte immunisation was present in 1 patient. The donors were AA in 7 cases and AS in 5. Eight donors were seropositive for CMV and 4 had a major ABO incompatibility with the recipient. The median follow-up of surviving patients is currently 36 months (range 9–120). Results: All patients engrafted (mean time for PMN > 0.5×109/L: 23days) and no rejection was observed. A patient who had successful engraftment but previous severe CNS vasculopathy and moya-moya, had a massive intracerebral haemorrhage at day 32 after transplant and died. Early toxicity included also a sub-dural haemorrhage successfully evacuated in a patient without any previous cerebral vasculopathy. Other early complications included seizures, viral pericarditis, bacteriemia and CMV reactivations, but no veno occlusive disease was observed. Four patients had acute GVHD grade II that responded to Prednisone treatment. Two patients had limited chronic GVHD, one with severe peripheral thrombocytopenia; however, both episodes resolved and the patients do not currently receive any immunosuppression. Only one death was observed, resulting in an overall survival and disease-free survival of 92% at 3 years. All survivors have the Hb electrophoretic profile of their donor and a normal Hb level. DNA-chimerism was characterized as full-donor. All patients have a normal quality of life, except for the sequelae of previous CNS disease in two patients and residual migraine after hematoma evacuation in one patient. Conclusion: This experience demonstrates that myeloablative allogenic BMT is feasible in selected young adults with SCD: The overall results are similar to those obtained in younger children. Early regimen-related toxicity does not appear to be increased as compared to BU-CY regimen used for other disease,but the early transplantation course has been more complicated in these patients than in younger children. Patients with severe CNS disease require special care. Of note, only one patient with significant renal involvement was included in this study. Acute and chronic GVHD have been moderate and easy to manage in these patients younger than 30 with an HLA identical sibling. A stable long-term full chimerism and cure has been achieved in all survivors. These results should encourage physicians to perform HLA typing in all young adults with SCD and their family in order to identify as early as possible potential candidates for transplantation. Although this experience is limited, it suggests that SCT could be proposed to young adults who had non-severe disease during childhood but developed severe criteria during adulthood such as a tricuspid regurgitant jet velocity at least 2.5 m/second. Count actuel 3359 (3800 autorisés) Disclosures: No relevant conflicts of interest to declare.

Abstract 3518 Background: Despite progress made in sickle cell anemia (SCA) management, such as the prevention of pneumococcal infections, introduction of hydroxyurea therapy and early cerebral vasculopathy detection with transcranial Doppler, SCA remains a disease with high risk of morbidity and early death. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCA; nevertheless, its use has been limited by the risk of transplant-related mortality (TRM). Our first experience, reported in Blood 2007, included 87 consecutive severe SCA- patients transplanted in France between 1988 and Dec-2004. We showed that the introduction of rabbit anti-thymocyte globulin (ATG) in the conditioning regimen in 2000 allowed a significant reduction of the rejection rate from 22.6% to 3% and that the outcome improved significantly with time as the DFS rate among the 44 patients transplanted after January 2000 was 95.3%. These data have justified continuing to transplant symptomatic young sickle cell patients having a geno-identical donor with the same conditioning regimen (CR) consisting of intravenous Busulfan (BU), Cyclophosphamide (CY) and rabbit ATG. Patients and Methods: In France, from 1992 to 2010, 144 SCA-patients (84M, 60F) have now been transplanted with a geno-identical donor using BU-CY-ATG as CR at the median age of 9.0 years (range:3.2-27.5). Transplants were performed in 16 different centers but 60 were performed in Hopital St-Louis and 21 in Hopital Debré in Paris. All recipients were SS or Sb0 and 76% of them were CMV+. All had been transfused and 47% had received more than 20 units. The source of cells was the bone marrow (BM) (n=121), cord blood (CB) alone (n=21), CSP (n=1) or BM+CB (n=1). GvHD prophylaxis consisted of the association of cyclosporine (CSA)-short MTX for BMT and CSA alone for CBT. Results: Engraftment was successful in 141/144; the time to absolute neutrophil count > 500/mm3 was significantly shorter after BMT compared to CBT (mean ± SD; 21.3 ± 6.7 vs 32.1 ± 9.8, respectively; p Conclusion: These results with 121 patients transplanted since 2000 confirm that it is possible to offer more than 95% chances of cure to SCA-children, indicating that HLA-geno-identical HSCT after myeloablative conditioning with ATG should be considered as standard of care for SCA children, not only for those at high risk of stroke but also for children experiencing crises or other complications requiring intensive therapy such as transfusion program or hydroxyurea. Sibling cord-blood cryopreservation should be systematically offered and pre-implantation genetic diagnosis coupled with HLA selection discussed with the parents. Disclosures: No relevant conflicts of interest to declare.

We previously reported that NK-cell generated after haploidentical hematopoietic stem-cell transplantation (SCT) in high risk AML patients are characterized, during the first 6 months post-SCT, by specific phenotypic features and impaired functioning having potential impact for transplantation outcome (Nguyen et al, Blood 2005). In the present study, we examined the impact of INF-γ on the impaired recognition of AML blasts by NK cells after haplo-SCT. We studied NK cell reconstitution at 1 to 6 months (M1–M6; n=21patients) or at 24 months (M24; n=3 patients) post transplant. Patients received SCT from haplo-mismatched related donor after a myeloablative conditionning regimen for high risk malignant hematological diseases. As previously described, the level of CD56brightCD94/NKG2A+ expression on NK cells was increased, around 3.5 higher at M1 and M6 as compared to the donors, and NK cytotoxicity against AML blasts was poorer than in the donors, suggesting an immature status of NK cells post haplo-SCT. However, at M24, similar level of CD56brightCD94/ NKG2A+ was observed in donors and patients and NK cells recovered normal cytolytic function. Blockading, ex vivo, the interaction between the inhibitory CD94/NKG2A receptor on NK cells at M3 and HLA-E on target cells restored the lysis. Higher level of intra-cellular IFN-γ production was detected by flow cytometry in CD94/NKG2A+NK from patients at M1 and M6 as compared to the donors and at M24 (mean 70.6%± 5.2% of CD94/NKG2A+NK cells produced INF-γ after incubation with IL-12+IL-18 at M6, versus 14.6%±5% in donors and at M24). These data were confirmed by Elisa. During the first 6-months, NK cells from the SCT patients, stimulated by IL2 or IL12 plus IL18, produced roughly 14.8 or 8.4 fold more IFN-γ than healthy controls, respectively. However at M24, the level of IFN-γ production was similar to the control, regardless the conditions. IFN-γ treatment led to higher surface expression of HLA-E on AML blasts (the mean Multiplicity Fluorescence Intensity (MFI) of HLA-E expression on AML blasts from 10 patients was 9 fold higher after adjonction of INF-γ), resulting in decreased killing by NK cells from healthy donors. Actually, adjonction of IFN-γ 1000 IU/ml on AML blasts induced an inhibition of lysis by healthy donor NK cells (12% ±2% specific lysis at a ratio E/T:20/1 with INF-γ, versus 37.5%±5% without INF-γ). Blockading CD94/NKG2A restored the lysis previously inhibited by INF-γ. Neutralization of INF-γ during the interaction of NK cells generated during the first months post haplo-SCT with mismatched AML blasts led to enhanced lysis (16.5%±4.1% specific lysis at a ratio E/T:20/1 without INF-γ neutralization versus 44.1%±10% after IFN-γ neutralization at M1). This effect was no more observed at M24 or in healthy donors. These data indicate that the increased secretion of INF-γ by immature CD94/NKG2A+/ CD56bright NK cells generated after haplo-SCT upregulate the cell surface expression of HLA-E on leukemic blasts. Increased HLA-E expression protects leukemic targets from NK-mediated lysis through the inhibitory CD94/NKG2A receptor which is overexpressed on NK cells during the first months post haplo-SCT. By contrast, at 2 years post-SCT, maturing NK cells were functionally active, as evidenced by high cytotoxicity and poor IFN-γ production. This suggests that HLA-E could be considered like a key component of an immune escape strategy against NK attack on AML blasts currently observed after haplomismathed SCT. Better understanding of how NK cells mature after SCT is necessary to improve immune response and transplatation outcome. Furthermore, the observation that mature NK cells are functionnally active against leukemic blasts is encouraging to develop clinical trials using adoptive transfer of healthy donor alloreactive NK cells.

Purpose: Objective was to assess indication, feasibility and efficacy of double allograft with reduced intensity conditioning. Patients and Methods: All double allogeneic hematopoietic stem cell transplantation (AHSCT) after reduced intensity regimen reported to Promise database were retrospectively studied. Because of the initial lack of clinical and biological data in the Registry, all centers were contacted to improve the database. Results: 66 double mini-allografts were realized in 25 french transplant centers. At this time, 35 mini-allografts presented complete informations corresponding to 14 transplant centers. Diagnosis was LAM (n=15), aplasic anemia (n=5), MDS and/or MPS (n=5), LAL (n=2), Myeloma (n=2), ovarian cancer (n=1), LNH (n=2), LLC (n=1), LMC (n=1) and Hodgkin lymphoma (n=1). Median age at first transplantation was 49.3 years [12.75–64.9] with a median time from diagnosis to first transplant of 22.9 months [3.6–163.7]. Disease status before transplant was progression (n=5), partial response (n=3), complete response (n=18), induction failure (n=2) and chronic phase (n=2). The 5 aplasic anemias were not evaluable for this status. Most patients received fludarabine (n=31), SAL (n=22), busulfan (n=15) based regimen. Graft source was PBSC in 74% of grafts. Median age of donor was 42 years [4–68]. HLA relation of donor with patient was identical sibling (n=22), match unrelated (n=10) or mismatch unrelated (n=3). Second allograft indication was relapse (n=22), lost of graft (n=7), no engraftment (n=5) or residual disease (n=1). Eihgty percents of patients underwent a different conditioning. It was similar for only 7 patients (same n=1, increase SAL dose n=1, SAL addition n=2, dose reduction n=1). Median time between the two allografts was of 7.6 month [1.1–61.0]. Source of stem cells and donor were changed in 13.6% and 37% of allograft respectively. The median time to reach an absolute neutrophil count of 0.5 × 109/l and platelet count of 50 × 109/l was shorter after second allograft (21.4 days [12.4–759.5] versus 16.8 days [6.2–105.4] and 16.3 days [9.3– 71.3] versus 14.26 [3.1–83.7]) with no significant difference. Overall incidence of acute GvH was similar after the two allografts (34% versus 43%). However, acute GvH grade III–IV proportion seems to be higher after second allograft (8% versus 40%). Similarly, proportion of chronic GvH was 23% versus 27% with an increase of extensive GvH after second allograft (50% versus 66%). Response was improved by second allograft in five patients (15%) and allowed to engraftment in 3 patients (7%). To date, 8 patients are still alive. 14 patients died from progression and 11 of TRM (3 multiple organ failure, 5 respiratory failure, 2 fungal infection and 1 rejection) giving a TRM proportion of 31%. With a median follow-up of 57.1 months [21.9–128.8], the median overall survival (OS) was of 24 months [2–71]. Delay greater than 7.7 months and same source of stem cell between two mini-allografts improved OS (50 months versus 9 months, p = 0.0085 and 28 months versus 14 months, p=0.11). Conclusions: Our preliminary results suggests response improvement after double AHSCT with reduced conditioning. However, TRM proportion was high.

Background: In acute leukemia (AL) patients, mortality rates of the most common IFI (i.e., aspergillosis and candidiasis) are well illustrated in the literature from the IFI diagnosis, as well as the rate of fungal relapse during subsequent periods at risk, such as neutropenic phases, or transplant. However, few data are available about the impact of IFI on the subsequent chemotherapy schedule and the indirect impact of IFI on the relapse-free and overall survival. Clinicians are usually reluctant to give the full chemotherapy doses on time, due to the risk of life-threatening fungal relapse during the subsequent courses. Even with secondary prophylaxis, they often delay or decrease the doses of chemotherapy. This may impact on the leukemia outcome. The aim of this case-control study was to assess the potential impact of proven or probable IFI onset on the application of the chemotherapy schedule in AL patients, and its consequences on the leukemia outcome, by comparing patients with and without IFI in a single institution. Delays and changes in chemotherapy doses and drug choices were evaluated and compared to the planned schedule in the protocol. Methods: All consecutive AL patients with a first episode of proven or probable IFI according to the EORTC-MSG criteria between 2000–2006 were reviewed. All patients have been treated in, or according to, clinical research protocols where timing and doses of chemotherapy were predefined. Patients who were planned for allogeneic transplant were excluded as those who were at their last consolidation course when they got IFI or in a palliative phase of the leukemia. Any delay, dose decrease, or dose change were defined as any difference compared to the planned schedule. We planned to include 3 control patients for each case, selected among AL patients without IFI, and matched for age, sex, type of AL, chemotherapy protocol, and year of treatment. 27 case and 76 control patients were finally included. The event-free survival (EFS) was defined as survival without evidence of relapse or progression, or death of any cause. Results: The mean age of the 27 case patients (26 myeloid and 1 lymphoblastic AL) was 52 y (± 13), the M/F ratio was 14/13 IFI (7 candidiasis, 19 aspergillosis, 1 zygomycosis) was proven for 10 patients (37%), and probable for 17 (63%). Twenty (71%) of these IFI occurred during the first induction phase. All patients were treated for their IFI with ≥ 1 antifungal, and 4 of them had a surgical resection of the main fungal lesion(s). These 27 patients were compared to 76 controls (73 myeloid and 3 lymphoblastic AL) without IFI. A delay of the next course of chemotherapy according to the planned protocol was significantly more frequent in the IFI group (16/27, 59%) than in the control group (16/76, 21%) (p=.001). Similarly, the dose or choice of the drugs was modified more frequently in the IFI group (7/27, 26%) than in the control group (6/76, 8%) (p=.037). Only 9 (33%) patients got their next chemotherapy course without any modification in time, dose, or choice of drug, vs. 19/76 (75%) in the control group (p Conclusion: In this single-institution case-control study, the occurrence of IFI significantly modified the application of chemotherapy courses, both on timing of the courses, and dose and choice of the drugs when compared to patients without IFI. Although the difference was not significant, there was a tendency for a lower EFS in the IFI group when compared to the control group.−

Background: Invasive fungal infections (IFIs) incur significant morbidity and mortality among neutropenic patients with hematological malignancies. Prevention and early antifungal treatment include prophylaxis where antifungal agent is started along with neutropenia; empirical therapy where antifungal agent is initiated in persistently febrile patients at least four days after neutropenia onset; preemptive therapy where antifungal agent is initiated later for a suspected IFI based on clinical symptoms, lung imaging, or biological markers. Without data from clinical trials comparing antifungal strategies, determining an optimal antifungal strategy for these patients is challenging. Objective: To determine the cost-effectiveness of all possible antifungal strategies. Target Population: Adult patients with hematological malignancies in induction chemotherapy at high risk for IFIs. Interventions: Prophylaxis with either fluconazole or posaconazole, empirical strategy, and preemptive strategy with fist-line antifungal therapy being amphotericin B, liposomal amphotericin B or caspofungin (Table). Design: Cost-effectiveness decision model. The study cohort encountered seven successive chance nodes during hospital stay: having fever; in those having fever, IFI incidence; Aspergillus or Candida species among IFI; admission in intensive care unit; dying from IFI or underlying hematological malignancy; in patients alive, having severe nephrotoxicity as defined by a twofold increase in baseline serum creatinin; dying from severe nephrotoxicity. Antifungal strategies modified the probabilities of IFI and severe nephrotoxicity that depended on both the duration and the type of antifungal drugs administered. Data Sources: PREVERT trial1, effectiveness data published to December 2007, probabilities of ICU admission and in-hospital mortality according to the occurrence of IFI in the French DRG database, life expectancy of French patients with acute myeloid leukemia, and actual French hospitalization costs (2007 euros). Time Horizon: Lifetime. Perspective: Societal. Outcome Measures: Incremental cost (euros) per discounted life-year saved averaged from 100 samples of 1000 patients (second-order probabilistic Monte Carlo simulations). Results (Table): Fluconazole prophylaxis followed by ampho-B treatment was the cheapest antifungal strategy. Posaconazole prophylaxis followed by ampho-B was nearly cost-effective (59,610 € per discounted year of life gained). Other strategies were either dominated or beyond usual societal thresholds of what may be worth it. Similar results were found in sensitivity analyses among plausible ranges. Conclusions: As compared to previous studies showing that new antifungal drugs were cost-effective within a single strategy, empirical and preemptive antifungal strategies were dominated by prophylaxis strategies. Table: Incremental cost-effectiveness ratio of antifungal strategies in high-risk neutropenic patients Total cost (2007 euros) Years of life gained ICER IFI (%) Aspergillus (%) Nephro-toxicity (%) Antifungals’ cost (2007 euros) Fluconazole then amphoB 35606 2.3448 -- 3.81% 2.61% 4.22% 909 Fluconazole then L-amphoB 36025 2.3457 (extended dominance) 3.81% 2.61% 3.67% 1430 Empirical amphoB 36033 2.3433 (dominated) 3.38% 2.3% 5.98% 1914 Posaconazole then amphoB 36065 2.3525 59,610 € 1.2% 0.81% 3.67% 2646 Preemptif amphoB 36160 2.3449 (dominated) 3.89% 2.67% 4.42% 1247 Posaconazole then L-amphoB 36389 2.3532 462,857 € 1.2% 0.81% 3.21% 3055 Fluconazole then Caspo 36557 2.3459 (dominated) 3.81% 2.61% 3.53% 1985 Preemptif L-amphoB 36616 2.3455 (dominated) 3.89% 2.67% 3.84% 1809 Posaconazole then Caspo 36828 2.3533 4,390,000 € 1.2% 0.81% 3.13% 3509 Empirical L-amphoB 37308 2.3462 (dominated) 3.38% 2.3% 4.39% 3239 Preemptif Caspo 37346 2.3458 (dominated) 3.89% 2.67% 3.55% 2597 Empirical Caspo 39123 2.3473 (dominated) 3.38% 2.3% 3.47% 5080

To the editor: The lymphopenic environment can induce T-cell expansion/activation, a phenomenon referred to as lymphopenia-induced proliferation that can be distinguished from (allogeneic) antigen-induced proliferation.[1][1] Dudley et al showed that a cyclophosphamide (Cy) and fludarabine (Flu)

We thank Dr Fitzhugh and colleagues for their careful reading of our paper. We agree with the authors that the 95% chance of cure reported for stem cell transplantation (SCT) in the most recent patient cohorts will elicit its recommendation to more patients and families. Therefore, documented data

Invasive fungal infection (IFI) occurs in 2–12% of acute leukemia patients, with different prevalences according to centers, and phases of treatment. The mortality rates of the main IFI (i.e., aspergillosis and candidiasis) are well illustrated in the literature, as well as the risk of fungal relapse during subsequent periods at risk, such as a new neutropenic phase, or transplant. However, few data are available about the impact of IFI on the subsequent chemotherapy schedule. Clinicians are usually reluctant to give the full chemotherapy doses on time, due to the risk of life-threatening fungal relapse during the subsequent courses. Even with secondary prophylaxis which is now widely given, they usually delay or decrease the doses of chemotherapy. This may impact on the leukemia outcome. Objective: The aim of this single-institution retrospective study was to look at the impact of proven or probable IFI onset on the application of the chemotherapy schedule as planned in the initial strategy in patients with acute leukemia. Delays, and changes in chemotherapy doses and drug choices were evaluated and compared to the planned schedule in the protocole. Methods: All consecutive acute leukemia patients with a first episode of proven or probable IFI according to the EORTC-MSG criteria between 2000–2006 were reviewed. All patients have been treated in, or according to, clinical research protocols where timing and doses of chemotherapy were predefined. Patients who were planned for allogeneic transplant were excluded as those who were at their last consolidation course when they got IFI. Any delay, dose decrease or dose change were defined as any difference compared to the planned schedule. Results: 28 patients (7 candidiasis, 20 aspergillosis, 1 zygomycosis) were included (M/F: 15/13; mean age: 54y), including 27 acute myeloid leukemia and one acute lymphoblastic leukemia. Eleven (39%) were proven, 17 (61%) were probable. Twenty (71%) of these IFI occurred during the first induction phase. All patients were treated for their IFI with ≥ 1 antifungal, and 4 of them had a surgical resection of the main fungal lesion (s). Seventeen of 28 (60%) had their next course delayed (median delay: 14 days) when compared to the planned protocole. The doses of the antineoplastic drug(s) were modified in 7 (25%) patients. Only 9 (32%) patients got their next chemotherapy course without any modification in time, dose, or choice of drug. Although the number of patients in our study does not allow definite conclusions, these changes should have an impact on leukemia-free and overall survivals. Conclusion: Due to the risk of fungal relapse during a subsequent period at risk, IFI has a practical impact on the dates, doses, and choices of chemotherapy in acute leukemia in more than two thirds of the patients, due to subjective decisions of the clinicians. Effective antifungal strategies are needed to avoid the onset of IFI in acute leukemia patients.

We have previously shown [1] that a calcineurin activity (CA) < 28 pmol RII/mg proteins/min was significantly associated with the absence of acute GVHD after allogeneic stem cell transplant (SCT). The CA is poorly correlated with cyclosporine (CsA) trough level which is not a good marker of CsA efficacy. Therefore, the in vitro assessment of CA could help in adapting the cyclosporine doses for GVHD prophylaxis, better than blood CsA levels. Objective: The aim of this prospective study was to evaluate the feasibility of adapting the CsA doses to the values of in vitro CA either until GVHD occurs, or until day 100, whichever occurs first. Methods: CA assessment methods in PBMC of transplant patients and monoclonal CsA level measurement methods were previously reported [1]. CA and CsA levels were measured once a week from d0–d15, twice a week from d16–d35, then once a week up to d100. The CsA doses were adapted in order to maintain a CA < 28 from transplant by 20% progressive changes of the CsA daily dose. Adaptation was stopped in case of clinical CsA blood levels ≥ 600 ng/ml, creatinine clearance < 40 ml/mn, serum bilirubin > 40 m/L, or severe clinical toxicity. Patients: 39 adult patients with malignant hemotologic diseases (acute leukemia: 28; chronic leukemia: 4; others: 4) or aplastic anemia (3), and undergoing myeloablative SCT were included (HLA-id donor: 20; unrelated donor: 19). GVHD prophylaxis consisted in short course Methotrexate and CsA (initial dose: 3 mg/kg IV). Results: Out of 39 patients, 2 were adapted until d100 and did not develop GVHD; 27 were adapted without CsA toxicity until they developed GVHD (grade I: n=2; grade II: n=18; grade III–IV: n=7). In 10 (26%) patients, adaptation was stopped before d100, due to neurologic (n=1), renal (n=2) or liver (n=2) toxicity, severe nausea (n=1), investigator decision (n=1) or protocol violation (no CsA increase despite high CA and no CsA toxicity: n=3). Conclusion: Our study shows that CsA dose adaptation is feasible on the basis of CA regular assessment within the safety limits defined in our protocole. This adaptation is, however, limited by CsA toxicity, due to the need to significantly increase the CsA dose to get a CA < 28 pmol in 26% of the patients. A prospective controlled study is needed to compare the routine use of CA to the use of CsA blood levels for managing CsA doses in GVHD prophylaxis.

Objective. Empirical antifungal therapy (Emp AF) is a standard of care for high-risk neutropenic patients with persistent or recurrent fever under broad-spectrum antibiotics. We conducted a cost-effectiveness analysis of four competitive strategies to reduce the burden of invasive fungal infection (IFI): the Emp AF, a preemptive antifungal strategy (PE AF) where AF is restricted to patients with clinical symptoms and/or mycological criteria of IFI, a prophylaxis with standard azole therapy (Fluconazole proph), and a prophylaxis with posaconazole (Posaconazole proph). Methods. A decision analytic model was developed to estimate costs and outcomes of the four strategies among patients with acute myeloid leukaemia at high risk of developing an IFI due to chemotherapy-induced neutropenia. The probabilities of receiving AF therapy, and experiencing IFI, either aspergillus or candidemia, were stratified according to induction and consolidation/autologous transplant phase of therapy.1,2 In the two prophylaxis strategies, patients could receive AF therapy, although at a lower rate than in the Emp AF. Length of stay and hospitalization costs depended on: IFI or no IFI; transfer into intensive care unit or not; dying or not (French national hospitalization database including 9,182 stays in 2005). AF drug costs were estimated as the product of the recorded drug consumption,1,2 and 2005 published mean average wholesale price from AP-HP central pharmacy, with exception of ambisome, voriconazole, caspofungine, and posaconazole where administrated national prices were taken. Second-order probabilistic Monte Carlo sensitivity was conducted to assess the effects of parameter uncertainty on the study findings. Results. In the induction phase of therapy, Emp AF (93.3% survival; 95.4% no IFI; €34,505 hospitalization costs) dominated (i.e., higher effectiveness, and lower hospitalization costs) PE AF and Fluconazole Proph strategies. Posaconazole Proph provided higher efficiency (93.5% survival; 97.3% no IFI) at higher hospitalization costs (€34,793) as compared to Emp AF. Results were sensitive to the relative risk of aspergillus invasive infection of posaconazole vs fluconazole (RR=0.11)2, and AF drug costs. In the consolidation/autologous transplant phases where the incidence of IFI is lower,1 all strategies provided similar effectiveness on survival and IFI prevention. Hospitalization costs and overall AF drug costs increased substantially from PE AF strategy (€24,761; €632) to Fluconazole Proph (€26,592; €1,457), Emp AF (€26,856; €1,419), and Posaconazole Proph (€28,094; €3,074). Conclusions. One AF strategy does not fit all high-risk neutropenic situations. In the induction phase of therapy, Emp AF and Posaconazole Proph provide the best outcomes with higher effectiveness and lower hospitalisation costs than PE AF and Fluconazole AF. In the consolidation/autologous phases of therapy, PE AF minimizes costs for similar outcomes in all AF strategies.

We previously demonstrated that natural killer (NK) cells generated after haploidentical stem-cell transplantation (SCT) are blocked at an immature state characterized by phenotypic features and impaired functioning, a blockage that may affect transplantation outcome (Nguyen et al. Blood 2005). Hypothesizing that the absence of mature donor T cells in the graft may affect NK cell differentiation and function, we examined NK cells from 21 patients who received haploidentical SCT from relatives for advanced malignant hematopoietic disease and underwent either partial (pTCD) (CD3+ in the graft >1x105/Kg, mean: 6.9x105/Kg; n=11) or extensive (e-TCD) (CD3+ in the graft

Empirical (E) antifungal therapy (ATF) is a standard of care in neutropenic patients with persistent or recurrent fever. However, the safety and cost-effectiveness of the E strategy are challenged by the development of better diagnostic methods and more effective therapies for invasive fungal infection (IFI). An economic analysis was conducted alongside a multicenter open-label randomized non-inferiority trial showing that a pre-emptive (PE) strategy based on clinical symptoms and GM Ag did not reduce the overall survival of prolonged neutropenic patients when compared to a E strategy (details provided in abstract 551005). Objective: The objective of the study was to compare hospital costs between the PE strategy and E strategy. Patients: 293 adult patients with hematologic malignancies and an expected neutropenia ( Methods: The economic analysis was conducted from the hospital perspective (€2005). Total medication costs were computed from individual records during hospital stay. Results: Overall, mean medication costs did not differ significantly between the PE and E groups (see Table). In patients in induction phase (n=151), mean medication costs were higher in the PE group than the E group (+921€, [95%CI, −1602 to +3444]) as explained by the significantly higher proportion of IFI in the PE group (16.4% vs. 3.9%, p Conclusion: Cost comparison between E and PE strategy showed opposite results in induction or consolidation/ASCT phases. This finding is mainly explained by different risks of developing IFI according to the therapeutic phase. (Grants: PRC 2002 AOR02028). Table: Comparison of mean(std) medication costs, antifungal therapy costs, and proportion of IFI between PE and E groups (€2005) PE strategy E strategy p Overall (n=293) Medication costs 3595 (7444), n=143 3745 (4768), n=150 ns Antifungal therapy costs 2218 (6969), n=143 2337(4093), n=150 ns Proportion of IFI 9.1% (13/143) 2.7% (4/150)

Between 1998 and 2004, 2141 allogeneic hematopoietic stem cell transplantations with unrelated donor (excluding cord blood) have been recorded on the SFGM-TC data base. The lack of unique behavior in regard of Anti Lymphocyte Globulin (ALG) use in unrelated donor transplants allowed us to perform a retrospective study to assess the influence of ALG in such grafts. To avoid biaises we excluded T cell depletion or monoclonal antibody use, reduced intensity conditioning and focused only on patients (pts) with acute leukemia (AL) and myelodysplasia (MDS). So we defined a study population of 553 pts. In addition, in order to analyze the actual impact of ALG on wellknown targets (engraftment, acute GvHD, relapse, survival) we choose to keep only the cases in which was available the allelic HLA typing (4 digits) for the recipient and the donor, i.e. 250 pts. There were 106 females and 144 males, allografted in 29 centers. The median age was 23 years (1–62) and there were 77 children under 15. There were 223 AL - myeloid n=120, lymphoid n=103 - and 27 MDS. The source of cells was bone marrow for 190 pts and peripheral mobilized stem cells for 60. One hundred and ninety-three pts received a conditioning regimen including Total Body Irradiation. Ninety-five pts received ALG (mainly Thymoglobulin Mérieux-Genzyme Lyon France at various doses) for a median of 3 days (group 1) and 155 did not (group 2). There were 189 donor/recipient pairs strictly HLA-matched 10/10, 49 with one allelic difference (HLA A n=13; HLA B n=8; HLA C n=13; HLA DRB1 n= 5; HLA DQB1 n=10 ), 11 with 2 allelic differences and 1 with 3 allelic differences. 238 pts have sustained engraftment at a median time of 20 days (9–41) without any influence of ALG use (97% vs 94% NS). Overall survival at 3 years was 45% in group 1 and 52% in group 2 (NS). The incidence of acute GvHD (grade II to IV) was similar in the two groups (59% vs 54%), but the incidence of acute GvHD (grade III to IV) was lower in group 1 (21% vs 32% p=0.03). Use of ALG did not affect rate relapse (18% vs 20% NS). In a multivariable analysis including HLA disparities, patient age, use of ALG, two factors appear as predictive for grade III–IV GvHD: HLA-identity (p=0.05) and use of ALG (p=0.02). These factors are not significant for survival in the same multivariable analysis but in this allelic matched population, the influence of ALG should be analyzed in regard of exact dose of ALG.

In hematology patients, HAI are a complex mixture of infections due to therapy, especially to transplant procedures, conditioning regimen, immunosuppressive drugs, and invasive procedures. These HAI may occur within the hospital and be related to the hospital environment (i.e., NI), or not. These different kinds of infections require very different preventive measures. The objective of our study was to assess the incidence, overlap, and causes of HAI and NI in our hematology ward over a 6 month period. Methods and Definitions: Infections were considered to be HAI, irrespectively of their site of occurrence (hospital or not) when they were directly related to the therapeutic procedures and subsequent immune deficiency. This included: febrile neutropenia (FN) subsequent to chemotherapy, catheter-related infections, herpes virus reactivations in patients previously sero+ to the virus, primary CMV infection in CMV− patients transplanted with a CMV + donor, toxoplasmosis reactivation, invasive fungal infections, and any infection occurring within 6 months of transplant, or later in case of immunosuppressive treatment after this date. Infections were considered to be NI when they occurred ≥ 48 h after hospitalization without any symptom of infection at hospitalization. Aspergillosis was considered to be NI when there was a delay of 7 days between hospitalization and first symptoms.We retrospectively assessed 223 consecutive infectious episodes occurring between 7–12/2000 in 137 patients, including 59 stem cell transplant recipients (allogeneic: 23; autologous: 36). Results : 204/223 infectious episodes (91,4%) were HAI while 94/223 (42%) were NI. 7/223 (3,6%) were causes of death. Among the 42 bacterial documented infections, 20 (47.6%) were due to potentially hospital-acquired strains: 16/23 (69.5%) of HAI-NIs, and 4/19 (21%) of the non NI-HAIs. Conclusion: In a preventive infection program, the assessment of NI clearly underestimates half of the HAI in hematology patients. Although hospital environment control is of paramount importance in transplant programs, we suggest that HAI should be a better quality indicator for hematology programs than NI.

Hematopoietic cell transplantation (HCT) is the only potentially curative treatment for sickle cell disease (SCD). The probability of event-free survival is currently between 85 and 90% after matched sibling-donor HCT. This intensive treatment conducts to a major change for patients and families. The purpose of the present study was to determine the psychological impact on individuals and their families of this recovery process after HCT. The hypothesis was that the recovery is a process with different aspects (medical, psychological, individual, familial and cultural) which could be discordant. This study was supported by the Etablissement Français des Greffes (EFG) and conducted by child and adolescent psychiatrists, psychologists and a medical anthropologist. The assessment consisted in two semi-structured interviews adressed to the patients and their families for studying their illness narratives and questionnaires for quality of life, post-traumatic stress disorder (PTSD), depression and anxiety. 28 patients (17 males, 11 females) with SCD treated in France by HCT (22 by bone marrow and 6 by cord blood transplantation) and their families were evaluated at least one year (median 5 years, range, 1–14 years) after HCT at the median age of 14 years (range, 4–24 years). Among these 28 patients, 7 had prior history of severe vasculopathy and 6 of stroke before the HCT. At evaluation, 27 patients were free from SCD symptoms (but 3 had moderate chronic GVHD), 1 had graft rejection. 9 patients (4 males, 5 females) (median age 16 years, range 7–23 years) presented psychiatric symptoms as inhibition, depression, hypomaniac symptoms, anxiety, stutter. A psychiatric care was indicated for these 9 subjects. No diagnosis of current PTSD was found. Psychological distress was reported by parents. 21 patients considered themselves as cured, 4 of them in an incomplete way (none with stroke before the HCT, 3 with moderate chronic GVHD), and 3 not cured at all (none with stroke before the HCT, 1 with graft rejection). The study of the narratives illustrated the major individual, familial and cultural changes after the HCT. The recovery needed complex adjustments. Patients had to cope with new potentialities and requirements: school and relationships with peers, identity issues (disease transmission, infertility) and family changes. This study underlies the importance of psychological interventions for improving the outcome of patients with SCD treated by HCT and their families.

RIC regimens are mainly used for patients who cannot benefit from the myelo-ablative (MA) approach, due to age, previous SCT, or comorbidities, based on an expected early toxicity of MA regimen. Some centers may also choose the RIC strategy in young patients because they have no facilities for the management of prolonged neutropenia. The development of RIC has been so encouraging that programs now compare MA and RIC in patients who could tolerate both approaches. However, RIC may fail, and MA SCT be secondary considered. Objective: The goal of this retrospective survey was to assess the toxicity and outcome of MA after RIC SCT. Patients: 17 patients (14–63 y; 6 CML, 4 AML, 2 CLL, 2 myeloma, 1 NHL, 1 CMML, 1 myelodysplastic syndrome) received a MA SCT after RIC, for relapse (n=12), graft rejection (n=4), or in a planned program (n=1), from an HLA-id sibling (n=12), a familial mismatched donor (n=1), an unrelated donor (n=3) or a cord blood (n=1). The reason for initial RIC approach was age in 4, a research program in 6, comorbidities in 3, and previous autologous SCT in 4 patients. RIC included Fludarabin (Flu)-Busulfan (BU) in 8, Flu-2Gy irradiation in 4, Cyclophosphamide (CPM)-antithymocyte globulin (ATG) in 1, and various Flu- regimens in 4. One patient had 2 consecutive RIC SCTs before MA SCT. The median delay between RIC and MA SCT was 9 months (range: 2.5–36 months). The same donor was used for the 1st and 2nd SCT in 12/17 cases. The MA transplant was conditioned with BU-CY (±VP16) in 9, Cyclo-TBI in 4, and other regimens in 4 patients. The median follow-up after MA SCT was 9 months Results: After the MA transplant, 1 patient developed veno-occlusive disease, 2 developed interstitial pneumonia. Five patients died from relapse between 8 and 21 mo after the MA SCT. Seven (41%) died from transplant-related causes at a median of 8 months (GVHD:3; infection:1; multi-organ failure:1; thrombotic microangiopathy:1; EBV proliferative disease:1). 5/17 (29.5%) patients are alive and well 4.5 to 26 months after MA transplant. Three of them have extensive chronic GVHD while they did not develop any GVHD after RIC. The delay between the 2 transplants did not influence the outcome. Conclusion: This survey shows that MA after RIC SCT is feasible, and considering that most of these patients have been initially eligible for RIC because of their age or comorbidities, MA SCT had an acceptable toxicity. It may be considered for patients who relapsed or rejected after RIC transplant.

DLI are an efficient treatment for CML in relapse after allogeneic stem cell transplantation, with 60 to 70% of complete response. However, major side effects such has graft-versus-host disease (GVHD) or pancytopenia are frequently observed after this treatment. Imatinib mesylate has also been shown to be effective in a such situation (Blood 2002, 100 (5): 1590–95). In the present study, we report the French experience, with a 3 years median follow-up of 26 patients in relapse of CML after allograft (21 sibling, 4 unrelated, 1 cord blood transplantations). At time of treatment with IM, twenty patients were in chronic phase and 5 only were in molecular relapse, while 4 patients were in blastic phase. Median time from graft to relapse was 36 months (3–132 months) and the median time from relapse to Imatinib was 15 months ( 1–96 months). Ten patients had already received DLI, 14 patients Interferon. Only 6 patients had no treatment prior to IM. With a median follow-up of 32 months (16–44 months), 19 patients are alive and 5 died. The majority of patients in chronic (19/20) phase are alive. At time of last follow-up : Three (11%) patients fail to respond to IM, 2 (8%) have a partial response, 12 (46%) are in CCR with 9/12 in molecular remission, and 4 (15%) patients present relapse (1 blastic phase, 3 molecular relapse). Eleven patients are evaluable for VNTR chimerism, 8(72%) achieved full donor chimerism at last follow-up. IM was well tolerated, and 2 GVHD reactivations were observed but 8 (42%) patients experienced cytopenias requiring drug modulation or withdrawal, and two grade IV toxicities (muscle cramps, cardiomyopathy). Six patients stopped IM (2 toxicities, 4 long term molecular remissions). Four of these patients experienced molecular relapse (3 after 12 months, 1 at 3 months). One patient achieved molecular remission after a new challenge with IM. In conclusion, Imatinib mesylate is efficient and safe treatment for CML in relapse after allogeneic HSCT without reactivating GVHD, and is able to restore full donor chimerism in a majority of responsive patients. A complete and sustained molecular response is obtained in about one third of patients, however molecular relapse is frequent after IM withdrawal. The place of IM and/or DLI for CML in relapse after allogeneic HSCT needs to be addressed prospectively and requires a randomized trail.

The Perugia group reported favourable outcome in patients under haploidentical hematopoietic stem cell transplantation (SCT) for acute myeloid leukemia (AML) when donor and recipient were KIR ligand mismatched. In our study, we observed contradictory results in 11 adults (median age 25 years, range 15–38) who received a full haplotype mismatch SCT from a related donor at the Pitie-Salpetriere Hospital between October 1998 and December 2003 for high risk AML according to modalities of conditioning described by the Perugia group. Despite a KIR ligand mismatch in the GvH direction for 10 pairs of 11, majority of patients died (10/11) due to relapse for 7/11 and TRM for 3/11. Only 1 patient is alive in remission 8 months post SCT. No GvHD arose since the graft was positively selected for CD34+ cells using Clinimacs, resulting in an extensive T depletion. These observations incited us to perform in vitro studies to analyze NK cells reconstitution during the early period post haplo-SCT, as all the relapses occurred during the first 4 months following SCT. Peripheral blood was collected from 7 patients each month after SCT, and compared with the donors. T cells recovered very lately, in contrast to NK cells, which reached a normal absolute count rapidly. Similar phenotypic NK profile was observed in all patients. The CD56bright immunoregulatory NK cells subset was increased, representing a median of 55% of circulating NK cells at one month (M1), and 25 % at 3 months (M3) post SCT, as compared to 4% in the donors. Expression of the activating receptor NKp30 was reduced. The ratio of recipient/donor NKp30 on NK cells was 0.6 at M1 and 0.7 at M3. All inhibitory KIRs, especially, KIR2DL1 were down- regulated after SCT with a ratio, for KIR2DL1, at 0.23 at M1 and 0.37 at M3. To counterbalance the low expression of KIRs, the inhibitory receptor CD94/NKG2A was strikingly over expressed by NK cells generated after SCT (median of circulating NK cells expressing NKG2A: 97% at M1 and 90% at M3 versus 43% in the donors). This unusual phenotype persisted during all the study and evocated an immature state of NK cells. Immaturity of NK cells in vitro was associated with impaired functions. NK cells generated after SCT had a less efficient lysis of K562 cell line, and no cytotoxicity against the primary mismatched AML blasts, as compared to the donors. Expression of NKG2A by most NK cells post SCT was correlated with this impaired lysis. The ligand for NKG2A is HLA-E, which was also expressed by AML blasts as indicated by Western Blot analyzes. NK cells generated after SCT were cytotoxic against the HLA class I negative LCL 721-221cell line. Cytotoxicity was reduced against the LCL derivated 721-221 AEH cell line, transfected by HLA-E, and the lysis was restored after inhibition of NKG2A. Similar results were obtained with primary AML blasts; NK cytotoxicity against AML blasts was restored after blockade of NKG2A. We conclude that NK cells generated after haploidentical SCT exhibited an immature phenotype that persisted several months after SCT. This immaturity was correlated in vitro with an impaired cytotoxicity due to a dominant inhibitory role of NKG2A, and in vivo with the absence of GvL effect despite the mismatch KIR ligand in the GvH direction.

We have used DNA sequence polymorphism analysis to document engraftment after T cell-depleted bone marrow transplantation (BMT), with a selected panel of four DNA probes. In contrast to nondepleted BMT recipients, the patients who received T cell-depleted marrow exhibited a mixed blood chimerism. This mosaicism was observed before graft failure or relapse in six patients. However, in five other patients, this mixed chimerism was not followed by these complications with a follow-up of 9 to 31 months after transplantation. Our results support the hypothesis that transplanted bone marrow T cells may help to maintain engraftment by eliminating host cells that can cause graft failure.