Search

Introduction:Severely injured patients develop a dysregulated inflammatory state characterized by vascular endothelial permeability, which contributes to multiple organ failure. To date, however, the mediators of and mechanisms for this permeability are not well established. Endothelial permeability in other inflammatory states such as sepsis is driven primarily by overactivation of the RhoA GTPase. We hypothesized that tissue injury and shock drive endothelial permeability after trauma by increased RhoA activation leading to break down of endothelial tight and adherens junctions. Methods:Human umbilical vein endothelial cells (HUVECs) were grown to confluence, whereas continuous resistance was measured using electrical cell-substrate impedance sensing (ECIS) Z-Theta technology, 10% ex vivoplasma from severely injured trauma patients was added, and resistance measurements continued for 2 hours. Areas under the curve (AUCs) were calculated from resistance curves. For GTPase activity analysis, HUVECs were grown to confluence and incubated with 10% trauma plasma for 5 minutes before harvesting of cell lysates. Rho and Rac activity were determined using a G-LISA assay. Significance was determined using Mann-Whitney tests or Kruskal-Wallis test, and Spearman ρ was calculated for correlations. Results:Plasma from severely injured patients induces endothelial permeability with plasma from patients with both severe injury and shock contributing most to this increased permeability. Surprisingly, Injury Severity Score (ISS) does not correlate with in vitrotrauma-induced permeability (−0.05, P> 0.05), whereas base excess (BE) does correlate with permeability (−0.47, P= 0.0001). The combined impact of shock and injury resulted in a significantly smaller AUC in the injury + shock group (ISS > 15, BE < −9) compared with the injury only (ISS > 15, BE > −9; P= 0.04) or minimally injured (ISS < 15, BE > −9; P= 0.005) groups. In addition, incubation with injury + shock plasma resulted in higher RhoA activation (P= 0.002) and a trend toward decreased Rac1 activation (P= 0.07) compared with minimally injured control. Conclusions:Over the past decade, improved early survival in patients with severe trauma and hemorrhagic shock has led to a renewed focus on the endotheliopathy of trauma. This study presents the largest study to date measuring endothelial permeability in vitrousing plasma collected from patients after traumatic injury. Here, we demonstrate that plasma from patients who develop shock after severe traumatic injury induces endothelial permeability and increased RhoA activation in vitro. Our ECIS model of trauma-induced permeability using ex vivoplasma has potential as a high throughput screening tool to phenotype endothelial dysfunction, study mediators of trauma-induced permeability, and screen potential interventions.

Background:Blood type O is the most common blood type and has lower von Willebrand factor (vWF) levels (25%–35% lower than non-O blood types). von Willebrand factor is important for initiating platelet attachment and binding factor VIII. We hypothesized that patients with type O blood are at an increased risk of trauma-induced coagulopathy and bleeding post injury. Study Design:Adult trauma activations with known blood type at a level I trauma center with field systolic blood pressure < 90 mm Hg were studied retrospectively. The relationships of blood group O versus non-O to coagulation assays, massive transfusion (MT), ventilator-free days, and mortality were adjusted for confounders. Hyperfibrinolysis (HF) was defined as thromboelastogram of percent lysis in 30 min > 3%, and fibrinolysis shutdown was defined as percent lysis in 30 min < 0.9%. von Willebrand factor activity was quantified on 212 injured patients using a STAGO apparatus. Results:Overall, 268 patients met criteria. Type O patients were more likely to develop HF than non–type O blood patients (43% vs. 29%, P= 0.06) and had significantly lower vWF activity (222% vs. 249%, P= 0.01). After adjustment for New Injury Severity Score and blunt mechanism, type O had higher odds of HF (odds ratio, 1.94, 95% confidence interval, 1.09–3.47) and increased odds of MT (odds ratio, 3.02; 95% confidence interval, 1.22–7.49). Other outcomes were not significantly affected. Conclusion:Type O patients with hypotension had increased HF and MT post injury, and these were associated with lower vWF activity. These findings have implications for the monitoring of HF in patients receiving type O whole-blood transfusions post injury.

ABSTRACTWe describe a project to formalize Galois theory using the Lean theorem prover, which is part of a larger effort to formalize all of the standard undergraduate mathematics curriculum in Lean. We discuss some of the challenges we faced and the decisions we made in the course of this project. The main theorems we formalized are the primitive element theorem, the fundamental theorem of Galois theory, and the equivalence of several characterizations of finite degree Galois extensions.

Greenhouse gases produced by livestock are important contributors to climate change. The ability to measure large-scale exhaled metabolic gases from cattle using GreenFeed systems will help farmers to reduce the enteric emissions while maintaining or increasing cow productivity. GreenFeed units are portable chamber systems that measure individual animal gas production in real-time. Thus, the machines generate large amounts of daily data that can be overwhelming for users to process. This challenge motivated us to develop an R-package named greenfeedrthat offers functions for downloading, processing, and reporting GreenFeed data. Herein, we describe all functions implemented in the greenfeedrR-package and present examples based on dairy cow data. The R-package has functions for downloading GreenFeed data (get_gfdata), for generating daily and final reports (report_gfdata), for processing daily and final records (process_gfdata), and extra functions that help to extract information regarding pellet intakes and daily visits (pellinand viseat). Using our example data with 32 lactating dairy cows, we demonstrated the capabilities of the different functions to generate easy-to-read reports and process large amount of data. Also, we included in the function process_gfdatasome parameters that will help users to define the best criteria to process their own GreenFeed data. Overall, the greenfeedrrepresents a significant advancement in the management and analysis of GreenFeed data, offering an efficient tool tailored to the needs of the user.

The postsynthetic processing of nanomaterials may allow researchers to reach specific properties, morphologies, or phase regimes that are not accessible by simple synthesis alone. Here, we take advantage of atomic interdiffusion at nanoparticle interfaces to fabricate core/alloy and core/alloy/shell nanoparticles. Modest temperature changes were found to have profound effects for the interfacial alloying of the confined nanosystem. The alloy formation and subsequent interdiffusion allowed us to tailor nanoparticle composition and ultrastructure, as well as surface plasmon response. This processing step, which involves the layer-by-layer formation of a core/alloy/shell morphology, utilizes hydrothermal annealing provided by automated microwave irradiation to control solute deposition, as well as alloy thickness. As a proof-of-principle system, we employed a Au/AuxAg1–x/Ag nanosystem, due, in large part, to its miscible phase diagram and rich plasmonic behavior. Nanostructure morphology was characterized by TEM and STEM, and compositional analysis was performed via selective area EDX and XPS. The resulting surface plasmon resonance signatures were modeled as a function of alloy or monometallic shell thickness, as well as alloy composition, using the discrete dipole approximation method. A proposed shell growth mechanism is described, which involves the competition between a classical ripening system at low temperature and a self-limiting growth at high temperature, the latter of which may be driven by an alloy order–disorder phenomena at the interface. These optical and growth models strongly correlate with the experimental results, namely, that the plasmon resonance is highly tunable on shell thickness and alloy composition and that alloy thickness and interdiffusion are highly tunable by the thermal processing.

In 1978, Lonza Group introduced hydantoin chemistry to the cosmetic/personal-care market. This hydantoin technology was the patented Glydant® preservative--designed and developed as a safer, odorless, controlled release form of formaldehyde. [...]

Despite advances in supportive therapy to prevent complications of sickle cell disease (SCD), access to care is not universal. Hematopoietic cell transplantation is, to date, the only curative therapy for SCD, but its application is limited by availability of a suitable HLA-matched donor and lack of awareness of the benefits of transplant. Included in this study are 1000 recipients of HLA-identical sibling transplants performed between 1986 and 2013 and reported to the European Society for Blood and Marrow Transplantation, Eurocord, and the Center for International Blood and Marrow Transplant Research. The primary endpoint was event-free survival, defined as being alive without graft failure; risk factors were studied using a Cox regression models. The median age at transplantation was 9 years, and the median follow-up was longer than 5 years. Most patients received a myeloablative conditioning regimen (n = 873; 87%); the remainder received reduced-intensity conditioning regimens (n = 125; 13%). Bone marrow was the predominant stem cell source (n = 839; 84%); peripheral blood and cord blood progenitors were used in 73 (7%) and 88 (9%) patients, respectively. The 5-year event-free survival and overall survival were 91.4% (95% confidence interval, 89.6%-93.3%) and 92.9% (95% confidence interval, 91.1%-94.6%), respectively. Event-free survival was lower with increasing age at transplantation (hazard ratio [HR], 1.09; P< .001) and higher for transplantations performed after 2006 (HR, 0.95; P= .013). Twenty-three patients experienced graft failure, and 70 patients (7%) died, with the most common cause of death being infection. The excellent outcome of a cohort transplanted over the course of 3 decades confirms the role of HLA-identical sibling transplantation for children and adults with SCD.

Transplantation of 2 unrelated cord blood (UCB) units instead of 1 has been proposed to increase the cell dose. We report a prospective randomized study, designed to compare single- vs double-UCB transplantation in children and young adults with acute leukemia in remission or myelodysplasia. Eligible patients had at least two 4-6 HLA-identical UCBs with >3 × 107 nucleated cells/kg for the first and >1.5 × 107 for the second. The primary end point was the 2-year cumulative incidence of transplantation strategy failure, a composite end point including transplant-related mortality (TRM), engraftment failure, and autologous recovery. Randomized patients who did not proceed to transplantation due to refractory disease were considered transplantation failures. A total of 151 patients were randomized and included in the intent-to-treat analysis; 137 were transplanted. Double-UCB transplantation did not decrease transplantation strategy failure (23.4% ± 4.9% vs 14.9% ± 4.2%). Two-year posttransplant survival, disease-free survival, and TRM were 68.8% ± 6.0%, 67.6% ± 6.0%, and 5.9% ± 2.9% after single-unit transplantation compared with 74.8% ± 5.5%, 68.1% ± 6.0%, and 11.6% ± 3.9% after double-unit transplantation. The final relapse risk did not significantly differ, but relapses were delayed after double-unit transplantation. Overall incidences of graft-versus-host disease (GVHD) were similar, but chronic GVHD was more frequently extensive after double-UCB transplantation (31.9% ± 5.7% vs 14.7% ± 4.3%, P = .02). In an exploratory subgroup analysis, we found a significantly lower relapse risk after double-unit transplantation in patients receiving total body irradiation without antithymocyte globulin (ATG), whereas the relapse risk was similar in the group treated with busulfan, cyclophosphamide, and ATG. Single-UCB transplantation with adequate cell dose remains the standard of care and leads to low TRM. Double-unit transplantation should be reserved for patients who lack such units. This trial was registered at www.clinicaltrials.gov as #NCT01067300.

The aim of our study was to analyze the factors contributing to heterogeneity of prognosis in patients with hyperdiploidy>50 chromosomes (HD>50), a group of B-cell precursor acute lymphoblastic leukemia with favorable outcome. The 541 HD>50 patients registered prospectively in the 58951 European Organisation for Research and Treatment of Cancer (EORTC) Children's Leukemia Group (CLG) trial, identified by karyotype (446 patients) and by DNA index (DI) (490 patients), had a 6-year event-free survival (EFS) of 89.0% (standard error [SE] = 1.5%) and a 6-year overall survival (OS) of 95.9% (SE = 0.9%). The strongest prognostic factor was the modal number of chromosomes (MNC): the 6-year EFS of 51-53, 54-57, and 58-66 MNC groups were 80%, 89%, and 99%, respectively (P< .0001). Ploidy assessed by DI was also a favorable factor: the higher the DI, the better the outcome. The 6-year EFS of the 3 subgroups of DI < 1.16/≥1.16-<1.24/≥1.24 were 83%, 90%, and 95%, respectively (P= .009). All usual combinations of trisomies (chromosomes 4, 10, 17, 18) were significant favorable factors but had lower EFS when MNC was lower than 58. In multivariate analysis, MNC remained the strongest factor. Consequently, the best indicator for excellent outcome was ploidy assessed by karyotype because patients with 58-66 chromosomes stood every chance of being cured (OS of 100% at 6-year follow-up) with less-intensive therapy. This trial was registered at www.clinicaltrials.govas #NCT00003728. Registered: http://www.eortc.org/, http://clinicaltrials.gov/show/NCT00003728.

The molecular mechanisms that underlie T-cell quiescence are poorly understood. In the present study, we report a primary immunodeficiency phenotype associated with MST1 deficiency and primarily characterized by a progressive loss of naive T cells. The in vivo consequences include recurrent bacterial and viral infections and autoimmune manifestations. MST1-deficient T cells poorly expressed the transcription factor FOXO1, the IL-7 receptor, and BCL2. Conversely, FAS expression and the FAS-mediating apoptotic pathway were up-regulated. These abnormalities suggest that increased cell death of naive and proliferating T cells is the main mechanism underlying this novel immunodeficiency. Our results characterize a new mechanism in primary T-cell immunodeficiencies and highlight a role of the MST1/FOXO1 pathway in controlling the death of human naive T cells.

Nonenzymatic glycation and oxidation of ubiquitous proteins in vivo leads to irreversible formation of advanced glycation end products (AGEs). Due to their relatively long half life and low clearance rate AGEs tend to accumulate within static tissues and the circulatory system. Spectra obtained using 830 nm near-infrared (NIR) excitation suggest that the so-called "autofluorescence" from all tissues has a finite number of sources but the fact that senior and diabetic subjects produce more than other members of the general population suggests that a significant portion of the total autofluorescence from all sources originates from AGEs. Using pentosidine generated in a reaction mixture as described by Monnier as representative, an in vitro study unveiled very similar fluorescence and photobleaching pattern as observed for autofluorescence in vivo. A series of oxygen, air and argon purging experiments on the pentosidine-generating reaction mixture suggests that pentosidine is a singlet oxygen sensitizer and secondary reactions between the pentosidine itself and/or other fluorophores and the photosensitized singlet oxygen explain the observed photobleaching. Ab initio Gaussian calculations on pentosidine reveal the existence of low-lying triplet excited states required for the sensitization of ground state oxygen. A commercially available product known as singlet oxygen sensor green (SOSG) that specifically serves as a singlet oxygen detection reagent confirms the generation of singlet oxygen from NIR irradiated pentosidine trimixture. This study provides one definite chemical mechanism for understanding in vivo human skin autofluorescence and photobleaching.

La meilleure compréhension des mécanismes de l’effet antitumoral allogénique et la volonté de minimiser la toxicité de la greffe, notamment dans la population âgée ont conduit ces dernières années au développement de conditionnements dits « réduits ». Ces conditionnements permettent une prise de greffe rapide avec un taux de toxicité liée à la chimiothérapie très faible. Ils sont actuellement largement utilisés chez l’adulte tant en condition géno- que phéno-identique et de nombreuses études ont montré leur faisabilité et leur efficacité. En pédiatrie, la faisabilité est maintenant établie mais la place de ces conditionnements à intensité réduite reste à déterminer. Cette option thérapeutique peut être proposée à deux types de population. La première est celle d’enfants porteurs de pathologies malignes, pour lesquels l’allogreffe est le seul traitement curateur potentiel reconnu, mais dont les traitements antérieurs reçus ou une comorbidité associée ne permettent pas la réalisation d’un conditionnement lourd myéloablatif. La seconde est celle d’enfants porteurs de tumeurs solides ou d’hémopathies malignes en échec des thérapeutiques de référence. Plus d’une centaine de cas pédiatriques ont actuellement été rapportés dans différentes pathologies telles que des hémopathies, leucémies aiguës et lymphomes (en particulier la maladie de Hodgkin) et des tumeurs solides. Les résultats intéressants publiés récemment dans plusieurs pathologies pédiatriques encouragent à la poursuite d’études multicentriques prospectives. Une meilleure compréhension des mécanismes immunitaires antitumoraux et l’utilisation de nouvelles thérapeutiques adjuvantes post-greffe permettront vraisemblablement de favoriser dans l’avenir le développement pédiatrique de l’allogreffe à conditionnement réduit.

TWISTand adenomatosis polyposis coli (APC) are critical signaling factors in normal bone development. In previous studies examining a homogeneously treated cohort of pediatric osteosarcoma patients, we reported the frequent and concurrent loss of both TWISTand APCgenes. On these bases, we created a related animal model to further explore the oncogenic cooperation between these two genes. We performed intercrosses between twist-null/+ and Apc1638N/+ mice and studied their progeny. The Apc1638N/+;twistnull/+ mice developed bone abnormalities observed by macroscopic skeletal analyses and in vivoimaging. Complementary histologic, cellular, and molecular analyses were used to characterize the identified bone tumors, including cell culture and immunofluorescence of bone differentiation markers. Spontaneous localized malignant bone tumors were frequently identified in Apc1638N/+;twist-null/+ mice by in vivoimaging evaluation and histologic analyses. These tumors possessed several features similar to those observed in human localized osteosarcomas. In particular, the murine tumors presented with fibroblastic, chondroblastic, and osteoblastic osteosarcoma histologies, as well as mixtures of these subtypes. In addition, cellular analyses and bone differentiation markers detected by immunofluorescence on tumor sections reproduced most murine and human osteosarcoma characteristics. For example, the early bone differentiation marker Runx2, interacting physically with hypophosphorylated pRb, was undetectable in these murine osteosarcomas, whereas phosphorylated retinoblastoma was abundant in the osteoblastic and chondroblastic tumor subtypes. These characteristics, similar to those observed in human osteosarcomas, indicated that our animal model may be a powerful tool to further understand the development of localized osteosarcoma.

The European Organisation for Research and Treatment of Cancer 58951 trial for children with acute lymphoblastic leukemia (ALL) or non-Hodgkin lymphoma (NHL) addressed 3 randomized questions, including the evaluation of dexamethasone (DEX) versus prednisolone (PRED) in induction and, for average-risk patients, the evaluation of vincristine and corticosteroid pulses during continuation therapy. The corticosteroid used in the pulses was that assigned at induction. Overall, 411 patients were randomly assigned: 202 initially randomly assigned to PRED (60 mg/m2/d), 201 to DEX (6 mg/m2/d), and 8 nonrandomly assigned to PRED. At a median follow-up of 6.3 years, there were 19 versus 34 events for pulses versus no pulses; 6-year disease-free survival (DFS) rate was 90.6% (standard error [SE], 2.1%) and 82.8% (SE, 2.8%), respectively (hazard ratio [HR] = 0.54; 95% confidence interval, 0.31-0.94; P = .027). The effect of pulses was similar in the PRED (HR = 0.56) and DEX groups (HR = 0.59) but more pronounced in girls (HR = 0.24) than in boys (HR = 0.71). Grade 3 to 4 hepatic toxicity was 30% versus 40% in pulses versus no pulses group and grade 2 to 3 osteonecrosis was 4.4% versus 2%. For average-risk patients treated according to Berlin-Frankfurt-Muenster–based protocols, pulses should become a standard component of therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003728.

Introduction: Partial splenectomy (PS) in children is a surgical option in haematological diseases and focal splenic tumours. The aim of this study was to describe the feasibility and the results of laparoscopic partial splenectomy in children in these two indications by a multicentric retrospective study. Methods: The authors reviewed the files of all children who underwent laparoscopic PS between March 2002 and September 2006 in two paediatric surgical centers. The data of 11 children were collected and included clinical presentation, age, gender, radiographic examinations, surgical procedure, need for blood transfusion and early complications. Results: From March 2002 to September 2006, laparoscopic PS had been performed on 11 children (6 boys, 5 girls) aged 23 months to 11 years (mean 7, 9). Four children had splenic focal tumours and seven had haematological diseases: six hereditary spherocytosis (HS) and one hemoglobinosis E. During the surgical procedure for haematological diseases 75–80% of the splenic tissue was removed. When PS was performed for focal splenic tumours, the splenic remnant was around 70%. No preoperative complications occurred (no bleeding, no diaphragmatic injury). Neither preoperative nor conversion was necessary. One postoperative complication occurred (left pleural effusion) but required no further treatment. The mean hospital stay was 7.7 days (range from 3 days to 10 days). No infectious postoperative complications occurred; the mean follow up was 21.1 months (range 3–52 months). Conclusion: Laparoscopic partial splenectomy is feasible and safe in children with hypersplenism or focal splenic tumours. Partial splenectomy is a good way to prevent postsplenectomy infections by preservation of the immune role of spleen in children with haematological diseases. This technique performed for focal splenic tumours allows the surgeon to choose the size of the splenic remnant.

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.

Cet article présente le cas d’un garçon de 8 ans atteint de ß-thalassémie majeure, affection qui représente une forme d’anémie héréditaire grave. Le traitement initial par transfusions répétées a entraîné le développement d’une hémochromatose sévère mettant en jeu le pronostic vital. Le patient a bénéficié d’une greffe allogénique de cellules souches hématopoïétiques, seul traitement curatif de la thalassémie majeure, mais il a rapidement développé une maladie du greffon contre l’hôte. Une prise en charge globale du patient a été réalisée dans le Département d’Odontologie Pédiatrique en raison des complications maxillo-faciales et bucco-dentaires importantes liées à ces deux affections. (Med Buccale Chir Buccale 2006 ; 12 : 163-70).

In a series of 153 children with T-cell malignancies enrolled in 2 consecutive European Organization for Research and Treatment of Cancer (EORTC) trials, we assessed the HOX11L2expression and/or the presence of a t(5;14)(q35;q32). Additionally, in 138 of these patients, HOX11expression and SIL-TALrearrangement were also assessed. These alterations were mutually exclusive, and their frequency was 23% (n = 35), 7% (n = 10), and 12% (n = 17), respectively. HOX11L2/t(5;14) positivity was more frequent in acute lymphoblastic leukemia (ALL) with cortical T immunophenotype and in children aged between 6 and 9 years. In contrast with previously reported data, patients positive and negative for HOX11L2/t(5;14) were comparable with regard to clinical outcome as well as to the response to a 7-day prephase treatment or to residual disease at completion of induction therapy. The 3-year event-free survival (EFS) rate (± SE percentage) for patients positive and negative for HOX11L2/t(5;14) was 75.5% (± 8.1%) and 68.3% (± 5.0%), respectively; the hazard ratio was 0.84 (95% confidence interval, 0.40-1.80). Patients with HOX11-high expression and those with SIL-TALfusion had low levels of residual disease at the end of induction and a favorable prognosis: the 3-year EFS rate was 83.3% (± 8.5%) and 75.3% (± 12.6%), respectively. The results obtained in HOX11L2/t(5;14) patients in this study do not confirm the unfavorable prognosis reported in previous studies.

Allogeneic bone marrow transplantation (BMT) from HLA-identical siblings is an accepted treatment for both thalassemia and sickle cell disease (SCD). However, it is associated with decided risk of both transplant-related mortality (TRM) and chronic graft-versus-host disease (GVHD). We analyzed 44 patients (median age, 5 years; range, 1-20 years) given an allogeneic related cord blood transplant for either thalassemia (n = 33) or SCD (n = 11). Thirty children were given cyclosporin A (CsA) alone as GVHD prophylaxis, 10 received CsA and methotrexate (MTX), and 4 patients received other combinations of immunosuppressive drugs. The median number of nucleated cells infused was 4.0 × 107/kg (range, 1.2-10 × 107/kg). No patient died and 36 of 44 children remain free of disease, with a median follow-up of 24 months (range, 4-76 months). Only one patient with SCD did not have sustained donor engraftment as compared with 7 of the 33 patients with thalassemia. Three of these 8 patients had sustained donor engraftment after BMT from the same donor. Four patients experienced grade 2 acute GVHD; only 2 of the 36 patients at risk developed limited chronic GVHD. The 2-year probability of event-free survival is 79% and 90% for patients with thalassemia and SCD, respectively. Use of MTX for GVHD prophylaxis was associated with a greater risk of treatment failure. Related CBT for hemoglobinopathies offers a good probability of success and is associated with a low risk of GVHD. Optimization of transplantation strategies could further improve these results.

Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and lymphoblastic lymphoma in children. It has minimal bone marrow toxicity. Its major side effects are anaphylaxis, pancreatitis, diabetes, coagulation abnormalities, and thrombosis, especially intracranial. It is derived from 2 different sources: Escherichia coli and Erwinia chrysanthemi. Nonrandomized clinical studies have suggested a similar efficacy of these 2 types of asparaginases and a lower toxicity for Erwinia-asparaginase. The European Organisation for Research and Treatment of Cancer–Children's Leukemia Group (EORTC-CLG) 58881 trial randomized 700 children with acute lymphoblastic leukemia or lymphoblastic lymphoma to eitherE coli– or Erwinia-asparaginase at the same dosage of 10 000 IU/m2 twice weekly to compare toxicity and efficacy. Coagulation abnormalities were more frequent in theE coli–asparaginase than in theErwinia-asparaginase arm of the study (30.2% versus 11.9%, P < .0001). The incidence of other toxicity was not significantly different. In the Erwinia-asparaginase arm, more patients failed to achieve complete remission (4.9% versus 2.0%; P = .038) and the relapse rate was higher, leading to shorter event-free survival (hazard ratio,1.59; 95% CI, 1.23-2.06; P = .0004). The estimate of event-free survival rate (SE) at 6 years was 59.8% (2.6%) versus 73.4% (2.4%). Overall survival rate at 6 years was also lower in theErwinia-asparaginase arm at 75.1% (2.3%) versus 83.9% (2.0%), P = .002. With the dose scheduling used in this protocol, E coli–asparaginase induced more coagulation abnormalities but was superior toErwinia-asparaginase for the treatment of childhood lymphoid malignancies.

This study was undertaken to show that a high survival rate can be obtained in B-cell (Burkitt and large B-cell) lymphoma and L3 leukemia with multiagent chemotherapy adapted to the tumor burden (stage, resection status, percentage of blasts in bone marrow, and central nervous system [CNS] involvement) and early response to chemotherapy, to investigate actual prognostic factors, and to see if large B-cell lymphoma can be treated with the same regimen as Burkitt lymphoma. Patients were classified into 3 risk groups. Group A (resected stage I and abdominal stage II) received 2 courses of vincristine, cyclophosphamide, doxorubicin, and prednisone. Group B (patients not eligible for groups A or C) received 5 courses of chemotherapy with, in addition, high-dose methotrexate, 3 g/m2 over 3 hours; infusional cytarabine; and intrathecal (IT) methotrexate. Group C (patients with CNS involvement and acute lymphoblastic leukemia with at least 70% of blasts in bone marrow) received 8 courses with, in addition, high-dose methotrexate, 8 g/m2; high-dose cytarabine; etoposide; and triple IT. Except in group A, treatment started with a prephase (COP, low-dose vincristine and cyclophosphamide). It was intensified for patients who did not respond to COP in group B and any patient with residual viable cells after the consolidation phase. A total of 561 patients were enrolled in the SFOP LMB89 protocol (July 1989-June 1996). Five-year survival is 92.5% (95% confidence interval [CI], 90%-94%) and event-free survival (EFS) 91% (95% CI, 89%-93%). EFS is 98% (95% CI, 90%-100%), 92% (95% CI, 89%-95%), and 84% (95% CI, 77%-90%) for group A, B, and C, respectively. In group B, multivariate analysis of prognostic factors showed that a lactate dehydrogenase level more than 2-fold the normal value, no response after COP, and age of at least 15 years were associated with a lower EFS. CNS involvement was the only prognostic factor in group C.

Abstract: To clarify the factors that may affect the peripheral blood stem cell (PBSC) collection in children weighing ≤ 15 kg, a consecutive registry of 109 leukapheresis procedures was analyzed. Collections were performed on a COBE Spectra separator. In 65.1% of the procedures, the peripheral vein, together with a central catheter inserted routinely at diagnosis, or 2 peripheral veins were used to access/return. For 84.4% of the procedures, the extracorporeal line was primed with red blood cells. The median granulocyte‐macrophage colony forming unit (CFU‐GM) number derived from 1 patient's blood volume processed was 13.8 times 104/kg. Six times, a collection series failed, al ways in children treated for ≥ 26 weeks and 4 of those times in children weighing ≤ 11 kg. The patient's age, diagnosis, duration of preleukapheresis treatment, and mobilization regimens did not significantly affect the collection yield. Twenty‐four transplantations were performed. The median times to neutrophils > 0.5× 109/L and platelets > 20 × 109/L were 13 and 20 days, respectively. We conclude that even in very small children, leukapheresis can be performed safely, allowing adequate PBSC collection for transplantation and/or in vitro manipulations.

Standard treatment of localized intracranial germinoma is focal irradiation of the primary tumor (45-50 grays [Gy]) combined with craniospinal radiotherapy (RT). To decrease late effects related to extensive fields of RT, the French Society of Pediatric Oncology decided in 1990 to replace prophylactic RT with chemotherapy (CT) and to deliver focal RT at 40 Gy. Twenty-nine patients with localized, biopsy proven germinoma were included in this study between January 1990 and December 1994. CT consisted of 2 cycles of carboplatin 600 mg/m² on Day 1, etoposide 150 mg/m² on Days 1-3, ifosfamide 1.8 g/m² on Days 22-26, and etoposide 150 mg² on Days 22-24, followed by RT delivered to the initial tumor volume (40 Gy). The median age of the 19 boys and 10 girls was 12.8 years; 25 patients had a unifocal tumor in the pineal (13), suprasellar (10), or thalamic (2) area, and 4 patients had a bifocal tumor. Three patients initially had complete surgery. Of the 26 patients evaluable for CT response, 11 had a small amount of tumor residue and 15 no residue; no patient underwent surgery after CT or RT. One patient recurred 3 years after diagnosis and is in his second complete remission. Twenty-eight patients are in their first complete remission after a median follow-up of 32 months (range, 7-68 months); 9 of the 28 have a small amount of tumor residue that is considered nonevolving. Overall survival at 4 years is 100% and event free survival is 93.3% (± 6%) after a median follow-up of 32 months. This treatment strategy avoids craniospinal RT and reduces focal RT, with results equivalent to those achieved with extensive RT. Thus, the authors consider it a valid treatment of nonmetastatic germinoma. Cancer 1997; 80:1792-7. © 1997 American Cancer Society.

Several genetic mechanisms have been shown to diversify the expressed antibody repertoire of commited B lymphocytes. These include V gene replacement, ongoing gene rearrangement and somatic hypermutation. These mechanisms may be operational at discrete points in the B cell differentiation pathway and generate idiotype diversity in various malignant B cell tumors. In particular, V region mutations have been established as a major mechanism of tumor escape from anti-idiotype immunotherapy in some lymphoma. On the other hand, previous studies on a few selected cases have shown that this mutation process does not affect the B cell clone during chronic lymphocytic leukemia. However, to what extent this intraclonal stability is a general phenomenon during B cell CLL is not clear. Therefore, we randomly selected 6 patients suffering from classical B cell CLL (sIgM (+), CD5 (+), CD19 (+)) at different stages of the disease and analysed the intraclonal variability of the expressed variable region of the heavy chain (VH). After PCR amplification of the cDNA corresponding to the rearranged VDJ regions, the products were cloned and sequenced. In five cases, multiple clone analysis did not show any intraclonal variability whatever the stage of the disease. Furthermore, in a single case, this intraclonal stability was confirmed during a three year period of time when the disease progressed. The sixth case behaved differently since we found multiple nucleotide substitutions, apparently accumulating as the malignant clone expanded. Besides the theoretical difficulties that these changes can induce during immunotherapy, two findings merit further discussion: 1) the distribution of the ongoing mutations affecting the VH region was not suggestive of an antigen driven selection, 2) this intraclonal variability was specific for the VH region, since the VL region showed no intraclonal variation.

Studies in adults have shown that an early molecular response to imatinib predicts clinical outcome in chronic myeloid leukemia (CML). We investigated the impact of the BCR-ABL1 transcript level measured 3 months after starting imatinib in a cohort of 40 children with CML. Children with a BCR-ABL1/ABL ratio higher than 10% at 3 months after the start of imatinib had a larger spleen size and a higher white blood cell count compared with those with BCR-ABL1/ABL ≤10%. Children with BCR-ABL1/ABL ≤10% 3 months after starting imatinib had higher rates of complete cytogenetic response and major molecular response at 12 months compared with those with BCR-ABL1/ABL >10%. With a median follow-up of 71 months (range, 22-96 months), BCR-ABL1/ABL ≤10% correlated with better progression-free survival. Thus, early molecular response at 3 months predicts outcome in children treated with imatinib for CML. This trial was registered at www.clinicaltrials.gov as #NCT00845221.

Background:Hematopoietic stem cell transplant (HSCT) from an HLA identical (HLAid) sibling donor is a well-established curative therapy for sickle cell disease (SCD). However, the ideal age to perform HSCT in SCD patients remains controversial. We report the outcomes after HLAid sibling HSCT for SCD according to patient's age at the time of HSCT as well as their pre-transplant characteristics.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Purpose

Bertrand: ERYTECH Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Walters: ViaCord and AllCells, Inc: Other: Medical director. Bertrand:ERYTECH Pharma: Consultancy. Peters:Medac: Research Funding; Fresenius: Research Funding; Amgen: Research Funding; Jazz: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Sanovi: Research Funding; Pierre-Fabre: Research Funding.

Hematopoietic stem cell transplantation (HSCT) is a therapeutic option for the treatment of relapsed anaplastic large cell lymphoma (ALCL) in children. To date, few pediatric reports have assessed its efficiency and tolerance. While processing the evaluation of new targeted drugs, the impact of HSCT have to be precised in this population.We retrospectively analyzed the data all patients (n=34, median age 7.4y [1.1-17]) registered in the SFGM-TC database, who underwent a HSCT for the treatment of an ALCL ALK+ between 1993 and 2011. Central histology review was carried out for 29/34 patients. Most of patients were treated in specific french protocols (HM, n=4 and ALCL99, n=27). Indications of HSCT were based on the european group EICNHL recommandations for 14 patients : on-therapy relapse (n=1) and CD3 positivity (n=13). For 14 other patients, the decision of HSCT relied on one or several of the following criteria : leukemic form (n=6), post autologous transplantation relapse (n=5) and medullar positivity of the NPM1-ALK fusion transcript at relapse (n=7). In the 6 last patients, none risk factor was identifed. At transplant, 26 patients (65%) were in complete remission (CR) (CR1 n=1, CR2 n=18, CR3 n=4, CR4 n=3) whereas 8 (23,5%) had a detectable disease. Five patients had an history of previous autologous transplantation while 1 patient received 2 subsequent HSCT. Of the 35 HSCT performed, donors were HLA-matched related in 11 cases, mismatched related n=1, matched unrelated n=9 and mismatch unrelated n=14 (including 10 cord blood units). Conditionning regimens were mostly myelo-abaltive (n=30) with the use of a total body irradiation of 12 Gy in 26 patients.Engraftment was observed in 32 patients (median time for neutrophil recovery : 22d [9-44]).With a median follow up of 2.9y [0,1-11,8], 2-year (2y) overall and event-free survivals were respectively 69,6%+-8% and 58,1%+-9%. Cumulative incidences of relapse and non-relapse mortality were 17,8%+-6% and 24,2%+-7% respectively. Six patients (including 3 without CR at transplant) relapsed (median time 108 days [35-235]). Prolounged CR was obtained in 4/6 patients after donor lymphocytes injection (n=1) or Vinblastine-corticosteroid treatment (n=3, with a follow-up of 6y, 2y and 8 months). Twenty-five patients (73.5%) presented an acute GVHD and 5 (14.7%) a chronic GVHD. Eventually, 10 patients died, including 8 of transplant toxicity (5 out of the 8 had been allografted before 2003).This report shows that HSCT is an efficient treatment in pediatric patients with high risk relapse of ALK+ ALCL. However, the high level of treatment-related mortality rises the question of the place of alternative treatments as Vinblastine, ALK or CD30 inhibitors in place of HSCT. When allograft is required, the use of reduced-intensity conditionning could be help reduce toxicity in these heavily pre-treated patients.No relevant conflicts of interest to declare.

Outcomes after RIC allo-SCT in children are rarely reported. The largest series published thus far included 47 children with hematologic malignancies (Pulsipher et al, Blood, 2009). There is thus currently no large series describing the results of RIC allo-SCT in children.

The French pediatric CML working group conducted a retrospective study analysing the growth of patients under 18 years with CML treated with imatinib front-line.Data from 78 patients receiving imatinib (260 mg/m2) for newly diagnosed CML in chronic phase between July 2001 and August 2012 were collected. Height was expressed as standard deviation score (SDS). Height SDS was calculated using French growth standard, as patient height minus mean height for age and sex, divided by standard deviation of height for age and sex. Pre-pubertal age was defined as being younger than 9 years for girls or 11 years for boys.A gradual decrease in the median of the height SDS score is observed over time since the start of imatinib. Sixty three patients were assessable for paired analysis at onset of imatinib and 12 months later. The median height-SDS in this group of patients was significantly lower (p < 10-4) 12 months after the start of imatinib. Variance analysis was performed in 36 children at onset of imatinib, 12 months and 24 months later. The median height-Z score in this group of patients was significantly lower 12 months and 24 months after the start of imatinib (p < 10-4) compared to initial diagnosis. The 63 patients assessable for paired data analysis at onset of imatinib and 12 months later were classified by sex and pubertal status. The median height Z score in boys and girls was significantly (p < 10-4) lower 12 months after the start of imatinib. When the patients were analysed according to the pubertal status at onset of imatinib, the median height SDS score was significantly (p < 10-4) lower 12 months after the start of imatinib in the prepurbertal group as well as in the postpubertal group. A similar finding was observed in the subgroups of boys and girls starting imatinib at a prebubertal or postpubertal age. No significant difference was observed when the median M12 height SDS score in patients who started imatinib at a prepubertal stage or a pospubertal stage were compared. Moreover, no significant difference was observed when the median M12 height-Z score in boys and girls were compared.This retrospective analysis revealed a statistically significant deceleration in growth during the first 2 years of imatinib treatment in children and adolescents with CML and an absence of influence of sex and pubertal status on the depth of growth deceleration.No relevant conflicts of interest to declare.

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.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 (p<0.001). DFS was similar whether the cell source was CB or BM. No lymphoma was observed, and only one post-transplant death at 5m was virus-related (adenoviral meningo-encephalitis). Incidences of AGVH ≥2, 3-4 and chronic-GVHD (cGVHD) were 19%, 6% and 8.7%, respectively. Univariate Cox analysis showed that significant risk factors affecting DFS were low dose of ATG, AGVH grade, and cGVHD, whereas cell source, ABO compatibility, donor’s and recipient’s (DR) age, and CMV status were not. Risk factors associated with cGVHD (Logistic regression) were ATG dose (20% incidence with no ATG, 15.6% with 5-15 mg/kg and only 6.4% with 20), DR age (even after exclusion of CBT), whereas DR genders, DR CMV status, and cell source doses (NC, CD34, CD3+) were not. Multivariate analysis only retained ATG dose (OR=0.9/mg/kg increase, 95% CI:0.8-1, p=0.007) and donor’s age>15 (OR=7.9, 95%CI: 2.5-25.6,p<0.001) as significant independent risk factors for cGVHD. Chimerisms (n=165) (Figure) were significantly associated with ATG dose and cGVHD occurrence. Among the 6 patients with low MC at 1yr, 3 had not received ATG and rejected at 2, 2.2 and 8yr post-HSCT whereas the 3 others who had received ATG remained stable. All patients with high MC at 1yr (n=54) and TDC (n=80) are doing well with the same electrophoretic profile as their donor, excepted one patient who experienced appearance of MC with hemolysis (D/R group U+/U-) 5yr post-CBT, requiring donor lymphocyte infusion (DLI), which reversed chimerism to TDC but induced cGVHD. 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.Bernaudin: Novartis: Research Funding. Bertrand:ERYTECH Pharma: Principal Investigator Other. Gluckman:Cord use: Honoraria; gamida: Honoraria.

Here we report a retrospective analysis based on BCR-ABL transcript level at 3 months after the start of imatinib assessing its impact on subsequent response and outcome in children and adolescents with chronic myeloid leukemia (CML) enrolled in the French prospective trial Glivec Phase 4 (Millot F et al, JCO 2011).

T-cell acute lymphoblastic leukemia (ALL) accounts for 15% of ALL cases in children and has been associated with a higher risk for central nervous system (CNS) relapse and a worse prognosis. In EORTC trials 58831 and 2, standard risk (SR) patients (pts) were not irradiated but received intermediate dose methotrexate (MTX) courses; for medium and high risk pts, high dose (HD) MTX was added to the treatment regimen and the administration of cranial radiotherapy (RT) was randomised. The omission of RT didn't result in an increase of CNS or systemic relapse and consequently, CNS-directed chemotherapy was substituted for RT in all following trials. The long-term outcome of T-ALL pts in the subsequent phase III trials (58881 and 58951) are presented here.The BFM backbone for ALL treatment was applied to all EORTC-CLG trials since 1983. As CNS treatment in study 58881, SR pts received 4 HD MTX courses (5 g/m2) in interval therapy and 10 IT MTX injections during the intensive treatment phases. Pts with CNS-3 status at diagnosis received 2 additional IT injections during induction, 2 during consolidation and 6 HD MTX courses + IT during maintenance. T-ALL pts with poor prephase response (PPR) at day 8 or who didn't achieve complete remission (CR) after induction were included in the very high risk (VHR) group. VHR CNS-directed chemotherapy included 10 IT MTX injections, 6 IT triple and 10 HD MTX courses during intensive treatment phases, followed by 4 IT MTX injections during maintenance (the latter for CNS-3 pts only). In the 58951 trial, all T-ALL pts had an intensified induction. The CNS-directed therapy of all average risk T-ALL pts was intensified to 11 HD MTX courses, 1 IT with MTX and 15 triple IT. MRD ≥ 1% at the end of induction was added as VHR criterium. All non-transplanted VHR pts received 1 IT MTX injection, 19 IT triple and 9 HD MTX courses. Several randomized questions were addressed in both trials of which most relevant for T-ALL pts: in study 58881 the comparison E.coli asparaginase (ASP) Medac versus (vs) “other ASP” (= Erwinia ASP or E.coli ASP Bayer); in trial 58951 1) the comparison dexamethasone (DEX 6 mg/m2/d) vs prednisolone (PRED 60 mg/m2/d) in induction and 2) conventional vs prolonged E.coli ASP for non-VHR pts.303 and 296 T-ALL pts were included in trials 58881 and 58951 resp, representing 14.5% and 15.2% of all pts. Outcome results and type of events for the entire 58881 and 58951 cohorts and according to several subgroups are presented in the table. The 8-year isolated and overall CNS relapse incidences were 6.8% and 10.9% in study 58881, 5.3% and 8.5% in study 58951. The 8-year EFS, DFS and OS improved remarkably in study 58951. In the latter trial, outcome improvement was particularly seen in pts with initial WBC<100x10E9/L and in the good prephase responders (GPR) which had a significant better outcome than those with PPR. 58881 pts assigned to the “other ASP” arm had an inferior outcome. Concerning the DEX/PRED comparison in the 58951 T-ALL cohort, no advantage was seen for EFS (hazard ratio (HR) (99%CI): 1.26 (0.70;2.27)) or OS. There even was a trend towards worse EFS for pts with initial WBC>100x10E9/L and for pts with PPR treated in the DEX arm (HR (99%CI): 1.52 (0.63;3.64) and 1.47 (0.64;3.35)). Prolonged ASP treatment did not improve outcome of the whole T-ALL 58951 cohort.Prophylactic and therapeutic RT can safely be omitted from frontline treatment of children with T-ALL. Adequate ASP therapy, intensified induction treatment and CNS directed therapy can result in a significant improvement of the outcome of at least 2/3rd of T-ALL pts, particularly those with initial WBC<100x10E9/L and GPR.No relevant conflicts of interest to declare.

Asparaginase (ASP) is an essential component in combination chemotherapy for childhood ALL and NHL, as indicated by several randomized trials. However, the optimal number of ASP administrations is still unknown. We conducted a randomized phase III trial comparing conventional E.coli ASP regimen (short-ASP, 12 doses) with prolonged E.coli ASP therapy (long-ASP, 24 doses).The European Organization for Research and Treatment of Cancer Children's Leukemia Group (EORTC-CLG) phase III 58951 trial was open to de novo ALL or NHL patients (pts) < 18 y. This BFM-based study addressed 2 main randomized questions. The first evaluated the value of dexamethasone (DEX, 6mg/m2/d) vs prednisolone (PRED, 60mg/m2/d) in induction for all pts. In the second question all non-very high risk (VHR) pts were randomized for either short- or long-ASP. All patients had to receive 8×10000 U/m2 in induction. In the short-ASP arm pts had to receive 4×10000 U/m2 in late intensification; pts in the long-ASP arm had to receive 8×5000 U/m2E.coli ASP injections in consolidation and 8 (4×10000 U/m2 + 4×5000U/m2) in late intensification. Patients with grade ≥2 allergy to E.coli ASP had to be switched to equivalent doses of Erwinia or PEG ASP. Central randomization was stratified by the 1st randomized arm, risk group (VLR, AR1, AR2) and center. The primary endpoint of the study was disease-free survival (DFS), secondary endpoints were overall survival (OS) and toxicity. Intention-to-treat analysis was performed.Between December 1998 and August 2008, 2038 patients were randomized for the 1st question and 1552 pts, ALL (n=1481) and NHL (n=71), were randomly assigned to receive long-ASP (n=775) or short-ASP (n=777). At a median follow-up of 7 years there were 97 vs 112 events in the long- vs short-ASP group (see table). The 8-year DFS rate was 87.0% in the long-ASP and 84.2% in short-ASP group (hazard ratio (HR) = 0.87, 95% CI 0.66–1.14, 2-sided logrank p=0.30). The 8-year OS rate was comparable in both treatment arms: 92.6% in the long-ASP group and 91.3% in the short-ASP group (HR = 0.89, 95% CI 0.61–1.29, 2-sided log rank p=0.53). Similar treatment differences were observed in each risk group, in randomized arm (PRED vs DEX), and B- and T-lineage ALL pts.The incidence of grade 3–4 infection was higher in the long- vs short-ASP group during consolidation (25.2% vs 14.5%) and late intensification (22.6% vs 15.9%). This difference was more pronounced in pts who were randomly assigned to DEX (see table). In the long- vs short-ASP group grade 2–4 allergy to ASP was 22.5% vs 0.3% in consolidation and 10.3% vs 21.5% in late intensification. During the whole treatment period, the incidence of grade 2–4 allergy was 30.5% in the long-ASP arm and 21.7% in the short-ASP arm. In the long- vs short-ASP arm approximately 67% vs 95% pts received at least the total number of E.coli or equivalent ASP administrations as planned according to the treatment arm.At long follow-up (median= 7 yrs) prolonged E.coli asparaginase therapy in consolidation and late intensification for VLR and AR pts did not improve significantly the outcome. Intensive ASP treatment did increase infection rate in consolidation and late intensification and resulted in more grade 2–4 allergic reactions. In the future, we aim to improve outcome rates by the use of PEG ASP and monitoring of asparaginase activity and antibody formation.No relevant conflicts of interest to declare.

Abstract 134

No relevant conflicts of interest to declare.

The first EORTC-CLG AML pilot study (58872) demonstrated the efficacy of Mitoxantrone (MTZ), substituted for daunorubicin, in the treatment of childhood AML. The subsequent trial (58921) aimed to compare MTZ and Idarubicin (IDA), an anthracycline with a favorable pharmacokinetic profile (such as good CSF penetration) and suggested to be more efficacious than daunorubicin in adult AML trials performed in the last two decades.Between March 1993 and December 2002, 227 eligible patients (pts) <18y of age with newly diagnosed AML (N=216) or high risk MDS (RAEB & RAEBt, N=11) were randomized in the EORTC phase III 58921 trial to receive either IDA or MTZ in induction and 1st intensification course, each at a dose of 10 mg/m2/d for 3 days in both courses. Concomitant chemotherapy consisted of standard dose Cytarabine (AraC) and Etoposide (Eto) in induction and high-dose AraC (18–36 g/m2) in 1st intensification. Allogeneic stem cell transplantation after 1st intensification was recommended for pts who achieved CR and had an HLA-identical sibling donor. Patients in CR without donor had to receive a 2nd (DCTER = continuous infusion of standard dose AraC, Daunorubicin 4x 20 mg/m2/d, Eto, Dexamethasone, 6-thioguanine) and 3rd intensification course (high dose AraC 12 g/m2 + Eto), followed by maintenance therapy (6-Thioguanine daily + AraC 4 days/month) for 12 months. CNS irradiation after the 3rd intensification for patients with initial WBC counts >=70×10E9/L was abandoned from November 1994 on. Randomization was done centrally. The primary endpoint was EFS; secondary endpoints were OS, CR rate after induction/1st intensification, DFS and toxicity. Intention-to-treat analysis was used.A total of 112 and 115 eligible pts were randomly assigned to receive IDA and MTZ, respectively. The rate of CR after two courses was 79.5% (IDA) vs 85.2% (MTZ). At an overall median follow-up of 9.9 y (range 0.25–16 y), there were 65 vs 59 events in the IDA vs MTZ group: failure to achieve CR (23 vs 17), relapse (35 vs 40), and death without relapse (7 vs 2). The 5-year EFS rate was 42.0% (SE 4.7%) in the IDA group and 48.4% (SE 4.7%) in the MTZ group (hazard ratio (HR) = 1.20, 95% CI 0.84–1.71, 2-sided log rank p=0.29). The 5-year OS rate was comparable in both treatment arms: 59.8% (SE 4.6%) in the IDA group and 57.5% (SE 4.7%) in the MTZ group (HR = 1.03, 95% CI 0.70–1.54, 2-sided log rank p=0.87).In CR patients with (N=46/187) or without (N=141/187) an HLA-identical sibling donor, the 5-year DFS rate from CR was 65.1% (SE 7.1%) and 51.5% (SE 4.2%) respectively (HR=0.65, 95% CI 0.37–1.11, 2-sided log rank p=0.11) and the 5-year OS rate 78.0% (SE 6.1%) and 60.7% (SE 4.1%) respectively (HR=0.53, 95% CI 0.28–1.01, 2-sided log rank p=0.048). This advantage for patients with a donor remained important regarding both DFS (HR=0.60, p=0.07) and OS (HR=0.49, p=0.03), even after adjustment for WBC count at diagnosis, age, cytogenetic features and randomized arm.The interval between start of induction and start of 1st intensification was similar in both arms (median 5.2 weeks). Grade 3–4 infection following the induction course was 37.5% (IDA) vs 25.4% (MTZ); incidence of fever grade 3–4 was 25% (IDA) vs 22.8% (MTZ). In this trial, the cumulative anthracycline dosage (conversion factor 5) was 380 mg/m2. Acute and late-onset cardiotoxicity was comparable in both treatment arms.There was no significant difference in efficacy and in toxicity between the two randomized treatment groups, IDA versus MTZ, although grade 3–4 infection rate following the induction course was slightly higher in the IDA arm. Patients who reached CR and who had a HLA compatible sibling donor had a longer DFS and OS than pts without a donor.No relevant conflicts of interest to declare.

In our previous EORTC 58881 trial in childhood ALL, we have shown the prognostic impact of the MRD, quantitated by polymerase chain reaction assay. Patients with TP1 MRD ≥10–2 (D35), had a higher risk of relapse than those with TP1 MRD<10-2 (Cavé et al, NEJM, 1998). For these patients hematopoietic stem cell transplantation (HSCT) with an HLA matched donor was recommended in our subsequent EORTC 58951 trial. Here we report on the patients with MRD ≥10-2 (TP1) in the 58951 trial.Between December 1998 and July 2008, 1955 children (1y-18y) with ALL were enrolled in the 58951 trial, 1459 being evaluable for the MRD (TP1) by PCR (rearrangements of the T-cell receptor and immunoglobulin heavy chain genes). Seventy-nine patients with Phi-negative ALL had a TP1 MRD ≥10-2, and 69 of them achieved a complete hematological remission after induction. Here we report on the outcome of these 69 patients.At a median follow-up of 7.6 years, the overall 5-years EFS and OS rates for the 69 patients were 56.1% (SE 6.0%) and 62.0% (SE 5.9%) respectively. Prephase good responders (PGR) (<1000 blasts/mm3 D8) (N=40) had a better OS rate as compared to prephase poor responders (PPR) (N=29): 72.2% (SE 7.1%) vs 48.0% (SE 9.3%) (hazard ratio=0.43, p = 0. 002). In B-lineage ALL patients the OS was similar between PGR (N=32) and PPR (N=6) subgroups, whereas in T-ALL patients there was a better OS for PGR (N=8) than for PPR (N=23) subgroup. Of note, all the patients with hyperdiploidy > 51 chromosomes were PGR.TP2 MRD (after consolidation) was assessed in 59 pts. All patients but one (1/13) with TP2 MRD ≥10-2 died. In PGR patients, those with TP2 MRD < 10-3 (N=18) had a significant better OS rate at 5 years than those with TP2 MRD ≥10-3 (N=17): 88.9% (SE 7.4%) vs 52.3 % (SE 12.3%), hazard ratio=0.17, p=0.009. We observed no relapses among patients with hyperdiploidy > 51 chromosomes and TP2 <10-3. PPR patients had a poor 5-year OS, independent of TP2 level (MRD <10-3 (N=8) or MRD ≥10-3 (N=16)).The prognosis of children ALL with TP1 MRD ≥10-2 has improved with early intensification of the treatment as compared to the one observed in our previous 58881 study. However the outcome of these patients was still heterogeneous and the prephase response was still of prognostic importance. Patients with GPR and TP2 <10-3 had a favourable prognosis and for those patients HSCT is not mandatory.For other patients with PPR and/or TP2 ≥10-3 the prognosis was worse and these patients may benefit from alternative therapy and/or intensification with HSCT.No relevant conflicts of interest to declare.

There is increasing information about reduced intensity conditioning regimen AlloHSCT (Allogeneic Hematopoietic Stem Cell Transplantation) in children. The safety of this approach is now well established but data regarding efficacy are limited and the role in pediatric cancer has yet to be defined.We report results of a French pediatric AlloHSCT protocol with ATG-fludarabine (180 mg/m2) - Busilvex (3.2 at 4.8 mg/kg/d for 2 days) conditioning regimen. Related, unrelated bone marrow (BM) and Peripheral blood stem cell (PBSC) donors and Cord blood units (CB) were allowed. In case of CB a TBI 2 grays, Cyclophosphamide 50 mg/kg, Fludarabine 100 mg/m2 conditioning regimen was recommended. GVH prophylaxis consists in cyclosporine alone. A rapid discontinuation of systemic immunosuppression and re-injecting donor lymphocytes to initiate graft-versus-tumor effect are based on tumor assessment and blood chimerism. Inclusion criteria are children with malignancies that can be potentially cure by allograft but a conventional conditioning regimen being impossible due to toxicity and children with solid tumor or hematological malignancy remaining unresponsive to the reference strategies according to French best practices in pediatrics.From April 2007 to April 2010, 40 RIC AlloHSCT were performed in 10 different French pediatric graft centers: 13 Hodgkin Lymphoma, 7 acute myeloblastic leukaemia, 2 acute lymphoblastic leukaemia, 6 neuroblastoma, 8 rhabdomyosarcoma, 3 desmoplastic tumor and 1 Ewing sarcoma. Median age at transplantation was 15 years and median time from diagnosis to transplant was 18 months. Before transplant, 15 patients are in complete response and 25 patients (14/18 solid tumors) have active disease (11 progressive, 14 partial response). 21 had already received a myeloablative therapy (18 autograft and 3 allograft). Graft source was PBSC in 17 cases (7 related and 10 unrelated), BM in 18 (10 related and 8 unrelated), and 5 CB. The RIC Bu-flu conditioning regimen permits rapid engraftment without major toxicity contrary to the Cy-TBI in CB. 1 patient had primary graft failure: 1 CB and 5 patients experienced secondary graft failure: 3 CB, 1 PBSC and 1 BM. Median time to reach an ANC of 0.5 109/l was 16 days. Median time to reach a platelet count of 20 × 109/l was 2 days. Platelet count did not decrease below 20 109/l in 10 allografts. At day 30 post-transplant, chimerism is mainly donor for 30 and partial for 6 children. At day 100 post transplant, 4 out of 6 with initial mixed chimerism were converted into full donor chimerism. 8 patients received DLI and 17 patients experienced acute graft versus host disease (GvH) (2 grade IV and 15 grade ≤ II). A low day 365 TRM of 5% is reported in these heavily pre-treated patients. With a median follow-up of 15 months, the estimated 2 yr overall survival (OS) was respectively 57 % (71% for hematological malignancies and 42% solid tumors) (fig 1) and event free survival (EFS) 36% (50% for hematological malignancies and 19% solid tumors). Univariate analysis of EFS and OS showed no effect of related versus unrelated stem cell sources and BM versus PBSC. Our analysis identified a group of patients, who had no measurable disease at transplant, with a 2 yr OS and EFS of 86%. In term of efficacy, we observe a graft versus lymphoma effect in patients with advanced active Hodgkin lymphoma. Concerning solid tumors, all children included had a very bad prognosis and detectable disease before transplant. Our results may suggest that an immune-mediated effect cannot be excluded in some refractory solid pediatric tumors particularly in neuroblastoma. The main cause of failure of this approach is disease progression. Immunologic approaches after transplantation may help cure more of these very-high-risk patients.Even if further follow up is needed, this prospective study suggest that RIC regimen provides promising outcome in children previously not eligible for myeloablative AlloHSCT.This study “RICE” was registered at www.clinicaltrials.gov as NCT 007 50 126No relevant conflicts of interest to declare.

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.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.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<0.001) and platelets reached 50,000/mm3 sooner after BMT (day 28.3 ± 16.6) than after CBT (day 48.5 ± 20.3; p<0.001). No engraftment occurred in 3 cases (1 BMT, 2 CBT) with gradual autologous reconstitution, and one rejection was observed 3 years post-transplant. GvHA grade ≥ II occurred in 23% of patients, GvHA ≥ III in 4.4%, chronic GvH in 9.6% (extensive in 3 cases). No GvHD ≥ II or chGvHD were observed after CBT. Death occurred in 6 cases (4 were GvHD-related, 1 hemorrhagic stroke in a patient with severe cerebral vasculopathy with Moya and 1 sepsis in aplasia). No veno-occlusive disease was observed, but hemorrhagic cystitis (n=4), EBV proliferative disease requiring anti-CD20 therapy (n=1), nephrotic syndrome (n=1), and cerebral vasculopathy not SCA-related (n=1) were. Despite preventive measures such as anticonvulsant prophylaxis, strict control of hypertension, swift magnesium replacement, and an increase in the red blood cell and platelet transfusion thresholds to 9 g/dL and 50,000/mm3, respectively, seizures and posterior leukoencephalopathy, albeit reversible, remained a particularly frequent adverse effect of CSA and steroid therapy. Replacing CSA in 2002 by mycophenolate mofetil in case of GvHD requiring steroid therapy resulted in a significant reduction of the rate of these complications. With a median follow-up of 3.1 years (range 0.2–15.5), the overall survival at 3 yr was 95% (95%CI:91-99%). Considering as events the non-engraftments, rejections and deaths, the event-free survival (EFS) was 92.9% (95%CI:88.3-97.5). However, comparing the results before (n=23) and after 2000 (n=121) showed significant improvement of EFS at 3 yr: 73.9% (95%CI: 55.5–92.3) for transplants performed before 2000 vs 96.8% (95%CI:93.2-100) after 2000.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.No relevant conflicts of interest to declare.