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Abstract Hypodense volumes (HDV) in mediastinal masses can be visualized in a computed tomography scan in Hodgkin lymphoma. We analyzed staging CT scans of 1178 patients with mediastinal involvement from the EuroNet-PHL-C1 trial and explored correlations of HDV with patient characteristics, mediastinal tumor volume and progression-free survival. HDV occurred in 350 of 1178 patients (29.7%), typically in larger mediastinal volumes. There were different patterns in appearance with single lesions found in 243 patients (69.4%), multiple lesions in 107 patients (30.6%). Well delineated lesions were found in 248 cases (70.1%), diffuse lesions were seen in 102 cases (29.1%). Clinically, B symptoms occurred more often in patients with HDV (47.7% compared to 35.0% without HDV (p = 0.039)) and patients with HDV tended to be in higher risk groups. Inadequate overall early-18F-FDG-PET-response was strongly correlated with the occurrence of hypodense lesions (p 40 ml (n = 80) had a 5 year PFS of 79.6% compared to 89.7% (p = 0.01) in patients with HDV

BackgroundSeronegative autoimmune hepatitis in children is a rare but potentially severe disease, sometimes requiring liver transplantation. This type of hepatitis may be associated with various immunological and hematological disorders, ranging from isolated lymphopenia to aplastic anemia. Precise pathophysiological mechanisms are still unknown, but the role of viruses cannot be excluded, either as directly pathogenic or as triggers, responsible for an inappropriate immune stimulation. Having the impression of an increasing number of seronegative autoimmune hepatitis since the beginning of SARS-CoV-2 pandemia period, we hypothesized that SARS-CoV-2 virus could be an infectious trigger.MethodsWe conducted a retrospective, observational, descriptive study about children with seronegative autoimmune hepatitis, in a tertiary care center, between 2010 and 2022.ResultsThirty-two patients were included. The overall incidence of seronegative autoimmune hepatitis increased 3.3-fold in 2020-2022, during the SARS-CoV-2 pandemia period (16 patients in 2.8 years) compared with 2010-2019 the pre pandemia period (16 patients in 9 years). Patients’ clinical and biochemical liver characteristics did not differ between the two periods. Hematological damages were less severe during the pandemia period. Immunological studies revealed a dysregulated immune response. The initiation of immunosuppressive therapy (corticosteroids ± cyclosporine) was earlier during the pandemia period than before.ConclusionIn cases of undetermined acute hepatitis, an immune-mediated origin should be considered, prompting a liver biopsy. If the histological aspect points to an immune origin, immunosuppressive treatment should be instituted even though autoimmune hepatitis antibodies are negative. Close hematological monitoring must be performed in all cases. The 3.3-fold increase of cases during the SARS-CoV-2 pandemia will need to be further analyzed to better understand the underlying immunological mechanisms, and to prove its potential involvement.

Not available.

Abstract Eosinophilic-related clinical manifestations are protean and the underlying conditions underpinning eosinophilia are highly diverse. The etiological workup of unexplained eosinophilia/hypereosinophilia can be challenging, and can lead sometimes to extensive, inappropriate, costly and/or invasive investigations. To date, guidelines for the etiological workup and management of eosinophilia are mainly issued by hematologists, and thus mostly cover the scope of clonal hypereosinophilic syndromes (HES). Here, thanks to an extensive literature review, and thanks to the joint work of a large panel of experts involving physicians from both adult and pediatric medicine and from various subspecialties (as well as a representative of a patients’ association representative), we provide recommendations for both the step-by step diagnostic workup of eosinophilia (whether unexplained or within specific contexts) as well as the management and follow-up of the full spectrum of eosinophilic disorders (including clonal, reactive, lymphocytic and idiopathic HES, as well as single-organ diseases). Didactic prescription summaries intended to facilitate the prescription of eosinophil-targeted drugs are also provided, as are practical diagnostic and therapeutic algorithms. Lastly, this set of recommendations also includes a summary intended for general practitioners, as well as an overview of the therapeutic patient education program set up by the French reference center for HES. Further updates will be mandatory as new validated information emerges.

Current immune thrombocytopenia (ITP) guidelines target children and adults, leading to oversimplification. Adolescents and young adults (AYAS) comprise a separate group with distinct health and psychosocial issues. This study aimed to describe the clinical presentation and therapeutic strategies of ITP among AYAS. We analyzed data from two large ITP registries (PARC-ITP; CARMEN-France) and included newly diagnosed ITP patients (aged 12–25 years) with an initial platelet counts of

Abstract This study quantifies the association between the COVID‐19 economic downturn and 2020 tropospheric ozone anomalies above Europe and western North America, and their impact on long‐term trends. Anomaly detection for an atmospheric time series is usually carried out by identifying potentially aberrant data points relative to climatological values. However, detecting ozone anomalies from sparsely sampled ozonesonde profiles (once per week at most sites) is challenging due to ozone's high temporal variability. We first demonstrate the challenges for summarizing regional trends based on independent time series from multiple nearby ozone profiling stations. We then propose a novel regional‐scale anomaly detection framework based on generalized additive mixed models, which accounts for the sampling frequency and inherent data uncertainty associated with each vertical profile data set, measured by ozonesondes, lidar or commercial aircraft. This method produces a long‐term monthly time series with high vertical resolution that reports ozone anomalies from the surface to the middle‐stratosphere under a unified framework, which can be used to quantify the regional‐scale ozone anomalies during the COVID‐19 economic downturn. By incorporating extensive commercial aircraft data and frequently sampled ozonesonde profiles above Europe, we show that the complex interannual variability of ozone can be adequately captured by our modeling approach. The results show that free tropospheric ozone negative anomalies in 2020 are the most profound since the benchmark year of 1994 for both Europe and western North America, and positive trends over 1994–2019 are diminished in both regions by the 2020 anomalies.

Primary mediastinal large B-cell lymphoma (PMLBL) is a rare entity predominantly affecting adolescents and young adults. Recently, an international phase II trial in pediatric patients using dose-adjusted etoposide, doxorubicin, and cyclophosphamide with vincristine and prednisone plus rituximab (DA-EPOCH-R) failed to reproduce excellent survival reported in some adult studies. The optimal therapy regimen needs to be determined in this disease. The French prospective LMB2001 trial included all patients ≤18 years with mature B-cell lymphoma treated in French centers. For patients with PMLBL, treatment included four to eight courses of Lymphomes Malins B (LMB)-based chemotherapy without radiotherapy. From 2008, rituximab was added before each chemotherapy course. From 09/2001 to 03/2012, 42 patients with PMLBL were registered. The median age was 15 years (range, 8-18). Twenty-one patients were treated with chemotherapy plus rituximab. The median follow-up was 7.1 years (interquartile range, 5.8-11.1). Five-year event-free and overall survival were 88.1% (95% confidence interval (CI): 75.0-94.8) and 95.2% (95% CI: 84.0-98.7) for the whole population. The 5-year EFS was 81.0% (95% CI: 60.0-92.3) and 95.2% (95% CI: 77.3-99.2) (hazard ratio =0.24; 95% CI: 0.03- 2.2) and 5-year overall survival was 90.5% (95% CI: 71.1-97.3) and 100% for patients treated without and with rituximab, respectively. Only one of 21 patients treated with rituximab and LMB-based chemotherapy had local early treatment failure but achieved prolonged complete remission with second-line chemotherapy and radiotherapy. Intensive LMBbased chemotherapy with rituximab achieved excellent survival in children/adolescents with PMLBL. Further international prospective studies are required to confirm these results in this population.

The Stratospheric Ozone Differential Absorption Lidar (DIAL) located at the Polar Environment Atmospheric Research Laboratory (PEARL) in Eureka, Nunavut (80°N, 86°W) has been a powerful tool for the measurement of stratospheric ozone vertical profiles in the Canadian High Arctic since 1993. The lidar ozone profiles measured during the 2017 Canadian Arctic Atmospheric Chemistry Experiment (ACE) and Optical Spectrograph and InfraRed Imaging System (OSIRIS) Validation Campaign were compared to coincident ozonesonde, ACE-FTS, ACE-MAESTRO and OSIRIS profiles. The results show the lidar overestimates the ozone by ∼10% in the 10 km–20 km altitude range on average in comparison with the ozonesonde. Above 20 km the profiles agree within 10%. The OSIRIS ozone agrees within 10% with DIAL ozone between 15 km and 41 km. A strong stratospheric ozone depletion event was seen on March 3, 2017, during which the ozone concentration dropped below 1012 molecules cm–3 at the 15.5 km and below 6 × 1012 molecules cm–3 at 16 km as measured by the ozonesonde and the lidar correspondingly. Laminated structures were observed in the ozonesonde profiles near the polar vortex edge regions. This together with non-optimal temporal and spatial coincidences between the measurements conducted by different instruments can affect validation accuracy.

Pediatric-onset Evans syndrome (pES) is defined by both immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) before the age of 18 years. There have been no comprehensive long-term studies of this rare disease, which can be associated to various immunopathological manifestations (IM). We report outcomes of the 151 patients with pES and more than 5 years of follow-up from the nationwide French prospective OBS’CEREVANCE cohort. Median age at final follow-up was 18.5 years (range, 6.8–50.0 years) and the median follow-up period was 11.3 years (range, 5.1–38.0 years). At 10 years, ITP and AIHA were in sustained complete remission in 54.5% and 78.4% of patients, respectively. The frequency and number of clinical and biological IM increased with age: at the age of 20 years, 74% had at least one clinical IM (cIM). A wide range of cIM occurred, mainly lymphoproliferation, dermatological, gastrointestinal/hepatic and pneumological IM. The number of cIM was associated with a subsequent increase in the number of second-line treatments received (other than steroids and immunoglobulins; hazard ratio 1.4, 95% Confidence Interval: 1.15–1.60, P=0.0002, Cox proportional hazards method). Survival at 15 years after diagnosis was 84%. Death occurred at a median age of 18 years (range, 1.7–31.5 years), and the most frequent cause was infection. The number of second-line treatments and severe/recurrent infections were independently associated with mortality. In conclusion, long-term outcomes of pES showed remission of cytopenias but frequent IM linked to high second-line treatment burden. Mortality was associated to drugs and/or underlying immunodeficiencies, and adolescents-young adults are a high-risk subgroup.

Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia primarily caused by pathogenic germline variants in ribosomal protein genes. DBA due to pathogenic RPL35A variants has been associated with large 3q29 deletions and phenotypes not common in DBA. We conducted a multi-institutional genotype-phenotype study of 45 patients with DBA associated with pathogenic RPL35A germline variants and curated the variant data on 21 additional cases from the literature. Genotype-phenotype analyses were conducted comparing patients with large deletions versus all other pathogenic variants in RPL35A. Twenty-two of the 45 cases had large deletions in RPL35A. After adjusting for multiple tests, a statistically significant association was observed between patients with a large deletion and steroid-resistant anemia, neutropenia, craniofacial abnormalities, chronic gastrointestinal problems, and intellectual disabilities (p

Abstract. The objective of this guideline is to aid clinicians in making individual salvage treatment plans for pediatric and adolescent patients with first relapse or refractory (R/R) classical Hodgkin lymphoma (cHL). While salvage with standard dose chemotherapy followed by high dose chemotherapy and autologous stem cell transplant is often considered the standard of care in adult practice, pediatric practice adopts a more individualized risk stratified and response adapted approach to salvage treatment with greater use of non-transplant salvage. Here, we present on behalf of the EuroNet Pediatric Hodgkin Lymphoma group, evidence and consensus-based guidelines for standardized diagnostic, prognostic and response procedures to allocate children and adolescents with R/R cHL to stratified salvage treatments.

Abstract: Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation–dependent DBA.

Abstract: T-cell acute lymphoblastic leukemia (T-ALL) expands in various bone marrow (BM) sites of the body. We investigated whether different BM sites could differently modulate T-ALL propagation using in vivo animal models. We observed that mouse and human T-ALL develop slowly in the BM of tail vertebrae compared with the BM from thorax vertebrae. T-ALL recovered from tail BM displays lower cell-surface marker expression and decreased metabolism and cell-cycle progression, demonstrating a dormancy phenotype. Functionally, tail-derived T-ALL exhibit a deficient short-term ex vivo growth and a delayed in vivo propagation. These features are noncell-autonomous because T-ALL from tail and thorax shares identical genomic abnormalities and functional disparities disappear in vivo and in prolonged in vitro assays. Importantly tail-derived T-ALL displays higher intrinsic resistance to cell-cycle–related drugs (ie, vincristine sulfate and cytarabine). Of note, T-ALL recovered from gonadal adipose tissues or from cocultures with adipocytes shares metabolic, cell-cycle, and phenotypic or chemoresistance features, with tail-derived T-ALL suggesting adipocytes may participate in the tail BM imprints on T-ALL. Altogether these results demonstrate that BM sites differentially orchestrate T-ALL propagation stamping specific features to leukemic cells such as quiescence and decreased response to cell-cycle–dependent chemotherapy.

From the earliest observations of ozone in the lower atmosphere in the 19th century, both measurement methods and the portion of the globe observed have evolved and changed. These methods have different uncertainties and biases, and the data records differ with respect to coverage (space and time), information content, and representativeness. In this study, various ozone measurement methods and ozone datasets are reviewed and selected for inclusion in the historical record of background ozone levels, based on relationship of the measurement technique to the modern UV absorption standard, absence of interfering pollutants, representativeness of the well-mixed boundary layer and expert judgement of their credibility. There are significant uncertainties with the 19th and early 20th-century measurements related to interference of other gases. Spectroscopic methods applied before 1960 have likely underestimated ozone by as much as 11% at the surface and by about 24% in the free troposphere, due to the use of differing ozone absorption coefficients. There is no unambiguous evidence in the measurement record back to 1896 that typical mid-latitude background surface ozone values were below about 20 nmol mol–1, but there is robust evidence for increases in the temperate and polar regions of the northern hemisphere of 30–70%, with large uncertainty, between the period of historic observations, 1896–1975, and the modern period (1990–2014). Independent historical observations from balloons and aircraft indicate similar changes in the free troposphere. Changes in the southern hemisphere are much less. Regional representativeness of the available observations remains a potential source of large errors, which are difficult to quantify. The great majority of validation and intercomparison studies of free tropospheric ozone measurement methods use ECC ozonesondes as reference. Compared to UV-absorption measurements they show a modest (~1–5% ±5%) high bias in the troposphere, but no evidence of a change with time. Umkehr, lidar, and FTIR methods all show modest low biases relative to ECCs, and so, using ECC sondes as a transfer standard, all appear to agree to within one standard deviation with the modern UV-absorption standard. Other sonde types show an increase of 5–20% in sensitivity to tropospheric ozone from 1970–1995. Biases and standard deviations of satellite retrieval comparisons are often 2–3 times larger than those of other free tropospheric measurements. The lack of information on temporal changes of bias for satellite measurements of tropospheric ozone is an area of concern for long-term trend studies.

Abstract. Despite significant progress in the treatment of pediatric acute myeloblastic leukemia (AML), relapse remains the commonest cause of death. Randomized ELAM02 trial questioned if maintenance therapy with interleukin-2 (IL2), for 1 year, improves disease-free survival (DFS). Patients aged 0 to 18 years, with newly diagnosed AML (excluding patients with acute promyelocytic leukemia or down syndrome AML) were enrolled. They received 1 course of induction treatment (cytarabine and mitoxantrone) and 3 courses of consolidation treatment (high-dose cytarabine in courses 1 and 3). According to the cytogenetics risk, patients not undergoing hematopoietic stem cell transplantation, still in complete remission (CR) after the third course of consolidation treatment, were eligible for randomization to 1 year of maintenance therapy with monthly courses of IL2 or no maintenance treatment. There were 438 evaluable patients, 154 of whom were randomized to the IL2/no maintenance groups. Relapse occurred in 28 patients from the IL2+ group and 29 patients in the IL2− group. Survival was similar in the 2 groups, with a 4-year DFS of 62% without IL2 and 66% with IL2 (P = 0.75). In the CBF population, 4-year DFS was 55% without IL2 and 78% with IL2 (P = 0.07). No deaths from toxicity or excess of serious adverse events related to IL2 treatment were recorded. Prolonged IL2 for maintenance therapy after intensive chemotherapy is feasible and safe in pediatric AML patients in their first CR. Such treatment did not improve DFS in this study, but a positive trend was observed in favor of IL2 maintenance therapy among core binding factor acute myeloblastic leukemia.

Abstract: Telomeres are repetitive hexameric sequences located at the end of linear chromosomes. They adopt a lariat-like structure, the T-loop, to prevent them from being recognized as DNA breaks by the DNA repair machinery. RTEL1 is a DNA helicase required for proper telomere replication and stability. In particular, it has been postulated that RTEL1 is involved in the opening of the T-loop during telomere replication to avoid sudden telomere deletion and telomere circle (T-circle) formation. In humans, biallelic RTEL1 mutations cause Hoyeraal-Hreidarsson syndrome (HH), a rare and severe telomere biology disorder characterized by intrauterine growth retardation, bone marrow failure, microcephaly and/or cerebellar hypoplasia, and immunodeficiency. To date, 18 different RTEL1 mutations have been described in 19 cases of HH with short telomeres. The impaired T-loop resolution has been proposed to be a major cause of telomere shortening in RTEL1 deficiency. However, the biological and clinical consequences of this disorder remain incompletely documented. Here, we describe 4 new patients harboring biallelic RTEL1 mutations, including 2 novel missense mutations located in the C-terminal end of RTEL1 (p.Cys1268Arg and p.Val1294Phe). Clinical characteristics from these 4 patients were collected as those from 4 other RTEL1-deficient patients previously reported. In addition, we assessed whether T-circles, the product of improper T-loop resolution, were detected in our RTEL1-deficient patients. Overall, our study broadens and refines the clinical and biological spectrum of human RTEL1 deficiency.

In this paper, we compare model calculations of ozone profiles and their variability for the period 1998 to 2016 with satellite and lidar profiles at five ground-based stations. Under the investigation is the temporal impact of the stratospheric halogen reduction (chemical processes) and increase in greenhouse gases (i.e., global warming) on stratospheric ozone changes. Attention is given to the effect of greenhouse gases on ultraviolet-B radiation at ground level. Our chemistry transport and chemistry climate models (Oslo CTM3 and EMAC CCM) indicate that (a) the effect of halogen reduction is maximized in ozone recovery at 1−7 hPa and observed at all lidar stations; and (b) significant impact of greenhouse gases on stratospheric ozone recovery is predicted after the year 2050. Our study indicates that solar ultraviolet-B irradiance that produces DNA damage would increase after the year 2050 by +1.3% per decade. Such change in the model is driven by a significant decrease in cloud cover due to the evolution of greenhouse gases in the future and an insignificant trend in total ozone. If our estimates prove to be true, then it is likely that the process of climate change will overwhelm the effect of ozone recovery on UV-B irradiance in midlatitudes.

Heterozygous germline GATA2 mutations strongly predispose to leukemia, immunodeficiency, and/or lymphoedema. We describe a series of 79 patients (53 families) diagnosed since 2011, made up of all patients in France and Belgium, with a follow up of 2249 patients/years. Median age at first clinical symptoms was 18.6 years (range, 0-61 years). Severe infectious diseases (mycobacteria, fungus, and human papilloma virus) and hematologic malignancies were the most common first manifestations. The probability of remaining symptom-free was 8% at 40 years old. Among the 53 probands, 24 had missense mutations including 4 recurrent alleles, 21 had nonsense or frameshift mutations, 4 had a whole-gene deletion, 2 had splice defects, and 2 patients had complex mutations. There were significantly more cases of leukemia in patients with missense mutations (n=14 of 34) than in patients with nonsense or frameshift mutations (n=2 of 28). We also identify new features of the disease: acute lymphoblastic leukemia, juvenile myelomonocytic leukemia, fatal progressive multifocal leukoencephalopathy related to the JC virus, and immune/inflammatory diseases. A revised International Prognostic Scoring System (IPSS) score allowed a distinction to be made between a stable disease and hematologic transformation. Chemotherapy is of limited efficacy, and has a high toxicity with severe infectious complications. As the mortality rate is high in our cohort (up to 35% at the age of 40), hematopoietic stem cell transplantation (HSCT) remains the best choice of treatment to avoid severe infectious and/or hematologic complications. The timing of HSCT remains difficult to determine, but the earlier it is performed, the better the outcome.

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

Few therapeutic options are available for patients with aplastic anemia who are ineligible for transplantation or refractory to immunosuppressive therapy. Eltrombopag was recently shown to produce trilineage responses in refractory patients. However, the effects of real-life use of this drug remain unknown. This retrospective study (2012–2016) was conducted by the French Reference Center for Aplastic Anemia on patients with relapsed/refractory aplastic anemia, and patients ineligible for antithymocyte globulin or transplantation, who received eltrombopag for at least 2 months. Forty-six patients with aplastic anemia were given eltrombopag without prior antithymocyte globulin treatment (n=11) or after antithymocyte globulin administration (n=35) in a relapsed/refractory setting. Eltrombopag (median daily dose 150 mg) was introduced 17 months (range, 8–50) after the diagnosis of aplastic anemia. At last followup, 49% were still receiving treatment, 9% had stopped due to a robust response, 2% due to toxicity and 40% due to eltrombopag failure. Before eltrombopag treatment, all patients received regular transfusions. The overall rates of red blood cell and platelet transfusion independence were 7%, 33%, 46% and 46% at 1, 3, 6 months and last follow-up. Responses were slower to develop in antithymocyte treatment-naïve patients. In patients achieving transfusion independence, hemoglobin concentration and platelet counts improved by 3 g/dL (interquartile range, 1.4–4.5) and 42×109/L (interquartile range, 11–100), respectively. Response in at least one lineage (according to National Institutes of Health criteria) was observed in 64% of antithymocyte treatment-naïve and 74% of relapsed/refractory patients, while trilineage improvement was observed in 27% and 34%, respectively. We found high rates of hematologic improvement and transfusion independence in refractory aplastic anemia patients but also in patients ineligible for antithymocyte globulin receiving first-line treatment. In conclusion, elderly patients unfit for antithymocyte globulin therapy may benefit from eltrombopag.

Abstract. Despite major treatment improvements over the past decades, pediatric acute myeloid leukemia (AML) is still a life-threatening malignancy with relapse rates up to 30% and survival rates below 75%. A better description of the pattern of molecular aberrations in childhood AML is needed to refine prognostication in such patients. We report here the comprehensive molecular landscape using both high-throughput sequencing focused on 36 genes and ligation-dependent RT-PCR in 385 children with de novo AML enrolled in the prospective ELAM02 trial and we evaluated their prognostic significance. Seventy-six percent of patients had at least 1 mutation among the genes we screened. The most common class of mutations involved genes that control kinase signaling (61%) followed by transcription factors (16%), tumor suppressors (14%), chromatin modifiers (9%), DNA methylation controllers (8%), cohesin genes (5%), and spliceosome (3%). Moreover, a recurrent transcript fusion was detected in about a half of pediatric patients. Overall, CBF rearrangements, NPM1 and double CEBPA mutations represented 37% of the cohort and defined a favorable molecular subgroup (3 years OS: 92.1%) while NUP98 fusions, WT1, RUNX1, and PHF6 mutations (15% of the cohort) segregated into a poor molecular subgroup (3 years OS: 46.1%). KMT2A-rearrangements (21% of the cohort) were associated with an intermediate risk. Despite some overlaps, the spectrum of molecular aberrations and their prognostic significance differ between childhood and adult AML. These data have important implications to contribute in refining risk stratification of pediatric AML and show the need for further validations in independent pediatric cohorts.

Abstract Development of novel therapies is critical for T‐cell acute leukaemia (T‐ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T‐ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T‐ALL cell samples cultured on stromal cells independently of NOTCH activation and maintained their ability to propagate in vivo. Similar results were obtained when T‐ALL cells were cultured with ERK1/2‐knockdown stromal cells or with conditioned medium from MEKi‐treated stromal cells. Microarray analysis identified interleukin 18 (IL‐18) as transcriptionally up‐regulated in MEKi‐treated MS5 cells. Recombinant IL‐18 promoted T‐ALL growth in vitro, whereas the loss of function of IL‐18 receptor in T‐ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL‐18R was activated by IL‐18 in blast cells. IL‐18 circulating levels were increased in T‐ALL‐xenografted mice and also in T‐ALL patients in comparison with controls. This study uncovers a novel role of the pro‐inflammatory cytokine IL‐18 and outlines the microenvironment involvement in human T‐ALL development.

Background The prognosis of patients with relapses of ETV6/RUNX1-positive acute lymphoblastic leukemia remains to be evaluated, particularly with regards to the frequency of late relapses. We performed a long-term, follow-up retrospective study to address the outcome of patients with ETV6/RUNX1-positive leukemia relapses.Design and Methods Among the 713 children tested for ETV6/RUNX1 enrolled into the FRALLE 93 protocol, 43 ETV6/RUNX1-positive patients relapsed (19.4%). Most were initially stratified in the low or intermediate risk groups. The median follow-up after relapse was 54.2 months. All but three received second-line salvage therapy and 16 underwent allogeneic transplantation.Results ETV6/RUNX1 had a strong effect on overall survival after relapse (3-year survival= 64.7% for positive cases versus 46.5% for negative cases) (P=0.007). The 5-year cumulative incidence of relapse was 19.4% and testes were more frequently involved in ETV6/RUNX1-positive relapses (P=0.04). In 81.4% of cases the relapses were late, early combined or isolated extramedullary relapses. The 5-year survival rate of patients with ETV6-RUNX1-positive acute lymphoblastic leukemia relapses reached 80.8% when the relapse occurred after 36 months (versus 31.2% when the relapse occurred earlier). In univariate analysis, female gender was associated with a poor survival, whereas site of relapse, age at diagnosis, leukocytosis and consolidation strategy had no effect. In multivariate analysis, only the duration of first remission remained associated with outcome.Conclusions We found an excellent outcome for patients with ETV6/RUNX1-positive leukemia relapses that occurred more than 36 months after diagnosis. The duration of first complete remission may, therefore, be a guide to define the treatment strategy for patients with relapsed ETV6/RUNX1- positive leukemia. Key words: ETV6/RUNX1, childhood leukemia, acute lymphoblastic, prognosis, relapse.

Background Patients with the Shwachman-Diamond syndrome often develop hematologic complications. No risk factors for these complications have so far been identified. The aim of this study was to classify the hematologic complications occurring in patients with Shwachman-Diamond syndrome and to investigate the risk factors for these complications.Design and Methods One hundred and two patients with Shwachman-Diamond syndrome, with a median follow-up of 11.6 years, were studied. Major hematologic complications were considered in the case of definitive severe cytopenia (i.e. anemia

Background Autoimmune hemolytic anemia is a rare condition in children. Little is known about its initial presentation and the subsequent progression of the disease.Design and Methods Since 2004, a national observational study has been aiming to thoroughly describe cases and identify prognostic factors. Patients from all French hematologic pediatric units have been included if they had a hemoglobin concentration less than 11 g/dL, a positive direct antiglobulin test and hemolysis. Evans’ syndrome was defined by the association of autoimmune hemolytic anemia and immunological thrombocytopenic purpura. Data from patients’ medical records were registered from birth to last follow-up. Autoimmune hemolytic anemia was classified as primary or secondary. Remission criteria, qualifying the status of anemia at last follow-up, were used with the aim of identifying a subgroup with a favorable prognosis in continuous complete remission.Results The first 265 patients had a median age of 3.8 years at diagnosis. In 74% of cases the direct antiglobulin test was IgG/IgG+C3d. Consanguinity was reported in 8% of cases and first degree familial immunological diseases in 15% of cases. Evans’ syndrome was diagnosed in 37% of cases. Autoimmune hemolytic anemia was post-infectious in 10%, immunological in 53% and primary in 37% of cases. After a median follow-up of 3 years, 4% of children had died, 28% were still treatment-dependent and 39% were in continuous complete remission. In multivariate analysis, IgG and IgG+C3d direct antiglobulin tests were associated with a lower rate of survival with continuous complete remission (adjusted hazard ratio, 0.43; 95% confidence interval, 0.21–0.86).Conclusions This nationwide French cohort is the largest reported study of childhood autoimmune hemolytic anemia. The rarity of this condition is confirmed. Subgroups with genetic predisposition and underlying immune disorders were identified.

Background β-thalassemia is a rare disease in France, encountered mainly in patients originating from Italy and North Africa. In the setting of the recent French plan for rare diseases, a National Registry for thalassemia has been developed since 2005. Epidemiological and clinical data have been collected on living patients with β-thalassemia major or intermedia, including those who underwent hematopoietic stem cell transplantation.Design and Methods A standardized questionnaire was sent to clinicians throughout the national professional networks involved in the management of thalassemic patients and data were updated every 18 months. A cross-sectional study was performed in February 2009.Results Data on 378 patients (267 with thalassemia major) with a median age of 20 were recorded. Hematopoietic stem cell transplantation was performed in 52 patients. Stature, rates of parenthood, splenectomy, and cholecystectomy were no different between non-transplanted thalassemia major and thalassemia intermedia patients, after adjustment for age. Among the 215 non-transplanted thalassemia major patients, the median serum ferritin level was 1240 ng/mL and the rates of iron-related complications were 10%, 6%, 10% and 48% for cardiac failure, diabetes, hypothyroidism, and hypogonadism, respectively. From 2005 to 2008, a dramatic switch in chelation treatment, from deferoxamine to deferasirox, was observed.Conclusions The rates of complications of iron overload in French thalassemia major patients appeared similar to those reported in other developed countries in which this condition is not endemic. There were no significant differences in height and parenthood rates between patients with the major and the intermedia forms of the disease, underlining the progress in clinical care. Future developments will focus on mortality and morbidity under oral chelation treatment.

Background Patients with bone marrow failure and undiagnosed underlying Fanconi anemia may experience major toxicity if given standard-dose conditioning regimens for hematopoietic stem cell transplant. Due to clinical variability and/or potential emergence of genetic reversion with hematopoietic somatic mosaicism, a straightforward Fanconi anemia diagnosis can be difficult to make, and diagnostic strategies combining different assays in addition to classical breakage tests in blood may be needed.Design and Methods We evaluated Fanconi anemia diagnosis on blood lymphocytes and skin fibroblasts from a cohort of 87 bone marrow failure patients (55 children and 32 adults) with no obvious full clinical picture of Fanconi anemia, by performing a combination of chromosomal breakage tests, FANCD2-monoubiquitination assays, a new flow cytometry-based mitomycin C sensitivity test in fibroblasts, and, when Fanconi anemia was diagnosed, complementation group and mutation analyses. The mitomycin C sensitivity test in fibroblasts was validated on control Fanconi anemia and non-Fanconi anemia samples, including other chromosomal instability disorders.Results When this diagnosis strategy was applied to the cohort of bone marrow failure patients, 7 Fanconi anemia patients were found (3 children and 4 adults). Classical chromosomal breakage tests in blood detected 4, but analyses on fibroblasts were necessary to diagnose 3 more patients with hematopoietic somatic mosaicism. Importantly, Fanconi anemia was excluded in all the other patients who were fully evaluated.Conclusions In this large cohort of patients with bone marrow failure our results confirmed that when any clinical/biological suspicion of Fanconi anemia remains after chromosome breakage tests in blood, based on physical examination, history or inconclusive results, then further evaluation including fibroblast analysis should be made. For that purpose, the flow-based mitomycin C sensitivity test here described proved to be a reliable alternative method to evaluate Fanconi anemia phenotype in fibroblasts. This global strategy allowed early and accurate confirmation or rejection of Fanconi anemia diagnosis with immediate clinical impact for those who underwent hematopoietic stem cell transplant.

Background The prognostic value of the ectopic activation of TLX3 gene expression, a major oncogenetic event associated with pediatric T-cell acute lymphoblastic leukemia, is controversial. Likewise, the frequency and the prognostic significance in pediatric T-cell acute lymphoblastic leukemia of the newly characterized NUP214-ABL1 fusion transcript is not yet clear.Design and Methods Two hundred children with T-cell acute lymphoblastic leukemia were treated in the French FRALLE-93 study from 1993 to 1999.The expression of TLX3, TLX1 and SILTAL1 genes was analyzed in samples from 92 patients by real-time quantitative reverse transcriptase polymerase chain reaction. Most of these samples were further studied for NUP214-ABL1 and CALM-AF10 fusion transcripts.Results The median follow-up was 7.9 years. At 5 years the overall survival (± standard deviation, %) was 62 (±3%) and leukemia-free survival was 58 (±3%). Patients with T-cell acute lymphoblastic leukemia positive for TLX3 had a poorer survival compared to those with T-ALL negative for TLX3 (overall survival: 45±11% vs. 57±5%, p=0.049). In multivariate analysis, TLX3 expression was an independent adverse risk factor predicting relapse with a hazard ratio of 2.44 (p=0.017) and an overall survival with a hazard ratio of 3.7 (p=0.001). NUP214-ABL1 was expressed in 16.6% of patients with TLX3-positive T-ALL (3 of 18); all of the patients with this association died before completion of the treatment. SILTAL expression did not significantly affect the prognosis of patients with T-cell acute lymphoblastic leukemia. Only three of 92 patients expressed the TLX1 gene and all three are alive.Conclusions TLX3 gene expression is an independent risk factor predicting poor survival in childhood T-cell acute lymphoblastic leukemia. When co-expressed with TLX3, NUP214-ABL1 transcripts may increase the risk of poor survival.

Background Mutations in the ribosomal protein S19 gene (RPS19) have been found in 25% of patients with Diamond-Blackfan anemia, a rare syndrome of congenital bone marrow failure characterized by erythroblastopenia and various malformations. Mechanistic understanding of the role of RPS19 in normal erythropoiesis and in the Diamond-Blackfan anemia defect is still poor. However, defective ribosome biogenesis and, in particular, impaired 18S ribosomal RNA maturation have been documented in association with various identified RPS19 mutations. Recently, new genes, all encoding ribosomal proteins, have been found to be mutated in Diamond-Blackfan anemia, adding further support to the concept that ribosome biogenesis plays an important role in regulating erythropoiesis. We previously showed variability in the levels of expression and subcellular localization of a subset of RPS19 mutant proteins.Design and Methods To define the mechanistic basis for this variability better, we studied a large number of mutant proteins and characterized both RPS19 expression level using a specific antibody against RPS19 and RPS19 subcellular localization after transfection of Cos-7 cells with various green fluorescent protein-RPS19 mutants. To investigate the role of the proteasome in RPS19 degradation, we examined the effect of various proteasome inhibitors, namely lactacystin, MG132, and bortezomib on RPS19 expression and subcellular localizationResults We found two distinct classes of RPS19 protein defects in Diamond-Blackfan anemia based on the stability of the mutant proteins: (i) slightly decreased to normal levels of expression and normal nucleolar localization and (ii) markedly deficient expression and failure to localize to the nucleolus. All the proteasome inhibitors tested were able to restore the expression levels and normal subcellular localization of several unstable mutant proteins.Conclusions Our findings demonstrate an important role for the proteasomal degradation pathway in regulating the expression levels and nucleolar localization of certain mutant RPS19 proteins in Diamond-Blackfan anemia.