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DDX41 germline mutations (DDX41MutGL) are the most common genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia (AML). Recent reports suggest that DDX41MutGL myeloid malignancies could be considered as a distinct entity, even if their specific presentation and outcome remain to be defined. We describe here the clinical and biological features of 191 patients with DDX41MutGL AML. Baseline characteristics and outcome of 86 of these patients, treated with intensive chemotherapy in 5 prospective Acute Leukemia French Association/French Innovative Leukemia Organization trials, were compared with those of 1604 patients with DDX41 wild-type (DDX41WT) AML, representing a prevalence of 5%. Patients with DDX41MutGL AML were mostly male (75%), in their seventh decade, and with low leukocyte count (median, 2 × 109/L), low bone marrow blast infiltration (median, 33%), normal cytogenetics (75%), and few additional somatic mutations (median, 2). A second somatic DDX41 mutation (DDX41MutSom) was found in 82% of patients, and clonal architecture inference suggested that it could be the main driver for AML progression. DDX41MutGL patients displayed higher complete remission rates (94% vs 69%; P < .0001) and longer restricted mean overall survival censored at hematopoietic stem cell transplantation (HSCT) than 2017 European LeukemiaNet intermediate/adverse (Int/Adv) DDX41WT patients (5-year difference in restricted mean survival times, 13.6 months; P < .001). Relapse rates censored at HSCT were lower at 1 year in DDX41MutGL patients (15% vs 44%) but later increased to be similar to Int/Adv DDX41WT patients at 3 years (82% vs 75%). HSCT in first complete remission was associated with prolonged relapse-free survival (hazard ratio, 0.43; 95% confidence interval, 0.21-0.88; P = .02) but not with longer overall survival (hazard ratio, 0.77; 95% confidence interval, 0.35-1.68; P = .5).

VEXAS (vacuoles, E1 enzyme, X-linked, auto-inflammatory, somatic) syndrome is an inflammatory disorder with hematological and systemic features. A recent study demonstrated that the dermal infiltrate in neutrophilic dermatosis from VEXAS patients is derived from the pathological UBA1-mutated myeloid clone. Neutrophilic dermatosis is, however, only one of the various skin involvements observed in VEXAS syndrome. We analyzed 10 formalin-fixed paraffin-embedded skin biopsies from genetically confirmed VEXAS syndrome. UBA1 mutation was found in the biopsies related to neutrophilic dermatitis but in none of the other histological patterns (leukocytoclastic vasculitis and septal panniculitis). This could lead to a distinction between clonal and paraclonal cutaneous involvements in VEXAS syndrome, which could in turn improve therapeutic outcomes.

International audience; Classical Philadelphia-negative myeloproliferative neoplasms include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are characterized by the presence of driver mutations of JAK2, CALR or MPL genes. Overexpression of WT1 is used as a marker of minimal residual disease in acute myeloid leukemia, especially after allogeneic stem cell transplantation (SCT). We investigated WT1 expression at diagnosis in 152 MPN patients and showed that the WT1 transcript was overexpressed in PMFs and PVs compared to controls. In particular, WT1 transcript levels were higher in PMF than in ET and PV. WT1 transcript levels were significantly increased during myelofibrotic transformation of ET or PV. Using multi-variate linear regression, high WT1 transcript levels in PMF were associated with age over 65, splenomegaly and thrombocytopenia. The ROC curve analysis showed that a level of WT1 transcript > 10 WT1 copies/10 4 ABL1 enabled the diagnosis of PMF with a specificity of 95.8% (PMF vs ET; ROC AUC = 0.91). In myelofibrosis, studying follow-ups of WT1 transcript showed that this marker is of interest after allogeneic SCT. These results demonstrate that WT1 overexpression is a simple marker of myelofibrosis in MPN and could be used during patient follow-up.

International audience; In this randomized phase 3 study, the FILO group tested whether the addition of 6 mg/m(2) of gemtuzumab ozogamycin (GO) to standard chemotherapy could improve outcome of younger patients with de novo acute myeloid leukemia (AML) and intermediate-risk cytogenetics. GO arm was prematurely closed after 254 inclusions because of toxicity. A similar complete remission rate was observed in both arms. Neither event-free survival nor overall survival were improved by GO in younger AML patients (

The benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with acute myeloid leukemia (AML) aged >60 years remains a matter of debate, notably when performed in first complete remission (CR1). To clarify this issue, the French Innovative Leukemia Organization (FILO) performed a 10-year real-world time-dependent analysis. The study enrolled patients between 60 and 70 years of age with AML in CR1 after intensive chemotherapy with intermediate (IR) or unfavorable (UR) risk according to the European LeukemiaNet (ELN) 2010 classification. The impact of allo-HSCT was analyzed through three models: (1) time-dependent Cox; (2) multistate for dynamic prediction; and (3) super landmark. The study enrolled 369 (73%) IR and 138 (27%) UR patients with AML, 203 of whom received an allo-HSCT. Classical multivariate analysis showed that allo-HSCT significantly improved relapse-free survival (RFS; hazard ratio [HR] [95% confidence interval (CI)], 0.47 [0.35-0.62]; P < .001) and overall survival (OS; HR [95% CI], 0.56 [0.42-0.76]; P < .001), independently of the ELN risk group. With the multistate model, the predicted 5-year probability for IR and UR patients to remain in CR1 without allo-HSCT was 8% and 1%, respectively. Dynamic predictions confirmed that patients without allo-HSCT continue to relapse over time. Finally, the super landmark model showed that allo-HSCT significantly improved RFS (HR [95% CI], 0.47 [0.36-0.62]; P < .001) and OS (HR [95% CI], 0.54 [0.40-0.72]; P < .001). allo-HSCT in CR1 is reported here as significantly improving the outcome of fit older patients with AML. Long-term RFS without allo-HSCT is very low (

International audience; Donor cell leukemia (DCL) is an infrequent complication after allogeneic hematopoietic stem cell transplantation (HSCT). Its true incidence is difficult to assess, although improvements in chimerism studies contributed to a better diagnosis of DCL. We report two rare cases of donor cell-derived acute promyelocytic leukemia (APL). To our knowledge, only two cases have been described in the literature. Here, we report one male and one female patients with acute myeloid leukemia (AML), who developed an APL in donor cells after HSCT. The latency between HSCT and DCL was 279 and 43 months, respectively. Fluorescent in situ hybridation and chimerism monitoring analysis proved the donor origin of APL. Surprisingly, donor lymphocyte infusion provided a hematological response during 19 months in the female patient. The mechanisms associated with pathogenesis of DCL are unclear and seem to be multifactorial. Increasing worldwide allogeneic hematopoietic stem cell transplantation activity and potentially the age of donor could explain the increasing incidence of DCL in the future. It is highlighted that long-term follow up of recipients will allow to report all cases of DCL, to clarify the genetic landscape and factors which contribute to DCL, to understand the response to DLI.

In 2020, accreditation of molecular tests according to ISO 15189 is a requirement for all French medical laboratories. For many years, the GBMHM group (French Group of Molecular Biologists in Hematology) supports this approach through organization of external quality evaluation campaigns, and by publishing recommendations that have allowed the accreditation of the most frequent molecular tests for most laboratories. However, some molecular abnormalities concerns very few patients (and sometimes a single patient), and therefore cannot be evaluated in the same way, because of the lack of external quality controls or inter-laboratory comparisons. In order to allow the accreditation of these rare analyzes, the GBMHM proposes recommendations, based on the fact that analyzes using the same methodology than those already accredited by an extensive validation process, may be accredited without the need for full analytical validation. In particular, assays based on quantitative PCR or endpoint PCR may be accredited after verification of primer specificity, repeatability and/or reproducibility, and the determination of detection or linearity limits. These recommendations, by defining the validation approach for rare molecular abnormalities, make it possible to extend the requirement of accreditation for rare tests, to provide the best patient care.

Relapse is a major complication of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (SCT). The objective of our study was to evaluate chimerism monitoring on the CD3-negative mononuclear cells by RQ-PCR to predict relapse of patients allografted for AML and to compare its performance with WT1 quantification. A cohort of 100 patients undergoing allogenic SCT for AML was retrospectively analyzed in a single institution. Patients without complete chimerism, defined as less than 0.01% of recipient’s DNA in CD3-negative cells, had a significantly higher risk of relapse and a lower overall survival (p

Aims Life expectancy of CML pts optimally responding to tyrosine kinase inhibitors (TKI) is close to that of the general population and recently, TFR has been acknowledged as a new goal of CML management. TKI discontinuation in the view of TFR requires the achievement of deep and long-lasting molecular responses (MR). The gold standard BCR-ABL mRNA quantification technology and MR definitions rely on internationally standardized (IS) RT-qPCR but atypical transcripts located outside the Major-BCR region, harbored by 1-2% of pts, cannot be expressed on the IS scale. Thus, most trials and clinical practice recommendations prevent such pts from attempting TFR. The Fi-LMC group retrospectively collected real-life observations to assess TFR likelihood in this rare population. Methods Data from CML pts with precise characterization of atypical transcripts in whom any line TKI was stopped for any reason but after at least 2 years of undetectable molecular residual disease (UMRD) by individualized non-standardized RT-qPCR were collected. RT-qPCR sensitivity varied depending on transcript type and local molecular biology laboratory. TFS was estimated by the Kaplan-Meier method. Relapse was analyzed using the cumulative incidence function, relapse being as UMRD loss at any time and any level during follow-up (FU). Results Our series comprised 16 adult CP CML pts with atypical BCR-ABL fusions including 12 males (75%). Median age at CML diagnosis was 56 years (range: 21-75) and that at TKI discontinuation was 67 years [range: 29-82]. Sokal score was low, intermediate and high in 7, 8 and 1 pts, respectively. ELTS score was low and intermediate in 10 and 4 pts, respectively and unknown in 2. Most pts expressed e19a2 (n=6) followed by e6a2 (n=4), b3a3 (n=3), b2a3 (n=2) and e8a2 (n=1). Seven pts discontinued imatinib, 4 stopped dasatinib, 4 nilotinib and 1 bosutinib. Number of lines of therapy was 2 in 8 pts, 1 in 5 pts and 3 in 3 pts. Median TKI treatment duration before discontinuation was 64 months (range: 31-218) and median duration of UMRD was 41 months (range: 21-168). The median FU after TKI discontinuation was 68 months (range: 3-149). Five pts experienced relapse leading to TKI resumption. Four relapses occurred within 3-6 months and included 2 loss of hematologic response in CP, 1 loss of hematologic response in accelerated phase CML and 1 molecular recurrence with BCR-ABL transcripts up to 1.5%. One relapse occurred at 49 months and consisted in loss of a complete cytogenetic response. These 5 pts resumed TKI and regained UMRD within 6 months, including 1 pt who died in UMRD from non-CML-related cause at the age of 82 years and 1 pt who rapidly failed a 2 nd TKI discontinuation attempt. In 1 additional pt, BCR-ABL transcripts became detectable intermittently with maximum transcript level of 0.15% and TKI was not resumed. The median FU of pts who remained treatment-free was 68 months (range: 8-149). Overall, the 5-year cumulative incidence of relapse regardless of whether TKI was resumed was 41.6% (95% confidence interval: 21.9%-78.7%) (Figure 1). The 5-year TFS rate was 65.2% (95% confidence interval: 40.3%-90.2%) (Figure 2). Conclusions Our observational study of TKI discontinuation in CML pts with atypical BCR-ABL transcripts is the largest reported so far. While effort must be made for proper assessment of deep MR, preliminary results suggest that TFS pattern might favorably compare with that obtained in pts with Major-type BCR-ABL transcripts. However, relapses may be more aggressive and caution is required in order to avoid loss of hematologic responses and progression. Whether the type of atypical fusion gene influences TKI discontinuation outcome, as well as other potential prognostic factors, need to be determined in a larger series. Figure 1 Figure 1. Disclosures Charbonnier: Novartis: Speakers Bureau; Incyte: Speakers Bureau. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Etienne: Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau. Rousselot: Incyte, Pfizer: Consultancy, Research Funding. Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria.

International audience

Introduction Citrulline, a non-essential amino acid produced exclusively by enterocytes in the small intestine and involved in the synthesis of L-arginine, is not metabolized by the liver. Therefore citrulline serum concentration is highly correlated with functional enterocyte mass, and decreases with digestive toxicity induced by conditioning therapy (radiotherapy and/or chemotherapy) for hematopoietic stem cell transplantation (HSCT) . Acute Graft-versus-host disease (GvHD), one of the major complications of HSCT, is correlated to conditioning-induced gut barrier damage and may be predicted by pre-transplant serum citrulline level (Rashidi, BBMT 2018). It could be interesting to know whether citrulline kinetics could also represent a biomarker for conditioning toxicity, non-relapse mortality (NRM), and GvHD. The aim of this study is thus to define group-based trajectory modeling, to identify clusters of individual serum citrulline kinetics in the early phase of allogeneic HCST, and to test whether these unsupervised trajectories were correlated with these early complications. Materials and Methods Serum citrulline was quantified by liquid chromatography in blood samples collected from consecutive patients who received an allogeneic HSCT in our institution between July 2014 and November 2019. These samples were drawn at different time-points: pre-transplant (D-7, D-3); day of transplant (D0), and post-transplant (D7, D15, D21). Distinct trajectories were identified for serum citrulline by using the semiparametric mixture model described by Nagin (Nagin, Stat Methods Med Res 2018). Results Among 161 patients (pts) included in the study, with a median age of 53 years (17-72), 98 pts (60.9%) received a reduced-intensity conditioning (RIC), 36 pts (22.4%) reduced-toxicity conditioning (RTC), 18 pts (11.1 %) sequential conditioning, and 9 pts (5.6%) myeloablative conditioning (MAC). Donor were identical sibling (22%), matched unrelated donor (52%) and haploidentical sibling (25%). Graft source was peripheral blood mononuclear cells in 144 pts (89.4%) and bone marrow in 17 pts (10.6%) respectively. HCT-CI score was low, intermediate and high-risk in 38%, 32%, and 30% of pts respectively. Disease-Risk Index (DRI) was low/intermediate in 111 pts (69%) and high/very-high in 50 pts (31%). With a median follow up of 29.1 month, 3-year overall survival (OS), disease-free survival (DFS), and NRM rates were 64.5%, 58.3%, and 18.9%, respectively. The median number of citrulline samples per patient was 7 [3-16]. Median citrulline concentrations before conditioning and at D-3, D0, D7 and D15 were statistically different during RIC, RTC, MAC, and sequential conditioning (p In the whole cohort, 3 citrulline trajectories were determined in an unsupervised method. Patients belonging to these 3 trajectories were different according to intensity of conditioning received with lower citrulline trajectories during MAC and sequential conditioning (p After restricting the analysis to pts who received RIC conditioning (n=98), higher pre-HSCT citrulline concentrations were associated with a lower NRM (p=0.042). Unsupervised analysis in this setting individualized 4 clusters of individual trajectories (figure 1), that did neither distinguish age (p=0.28), DRI (p=0.87), HCT-CI score (p=0.81) nor the incidence of acute (p=0.6) or chronic (p=0.4) GvHD. However, the lowest citrulline trajectory contained significantly more haploidentical transplantations (p=0.004) and less pts who received antithymocyte globulin for GvHD prophylaxis (p=0.005). Interestingly in this RIC cohort, cumulative incidence of NRM at 12 months was 23%, 21%, 8%, and 0% respectively according to the 4 citrulline trajectories (figure 2). Conclusion In patients receiving allogeneic HSCT, the variation of serum citrulline concentrations depends on the intensity of the conditioning regimen. In patients who received RIC conditioning, lower plasma citrulline trajectories are associated with higher NRM. In this setting, citrulline may be an attractive biomarker for predicting conditioning toxicity and NRM. Disclosures Hunault: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Diachi: Membership on an entity's Board of Directors or advisory committees; Jansen: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Thepot:astellas: Honoraria; novartis: Honoraria; sanofi: Honoraria; celgene: Honoraria.

Introduction: The prognosis of older patients treated for acute myeloid leukemia (AML) relies on cytogenetic/molecular classifications as well as their ability to tolerate intensive induction therapy for which comorbidities have an important impact. A prognostic model has been elaborated to incorporate these two variables, cytogenetic/molecular risk and comorbidities evaluated by the Hematopoietic Cell Transplantation - Comorbidity Index (HCT-CI) (Sorror et al. 2017). The AML-composite model (AML-CM), which also incorporates hypoalbuminemia and high LDH levels at diagnosis, has been recently updated to incorporate the new European LeukemiaNet (ELN) 2017 classification (Sorror et al. 2019). In this study, we aimed to confirm the predictive impact of the revised AML-CM in an independent cohort and to explore which parameters were the most relevant for predicting early mortality and relapse. Patients and Methods: All patients (pts) older than 60 years diagnosed with AML who received intensive induction therapy in our department between 2004 and 2017 were included. Patients with acute promyelocytic leukemia were excluded. They received induction therapy with idarubicin 8 mg/m2 for 5 days and cytarabine 100 mg/m2 for 7 days with or without lomustine 200 mg/m2 on day 1. Patients in first complete remission (CR1) were to receive six consolidation courses with idarubicin 8 mg/m2 for one day and cytarabine 100 mg/m2 for 5 days and maintenance therapy with oral methotrexate and mercaptopurine. The HCT-CI and the revised AML-CM were calculated as previously described (Sorror et al. 2005; Sorror et al. 2019) using individual patient medical records. Early mortality was defined as death within one month after the start of induction therapy. The ELN 2017 classification, the HCT-CI, and the revised AML-CM were considered to determine which parameters - comorbidities or cytogenetic/molecular risk - were associated with each outcome. Results: Ninety-nine pts were included in the study with a median age of 66 years-old (range: 60 - 82). Twenty-seven pts (27%) had secondary AML (prior solid tumor requiring chemotherapy and/or radiation therapy in 11 pts and prior hematological malignancy in 16 patients). According to the ELN 2017 classification, 24 (24%) were favorable, 53 (54%) were intermediate, and 22 (22%) were adverse. The most frequent comorbidities included liver disease (30 pts, low/moderate, 5 pts, severe), previous cancer (22 pts), and arrythmia (22 pts). Thus, 13 (13%), 25 (25%), 44 (45%), and 17 (17%) pts had a revised AML-CM score of 1-4, 5-6, 7-9, and >10, respectively. 78 pts (79%) achieved CR1, with 10 early deaths (10%) due to toxicity (early mortality) and 11 induction failures (11%). Fifteen pts received allogeneic SCT. 54 (55%) relapsed, and 72 (73%) died with a median follow-up of 18 months (range: 0 - 167). Both the HCT-CI and the AML-CM were associated with increased early mortality (OR: 1.4, 95% CI: 1 - 1.8, p=0.03 for HCT-CI and OR: 1.3, 95% CI: 1-1.7, p=0.03 for AML-CM) whereas the ELN 2017 classification had no impact. The predictive value of the AML-CM for early mortality was not superior to the HCT-CI (area under the curve - AUC: 0.76, p=0.01, and 0.76, p=0.01, respectively). The risk of relapse was only associated with an unfavorable ELN 2017 classification (OR: 8.8, 95% CI: 1.1 - 70, p=0.04). It was the most predictive parameter for relapse, in comparison to the HCT-CI and the revised AML-CM, but this did not reach statistical significance (AUC: 0.62, p=0.08). The AML-CM clearly distinguishes 4 groups with different prognosis (Figure, p 10, respectively (OR: 1.4, 95% CI: 1.2 - 1.8, p Conclusion: The revised AML-CM is an effective tool for predicting overall survival in older pts with AML receiving intensive induction therapy. It is a better prognostic system compared to the HCT-CI and the ELN 2017 classification as it combines the evaluation of comorbidities, which predicts early mortality, and cytogenetic/molecular risk, which predicts relapse. Figure Disclosures Orvain: Novartis: Honoraria; Incyte: Honoraria.

International audience

International audience; no abstract

The University at Buffalo (UB) and Empire State College (ESC) designed a project that demonstrates the viability of shipping materials directly to the patron, the cost-effectiveness of using UPS for delivering and returning materials, the value of expanded library service to ESC patrons, and the practicality of purchasing-on-demand as a service and collection development model. UB staff provide the service and have access to both institutions’ ILLiad clients. Articles are sent via Odyssey. Books not owned by UB are purchased and shipped directly to the patron. The purchased books are added to the UB collection upon return.

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.

A cell permeabilization method for the release of intracellular proteins from microbial cells was developed. The method was applied to the recovery of recombinant botulinum neurotoxin fragments, expressed intracellularly in the yeast Pichia pastoris, by suspending the cells in an aqueous solution containing N,N-dimethyltetradecylamine. For the botulinum neurotoxin serotype B C-terminal heavy chain fragment, 1.8 mg g(-1) biomass were recovered. For the botulinum neurotoxin serotype A C-terminal heavy chain fragment, 3.7 mg g(-1) biomass were recovered. The concentration of recombinant protein in the cell extracts remained stable for up to 48 and 24 h for the serotype B and serotype A fragments, respectively. The permeabilization method was compared with high-pressure homogenization; the permeabilization method proved to be both more selective and more efficient.

Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(Hc)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is approximately 3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(Hc) proteins.

An absolute kinetic scale for amidyl radical reactions was established by a combination of direct laser flash photolysis (LFP) and indirect competition kinetic studies. Six amidyl radicals were studied by LFP. Arrhenius parameters were determined for 6-exo cyclizations of the N-butyl-6,6-diphenyl-5-hexenamidyl radical (2) and the N-butyl-6-(trans-2-phenylcyclopropyl)-5-hexenamidyl radical (5) and for 1,5-hydrogen transfer in the N-(6,6-diphenyl-5-hexenyl)acetamidyl radical (3); rate constants for these reactions at 20 °C are 3.0 × 107 s-1 (2), 5.5 × 106 s-1 (3), and 1 × 107 s-1 (5). Kinetic limits were established by LFP for the fast cyclizations of the N-methyl-5,5-diphenyl-4-pentenamidyl radical (1) and the N-methyl-5-(trans-2-phenylcyclopropyl)-4-pentenamidyl radical (4) (k > 2 × 108 s-1 at ambient temperature) and for the slow fragmentation of the N-(2,2-diphenylethyl)acetamidyl radical (6) (k = 2 × 104 s-1 at 48 °C). Rate constants for amidyl radical reactions with Bu3SnH and PhSH were determined by ...

Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these “half-molecules” could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14–16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.

The Sokal, Hasford and EUTOS scoring systems have been successively proposed to predict the outcome of CML patients in chronic phase (CP-CML), both in terms of disease response and survival. The EUTOS score, which is the only one developed for patients treated with tyrosine kinase inhibitors, considers 2 risk groups based on spleen size and peripheral blood basophil percentage. No clear link has been identified between the covariates used to calculate those scores and CML molecular pathophysiology. To better delineate the molecular mechanisms underlying those scoring systems, a gene expression profiling study was performed on 48 CP-CML patients included in the Novartis ENEST1st study, for whom good quality peripheral blood total RNA was available at diagnosis. First, patients with low-risk (n=21) and high-risk (n=12) Sokal scores were compared, leading to identify 13 genes differentially expressed between those groups (q Disclosures Gardembas: Novartis: Speakers Bureau. Spentchian:Novartis: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Radich:Gilliad: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Rousselot:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Speakers Bureau.

Escherichia coli mutants in the beta subunit of the F1F0-ATPase can be complemented with the beta subunit from the obligate aerobe Bacillus megaterium. It has been shown that cells carrying such hybrid ATPases have an unusual energy-coupling phenotype. Although they are able to grow on minimal succinate medium, and therefore carry a functional ATP synthase, they are defective in the ability to grow anaerobically, indicating some defect in ATP-driven proton pumping (Scarpetta, M., Hawthorne, C. A., and Brusilow, W. S. A. (1991) J. Biol. Chem. 266, 18567-18572). In this study, chimeric beta subunits were constructed consisting of the E. coli or the B. megaterium beta subunit carrying the C-terminal 18% of the other's beta subunit. The phenotypes of an E. coli beta mutant complemented with these chimeric subunits showed that the energy-coupling defect was located in this C-terminal region. The E. coli beta subunit carrying the B. megaterium C-terminal region displayed the energy-coupling defect, while the B. megaterium beta subunit carrying the E. coli C-terminal region did not. In ATP-dependent fluorescence quenching assays, membranes isolated from cells displaying the energy-coupling defect also pumped protons less well than membranes isolated from cells that were able to grow anaerobically. These results demonstrate that the C terminus of the beta subunit is involved in the conformational coupling pathway, which, through the polypeptide backbone of the beta subunit, physically links ATP synthesis or hydrolysis to the energy of proton translocation.

Background: EVI1 gene overexpression is found in approximately 10% of acute myeloid leukemia (AML) patients, with a higher frequency seen in AML carrying chromosome 3q26 abnormality or MLL gene rearrangement, and associated with a dismal prognosis. Deregulation of EVI1 expression has also been reported in ALL, but its prognostic impact is unclear. Here, we retrospectively analyzed EVI1 expression in a large cohort of adult ALL patients, its correlation with ALL subsets, and its impact on patient outcome. Patients and Methods: EVI1 gene expression was measured by RQ-PCR detecting all splicing variants, with PBGD as control gene. We used dilutions of EVI1+ SKOV3 (kindly provided by Peter Valk, Rotterdam, The Netherlands) and EVI1-neg HL-60 cell line cDNA to build EVI1 and PBGD standard curves. Results were expressed as EVI1/PBGD ratio x 100. Blast samples from 354 patients treated in the GRAALL-2003/2005 and GRAAPH-2005 trials (191 B-cell precursor [BCP]-ALL, including 138 Ph-neg and 53 Ph+; 163 T-ALL) and 62 controls were analyzed. Immunophenotype results were centrally reviewed. In controls, median EVI1 expression level was 0.33% (Q1-Q3, 0.20-0.69). For prognostic analysis, we used the 1st and 99th percentiles of the controls (0.05% and 1.65%) to define patients with low and high EVI1 expression, respectively. Clinical endpoints were cumulative incidence of failure (CIF, failure meaning primary refractoriness or relapse) and event-free survival (EFS). Results: As illustrated in Figure 1, we observed that, as in one AML series, EVI1 expression may be up or down regulated in adult ALL. When compared to controls, the proportions of low and high EVI1 patients were 21 and 23% in Ph-neg BCP-ALL, 9 and 42% in Ph+ ALL, and 21 and 18% in T-ALL, respectively. In BCP-ALL patients, median EVI1 expression was similar to controls (0.53%; Q1-Q3, 0.11-1.88; p=0.15), but higher in the Ph+ as compared to the Ph-neg subgroup (0.93% versus 0.36%; p Conclusion: Overall, these results confirm that EVI1 gene expression is frequently deregulated in adult ALL. In BCP-ALL, down-regulation is observed in t(4;11) and up-regulation in BCR-ABL cases. Further studies are needed to confirm that, in T-ALL, a lower expression is associated with the early, mature and ETP phenotypes and independently predictive of a worse patient outcome. Figure 1 Figure 1. EVI1 gene expression in ALL and control samples. Disclosures No relevant conflicts of interest to declare.

In this publication, we have intended to make out how the characters in the fiction of Claude Simon may function and develop. We have made use of a critical apparatus based on semiology and narratology; we have studied the characters as consistent semantic wholes, spread out all along the text, conveyed by designators, built in the overall architecture of the novel, with a structuring role. From Gulliver to les géorgiques the characters with the work move through several periods. First, they strive with influences and tradition and have already lost their psychological or social balance. Then, they break up, caught in the discourse of a narrator or in the point of view of a character, who vainly try to reach reality through language. Designation is no longer sufficient to define them and them ara outlined through metaphors and recurring meaning units. In the following novels, as proper nouns disappear, they evolve into numerous signifies, which become harmoniously integrated into a text built in a musical or pictorial way. as a point of view, they remain a basic element bringing information and focusing on them, and thus, making the text rich. In les géorgiques the characters reappear as "heroes»: they act, writ, remember... and so, become once again one of the essential elements in a work focused on its actors. Sensitive to some influences, refusing to cope with the balzacian standard or the greimasian analysis, the characters of Simon are framed through metaphors a semantic consistency prodiding them with an internal balance and connecting them with the other actors and novelistic features. No longer appearing through a classical and univocal designation, they remain a pattern bringing their specificity and richness to the whole work., L’objectif de ce travail est de déterminer le fonctionnement et l'évolution du personnage dans l'œuvre romanesque de Claude Simon. Nous utilisons un appareil critique fonde sur la sémiologie et la narratologie; nous considérons le personnage comme un ensemble sémantique cohérent réparti tout au long du texte, manifeste par des désignateurs, intégré dans l'architecture générale du roman et y jouant un rôle structurant. De Gulliver aux géorgiques, le personnage, avec l'œuvre, traverse plusieurs périodes. D’abord, il est aux prises avec les influences et la tradition, et à déjà perçu sa stabilité psychologique ou sociale. Ensuite, il éclate, pris dans le discours d'un narrateur ou le point de vue d'un personnage qui tente en vain d'atteindre le réel avec le langage. La désignation ne permet plus de le cerner et il est structuré par des métaphores et des scènes récurrentes. Dans les romans suivants, avec la disparition du nom propre, il devient une constellation de signifies qui entre en jeu avec un texte construit sur le mode musical ou pictural. Comme point de vue, il reste un élément central qui apporte les informations et les focalise, donnant ainsi sa richesse au texte. Dans les géorgiques, le personnage resurgit comme "héros" : il agit, écrit, se souvient... et redevient donc l'un des éléments essentiels d'une œuvre focalisée sur ses acteurs. Sensible a certaines influences, rétif au modèle balzacien, ou a l'analyse grelmasienne,... cohérence sémantique qui lui confèrent son équilibre interne et le mettent en relation avec les autres acteurs et les autres données romanesques. N’étant plus manifeste par une désignation classique et univoque, il demeure un motif qui apporte sa particularité et sa richesse a l'ensemble de l'œuvre.