Stenotrophomonas maltophilia susceptibility to ceftazidime-avibactam combination versus ceftazidime alone.

OBJECTIVE: To compare the minimum inhibitory concentrations (MIC) of the ceftazidime-avibactam (CZA) combination versus ceftazidime alone (TZ) for Stenotrophomonas maltophilia. PATIENTS AND METHODS: MIC comparison was performed by E-tests. We assumed that CZA was more effective in vitro than TZ alone when CZA led to a category change from "Resistant" with TZ alone to "Susceptible" or "Intermediate" with CZA, or if the MIC of CZA was at least 4-fold lower than the MIC of TZ for TZ-susceptible isolates. RESULTS: For the 54 clinical isolates included in the study, CZA showed better results in terms of the proportion of susceptible isolates (66.7% vs. 38.9%, P<0.01), MIC50 (2µg/mL vs. 12µg/mL, P<0.05), and MIC distribution. According to our definition, CZA was also more effective in vitro than TZ alone for 50% of the isolates. CONCLUSION: Using CZA for empirical treatments in severe or polymicrobial infections with S. maltophilia seems appropriate.

[CAR-T cells: Lymphocytes that express a chimeric antigen receptor]. / CAR-T cells : lymphocytes exprimant un récepteur chimérique à l'antigène.

CAR-T cells are genetically modified human lymphocytes and gene therapy medicinal products. They are developed to treat cancers that express a membrane antigen targeted by the CAR. The FDA approved the two first-in-class medicinal products in 2017 and EMA in August 2018; both are autologous CAR-T cells targeting CD19 that is expressed at the surface of normal B-cells throughout their differentiation, and on B-cell lymphoid malignancies. Clinical efficacy was demonstrated for B-cell acute lymphoblastic leukemias, non-Hodgkin's lymphoma and chronic lymphocytic leukemia, although the marketing authorizations are less liberal in terms of indications. Manufacturing of these personalized treatments necessitates that a novel organization and supply chain be set in place, to ensure product preservation, patient safety and compliance with complex regulatory requirements. Side effects are commensurate with clinical efficacy and can be life-threatening: proper management imposes tight coordination between various specialists, particularly between hematologists and intensive care practitioners. High pricing for these treatments is part of a long-term trend for increasing costs of innovations in hematology and oncology; it questions the ability of healthcare systems to sustain their reimbursement.

Corrigendum: Disease status is a more reliable predictive factor than histology in lymphoma patients after reduced-intensity conditioning regimen and allo-SCT.

This corrects the article DOI: 10.1038/bmt.2012.225.

Assessment of the automated multiplex-PCR Unyvero i60 ITI® cartridge system to diagnose prosthetic joint infection: a multicentre study.

OBJECTIVES: Prosthetic joint infections (PJI) are responsible for significant morbidity and mortality and their number continues to rise. Their management remains complex, especially the microbiological diagnosis. Besides 'homemade' tests developed by several teams, new molecular biology methods are now available with different analytical performance and usability. METHODS: We studied the performances of one of these tests: ITI® multiplex PCR (mPCR) by the Curetis® company and compared it to either 'optimized' culture or 16S rRNA PCR. We performed a retrospective multicentre study to assess the contributions of mPCR in the diagnosis of PJI. We randomly selected 484 intraoperative specimens among 1252 of various types (biopsy, bone, tissue around the prosthesis, synovial fluid) from 251 patients in seven different hospitals. Each sample was treated according to the recommendations of the manufacturer. RESULTS: In all, 154 out of 164 (93.9%) samples negative in culture were negative with the mPCR. Among the 276 positive samples in culture, 251 (90.9%) were monomicrobial, of which 119 (47.4%) were positive with the mPCR, and 25 (9.1%) were polymicrobial, of which 12 (48%) were positive with the mPCR. The concordance rate of mPCR with culture was 58.1% (53.6%-62.7%) and the concordance rate with 16S rRNA PCR was 70.1% (65.5%-74.6%). CONCLUSION: This new standardized molecular test showed a lack of detection when the bacterial inoculum was low (number of positive media per sample and number of colonies per media) but can be useful when patients have received antibiotic therapy previously.

Highly favorable outcome in BRCA-mutated metastatic breast cancer patients receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation.

Breast cancer carrying BRCA mutation may be highly sensitive to DNA-damaging agents. We hypothesized a better outcome for BRCA-mutated (BRCA(mut)) metastatic breast cancer (MBC) patients receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation (HDC AHSCT) versus unaffected BRCA (BRCA wild type; (BRCA(wt))) or patients without documented BRCA mutation (BRCA untested (BRCA(ut))). All female patients treated for MBC with AHSCT at Institut Paoli-Calmettes between 2003 and 2012 were included. BRCA(mut) and BRCA(wt) patients were identified from our institutional genetic database. Overall survival (OS) was the primary end point. A total of 235 patients were included. In all, 15 patients were BRCA(mut), 62 BRCA(wt) and 149 BRCA(ut). In multivariate analyses, the BRCA(mut) status was an independent prognostic factor for OS (hazard ratio (HR): 3.08, 95% confidence interval (CI): 1.10-8.64, P=0.0326) and PFS (HR: 2.52, 95% CI :1.29-4.91, P=0.0069). In this large series of MBC receiving HDC AHSCT, we report a highly favorable survival outcome in the subset of patients with documented germline BRCA mutations.

Growth arrest-specific 6 regulates thrombin-induced expression of vascular cell adhesion molecule-1 through forkhead box O1 in endothelial cells.

BACKGROUND: Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase-Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE. OBJECTIVES: To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression. METHODS: Thrombin was used to stimulate endothelial cells (ECs). Wild-type (WT) and Gas6(-/-) ECs were transfected with small interfering RNA targeting Axl or FoxO-1, a luciferase-coupled plasmid containing the FoxO-1 consensus sequence, and a phosphorylation-resistant FoxO-1 mutant, or treated with an Akt inhibitor. VCAM-1 mRNA expression was measured by real time-qPCR. VCAM-1 protein expression and FoxO-1 and Akt phosphorylation were assessed by western blot analysis. FoxO-1 localization was assessed by immunofluorescence. Adhesion of bone marrow mononuclear cells (BM-MCs) on ECs was assessed by fluorescence. RESULTS AND CONCLUSIONS: Thrombin induces both VCAM-1 expression and FoxO-1 phosphorylation and nuclear exclusion in WT ECs only. Silencing of FoxO-1 enhances VCAM-1 expression in both WT and Gas6(-/-) ECs. Inhibition of Akt or FoxO-1 phosphorylation prevents VCAM-1 expression in WT ECs. These data show that Gas6 induces FoxO-1 phosphorylation, leading to derepression of VCAM-1 expression. BM-MC-EC adhesion is increased by thrombin in WT ECs. BM-MC-EC adhesion is further increased when FoxO-1 is silenced, but decreased when FoxO-1 phosphorylation is inhibited. These results demonstrate that the Gas6-FoxO-1 signaling axis plays an important role in VCAM-1 expression in the context of VTE by promoting BM-MC-EC adhesion.

Quality controls on cord blood unit contiguous segments: recommendation of the SFGM-TC.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) set up its fourth annual series of workshops which brought together practitioners from all of its member centers. These workshops took place in September 2013 in Lille. Literature and intra-laboratories studies suggest that attached segment is representative of cord blood unit (CBU). Nevertheless, some discrepancies have been observed when analyzing large data registries. To address these issues, we have listed recommendations to increase the standardization of segment processing and quality control (QC), information on units of measurement and specifications and action to be taken in case of out of specifications QC results on segment.

Gas6-induced tissue factor expression in endothelial cells is mediated through caveolin-1-enriched microdomains.

BACKGROUND: Gas6 has been shown to interact with Axl in endothelial cells and to induce several signaling pathways involved in cell survival and proliferation. However, the interaction of Gas6/Axl with lipid raft/caveolin-1 in endothelial cells and its role in thrombosis are unknown. OBJECTIVES: We tested whether Axl and/or caveolin-1 is involved in Gas6-induced Akt, ERK1/2, and c-Src activation leading to altered tissue factor expression in endothelial cells. METHODS: Gas6-treated endothelial cells were transfected with small interfering RNA (siRNA) for Axl, caveolin-1, c-Src, and Akt or treated with pharmacological inhibitors of c-Src and ERK1/2. Sucrose gradient centrifugation and confocal microscopy were used to study lipid raft/caveolin-1-enriched fractions. Akt, ERK1/2, p38, and c-Src activation was analyzed by Western blot analysis. Tissue factor expression was assessed by real-time quantitative polymerase chain reaction and immunofluorescence. RESULTS AND CONCLUSION: Gas6 induced Axl and c-Src localization into lipid raft/caveolin-1-enriched fractions. Gas6 increased the phosphorylation of Akt, ERK1/2, and c-Src but not p38. Using siRNA, we demonstrated that Axl is required for Akt, ERK1/2, and c-Src activation after Gas6 stimulation. siRNA for caveolin-1 blocked Gas6-induced phosphorylation of Akt, ERK1/2, and c-Src. c-Src downregulation inhibited Gas6-induced Akt but not ERK1/2 phosphorylation. Finally, Gas6 increased tissue factor mRNA and protein expression in endothelial cells. Tissue factor expression was blocked by siRNA for Axl, caveolin-1, or Akt as well as c-Src inhibition. These data demonstrate that the signaling pathway Gas6/Axl/caveolin-1/c-Src/Akt is required for tissue factor expression in endothelial cells, providing mechanistic insight into how Gas6 exerts its prothrombotic role in the vasculature.

[Hematopoietic Stem Cell Transplantation using unrelated cord blood: which unit to choose? A report of the SFGM-TC]. / Allogreffe de cellules souches hématopoïétiques à partir d'un sang cordon non apparenté: quelle unité choisir?

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. Here we report our results and recommendations regarding the choice of optimal unrelated cord blood unit in terms of cell dose, HLA-matching and other characteristics.

Disease status is a more reliable predictive factor than histology in lymphoma patients after reduced-intensity conditioning regimen and allo-SCT.

We analyzed 113 patients with lymphoma who underwent allogeneic transplantation with reduced-intensity conditioning (allo-RIC) regimens at a single institution, from February 2001 through November 2009, searching for factors predictive of the outcome. At the time of transplantation, 60% of patients were in CR, 29% in PR and 11% had progressive or stable disease. At a median follow-up of 34 months (confidence interval (CI) 17-45), the 3-year OS and PFS were 59% (CI 48-68%) and 51% (CI 41-61%), respectively. The 100-day and 2-year nonrelapse mortalities (NRM) were 6% and 28% (CI 20-35%), respectively. Grade II-IV acute GVHD (aGVHD) incidence was 38%, and the global incidence of chronic GVHD was 33%. In univariate analysis, OS was influenced by disease status before allo-RIC; aGVHD negatively affected on survival. Similarly, PFS was influenced only by disease status. Histological subtype did not affect OS or PFS. We conclude that disease status at the time of transplantation significantly influences survival in patients receiving allo-RIC for lymphoma, whereas histological subtype does not. This reinforces the need to administer more effective debulking treatments to lymphoma patients, for optimal benefit of allogeneic immune recognition of minimal residual disease, independently from lymphoma histology.

[Quality control of defrosted cord blood units: results from an inter-laboratory study]. / Contrôle de qualité des greffons de sang placentaire décongelés : résultats d'une étude multicentrique.

PURPOSE: Today, haematopoietic stem cell graft from placental blood concerns more than 15 % of allogeneic grafts. An inter-laboratory study of the quality control of defrosted cord blood units has been coordinated by the French society for cell and tissue bioengineering (SFBCT), with the cord blood bank of Bourgogne Franche-Comté and controlled by the French health products safety agency (Afssaps). The aim of this study is to ensure the inter-laboratory reproducibility of the quality controls practised by the banks during defrosting. The cellular outputs were analyzed according to the defrosting techniques, according to the method used in flow cytometry: single-platform (SP) versus double-platform (DP), or the product nature, i.e. in total blood or miniaturized. METHODS: Forty-two units of placental blood (USP), which were out of range were provided for defrosting to 14 participating sites. USP were defrosted and controlled according to the procedures of each bank. Once the USP is defrosted, a part of the product was controlled by the site and the other part by Afssaps. Following controls were carried out: numeration of the total nucleated cells (TNC) and of CD34+ cells (made by a SP method in Afssaps) and functional assay. RESULTS: Concerning TNC, the defrosting sites obtained a cellular output of 94 %+/-28 in day 0 compared with an output of 72 %+/-24 in Afssaps showing a rather good stability of the USP transmitted with an average deviation of 23 %+/-22. The freezing process with or without reduction of volume does not affect this variation. Concerning the numeration of CD34+ cells, the average deviation between the participating sites and Afssaps was 29 %+/-23 compared with 21 %+/-16 for the sites using a SP method against 47 %+/-25 for those using a DP method. The CD34+ outputs are equal to 82 % +/- 60 in day 0 for the participating sites against 52 %+/-20 for Afssaps. For the sites using a DP method, it is stressed that this output is particularly high with a rate of 126 %+/-90 (n=15) whereas it is 62 %+/-20 (n=32) for the sites using a SP method. CONCLUSION: These results underline a good stability of viable CD34+ cells and a greater reliability of the SP methods for the CD34+ cell numeration for these defrosted USP. Lastly, the results of the functional assay regarding the average clonogenicities (equal to 15 %) reinforce the conclusions on the quality of the defrosted products.

CD34(+) progenitors are reproducibly recovered in thawed umbilical grafts, and positively influence haematopoietic reconstitution after transplantation.

Cord blood (CB) units are increasingly used for allogeneic transplantation. Cell dose, a major factor for CB selection, is evaluated before freezing by each CB bank, using various techniques. This may introduce variability and affect the prediction of cell recovery after thawing, or haematopoietic reconstitution. Forty-two children were transplanted at the same institution with unrelated CB units. All units were thawed and evaluated at the same cell therapy facility, using standard procedures. We investigated: (i) factors that affect cell loss after thawing, and (ii) the importance of CD34(+) cell doses. Prefreeze and post-thaw CD34(+) cell doses were statistically correlated, thus suggesting that variability in numeration techniques used by different CB banks does not compromise the biological and clinical value of these figures. CD34(+) cell recovery appeared to be correlated with the absolute number of CD34(+) cells per frozen bag. Infused CD34(+) is the cell dose that better correlates with platelet reconstitution delay; in addition, when using a quartile comparison, haematopoietic recovery appeared to be related with prefreeze and post-thaw CD34(+) cell doses. We conclude that enumeration of CD34(+) cells in CB units is of biological significance, and may help select CB units and identify patients at risk of delayed recovery.