Publications by authors named "Marie-Christine Jacob"

43 Publications

The metabolic reprogramming in acute myeloid leukemia patients depends on their genotype and is a prognostic marker.

Blood Adv 2021 01;5(1):156-166

University Grenoble Alpes (UGA)/INSERM U1209/National Center for Scientific Research 5309, Institute for Advanced Biosciences, Grenoble, France.

Leukemic cells display some alterations in metabolic pathways, which play a role in leukemogenesis and in patients' prognosis. To evaluate the characteristics and the impact of this metabolic reprogramming, we explore the bone marrow samples from 54 de novo acute myeloid leukemia (AML) patients, using an untargeted metabolomics approach based on proton high-resolution magic angle spinning-nuclear magnetic resonance. The spectra obtained were subjected to multivariate statistical analysis to find specific metabolome alterations and biomarkers correlated to clinical features. We found that patients display a large diversity of metabolic profiles, according to the different AML cytologic subtypes and molecular statuses. The link between metabolism and molecular status was particularly strong for the oncometabolite 2-hydroxyglutarate (2-HG), whose intracellular production is directly linked to the presence of isocitrate dehydrogenase mutations. Moreover, patients' prognosis was strongly impacted by several metabolites, such as 2-HG that appeared as a good prognostic biomarker in our cohort. Conversely, deregulations in phospholipid metabolism had a negative impact on prognosis through 2 main metabolites (phosphocholine and phosphoethanolamine), which could be potential aggressiveness biomarkers. Finally, we highlighted an overexpression of glutathione and alanine in chemoresistant patients. Overall, our results demonstrate that different metabolic pathways could be activated in leukemic cells according to their phenotype and maturation levels. This confirms that metabolic reprogramming strongly influences prognosis of patients and underscores a particular role of certain metabolites and associated pathways in AML prognosis, suggesting common mechanisms developed by leukemic cells to maintain their aggressiveness even after well-conducted induction chemotherapy.
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http://dx.doi.org/10.1182/bloodadvances.2020002981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805315PMC
January 2021

Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression for ruling out myelodysplastic syndromes: a prospective validation study.

Ann Hematol 2021 May 10;100(5):1149-1158. Epub 2021 Feb 10.

Quality of Care Unit, INSERM CIC 1406, Grenoble University Hospital, F-38043, Grenoble, France.

Suspicion of myelodysplastic syndromes (MDS) is the most common reason for bone marrow aspirate in elderly patients. This study aimed to prospectively validate the accuracy for flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression in ruling out MDS. We enrolled 62 consecutive patients who were referred for suspected MDS, based on medical history and peripheral blood cytopenia. The accuracy of intra-individual robust coefficient of variation (RCV) for peripheral blood neutrophil myeloperoxidase expression was assessed with a prespecified 30% threshold. Cytomorphological evaluation of bone marrow aspirate performed by experienced hematopathologists confirmed MDS in 23 patients (prevalence, 37%), unconfirmed MDS in 32 patients (52%, including 3 patients with idiopathic cytopenia of undetermined significance (ICUS)), and was uninterpretable in 7 patients (11%). The median intra-individual RCV values for neutrophil myeloperoxidase expression in peripheral blood were 37.4% (range, 30.7-54.1), 29.2% (range, 28.1-32.1), and 29.1% (range, 24.7-37.8) for patients with confirmed suspicion of MDS, ICUS, and unconfirmed suspicion of MDS, respectively (P<0.001). The area under the ROC curve was 0.92 (95% confidence interval, 0.86-0.99). An intra-individual RCV value lower than 30% ruled out MDS for 35% (i.e., 19/55) patients referred for suspected disease, with 100% sensitivity (95% CI, 85-100%) and 100% negative predictive value (95% CI, 82-100%) estimates. This study shows that flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression might obviate the need for bone marrow aspirate for 35% of patients with suspected MDS. Trial registration: ClinicalTrials.gov identifier: NCT03363399 (first posted on December 6, 2017).
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http://dx.doi.org/10.1007/s00277-021-04446-7DOI Listing
May 2021

Myelodysplastic Syndromes.

N Engl J Med 2020 12;383(26):2590

University Grenoble Alpes, Grenoble, France

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http://dx.doi.org/10.1056/NEJMc2032391DOI Listing
December 2020

Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape.

Haematologica 2020 10 13;Online ahead of print. Epub 2020 Oct 13.

Service de Biologie Médicale, Centre Hospitalier de Versailles A. Mignot, Le Chesnay.

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs are associated with myeloid malignancies: most frequently Chronic MyeloMonocytic Leukemia (CMML) but also Acute Myeloid Leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs in pDC-AML harbor a clearly different phenotype from BPDCN: CD4+ CD56- in 100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a pre-pDC stage. In all cases, pDCs, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.
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http://dx.doi.org/10.3324/haematol.2020.253740DOI Listing
October 2020

Prognostic value of high-sensitivity measurable residual disease assessment after front-line chemoimmunotherapy in chronic lymphocytic leukemia.

Leukemia 2021 06 15;35(6):1597-1609. Epub 2020 Sep 15.

Laboratoire d'Hématologie, APHP CHU Avicenne, Bobigny, France.

Measurable residual disease (MRD) status is widely adopted in clinical trials in patients with chronic lymphocytic leukemia (CLL). Findings from FILO group trials (CLL2007FMP, CLL2007SA, CLL2010FMP) enabled investigation of the prognostic value of high-sensitivity (0.7 × 10) MRD assessment using flow cytometry, in blood (N = 401) and bone marrow (N = 339), after fludarabine, cyclophosphamide, and rituximab (FCR)-based chemoimmunotherapy in a homogeneous population with long follow-up (median 49.5 months). Addition of low-level positive MRD < 0.01% to MRD ≥ 0.01% increased the proportion of cases with positive MRD in blood by 39% and in bone marrow by 27%. Compared to low-level positive MRD < 0.01%, undetectable MRD was associated with significantly longer progression-free survival (PFS) when using blood (72.2 versus 42.7 months; hazard ratio 0.40, p = 0.0003), but not when using bone marrow. Upon further stratification, positive blood MRD at any level, compared to undetectable blood MRD, was associated with shorter PFS irrespective of clinical complete or partial remission, and a lower 5-year PFS rate irrespective of IGHV-mutated or -unmutated status (all p < 0.05). In conclusion, high-sensitivity (0.0007%) MRD assessment in blood yielded additional prognostic information beyond the current standard sensitivity (0.01%). Our approach provides a model for future determination of the optimal MRD investigative strategy for any regimen.
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http://dx.doi.org/10.1038/s41375-020-01009-zDOI Listing
June 2021

Multicentric MFI30 study: Standardization of flow cytometry analysis of CD30 expression in non-Hodgkin lymphoma.

Cytometry B Clin Cytom 2021 Jul 17;100(4):488-496. Epub 2020 Aug 17.

Laboratoire d'Hématologie, Groupe Hospitalier de la région Mulhouse Sud Alsace, Mulhouse, France.

CD30 transmembrane receptor, a member of the tumor necrosis factor receptor family, is expressed in different lymphomas. Brentuximab vedotin (BV), a CD30 monoclonal antibody (Ab)-drug conjugate, is effective in CD30-positive lymphomas. However, the response to BV is not always correlated to CD30 expression detected by immunohistochemistry (IHC). The objectives of this study were to standardize and evaluate CD30 intensity by flow cytometry (FCM) in non-Hodgkin's lymphomas. Twelve centers analyzed 161 cases on standardized cytometers using normalized median fluorescence intensity (nMFI30) of three different Abs, of which one clone can recognize the same epitope as BV. FCM distinguished four groups of cases: negative group (n = 110) which showed no expression with the three clones; high positive group (n = 13) which gave nMFI30 > 5% with all tested clones; dim positive group (n = 17) which showed nMFI30 > 1% with all tested clones and <5% for at least one; discordant group (n = 21) with positive and negative expression of the different clones. In consistency with the literature, CD30 was positive in all anaplastic large cell lymphomas, in some diffuse large B-cell lymphomas (DLBCL), and in other rare lymphomas. FCM results were concordant with those of IHC in 77% of cases. Discrepancies could be explained by clones-related differences, microenvironment, or intracytoplasmic staining. Interestingly, FCM was more sensitive than IHC in 11% of cases, especially in DLBCL. Multicenter standardized FCM of specific CD30 could improve case detection and extend the treatment of BV to various CD30-positive lymphomas.
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http://dx.doi.org/10.1002/cyto.b.21940DOI Listing
July 2021

Transformation of a low-grade follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing Terminal deoxynucleotidyl Transferase: a case report.

J Med Case Rep 2020 Jul 27;14(1):117. Epub 2020 Jul 27.

Department of Pathology, CHU de Grenoble, Grenoble, France.

Background: High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 is an aggressive mature B-cell neoplasm, whereas B-lymphoblastic lymphoma is immature cell proliferation, with a frequent positivity for terminal deoxynucleotidyl transferase. The transformation of a low-grade follicular lymphoma into a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase is a very rare event.

Case Presentation: A 55-year-old Caucasian man was followed for a grade 1-2 follicular lymphoma carrying a t(14;18) IGH/BCL2+ and was initially treated with R-CHOP. The follicular lymphoma presented two relapses. In the third relapse, the patient had multiple lymphadenopathy and ascites, which motivated a retroperitoneal biopsy and an ascitic tap. These samples were analyzed by histological, cytological, flow cytometric, cytogenetic, and molecular assessments. The patient died of a multiple organ dysfunction syndrome 2 weeks after his third relapse. The biopsy revealed a diffuse proliferation made up of two types of tumor cells: centroblasts (Bcl-6-positive) and immature cells (terminal deoxynucleotidyl transferase-positive). Flow cytometric analysis confirmed the immature phenotype, with an expression of terminal deoxynucleotidyl transferase, combined with a loss of membrane immunoglobulins. The cytogenetic analysis performed on the ascites revealed a clonal evolution characterized by a t(8;22)(q24;q11) MYC+ translocation not previously detected in follicular lymphoma. Fluorescence in situ hybridization confirmed the double rearrangement of the BCL2 and MYC genes. Polymerase chain reactions and sequencing were used to study the clonal relationship between follicular lymphoma and the secondary tumors. The IGVH gene rearrangement revealed a unique clonal rearrangement involving an IGVH4-59 subset in all three specimens.

Conclusion: These findings suggest a clonal relationship between the two types of lymphoma cells. Furthermore, they support the transformation of an acute follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase. This case report highlights the possible transformation of follicular lymphoma into a highly aggressive and immature proliferation.
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http://dx.doi.org/10.1186/s13256-020-02433-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384216PMC
July 2020

Baseline anti-CMV cellular immunity is similar between patients with a kidney transplant or receiving hemodialysis.

Transpl Int 2020 08 14;33(8):961-962. Epub 2020 May 14.

Nephrology, Hemodialysis, Apheresis, and Kidney Transplantation Department, University Hospital of Grenoble-Alpes, Grenoble, France.

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http://dx.doi.org/10.1111/tri.13620DOI Listing
August 2020

Increased S100A8 expression in bone marrow plasma by monocytic cells from acute myeloid leukemia patients.

Hematol Oncol 2020 Feb 6;38(1):114-118. Epub 2020 Jan 6.

Institute for Advanced Biosciences, University Grenoble Alpes /INSERM U1209/CNRS 5309, Grenoble, France.

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http://dx.doi.org/10.1002/hon.2707DOI Listing
February 2020

How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients?

Blood Adv 2019 12;3(24):4238-4251

Laboratoire d'Hématologie, CHU Estaing 1, Clermont-Ferrand, France.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)-like, acute lymphoid leukemia (ALL)-like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])-like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.
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http://dx.doi.org/10.1182/bloodadvances.2019000647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929390PMC
December 2019

Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience.

Cytometry A 2019 09 31;95(9):1008-1018. Epub 2019 Jul 31.

Groupe Hospitalier Pitié-Salpêtrière, UF Phénotypage des Hémopathies, Centre d'Ecologie Cellulaire, Service d'hématologie Biologique, Paris, France.

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23844DOI Listing
September 2019

Switching renal transplant recipients to belatacept therapy: results of a real-life gradual conversion protocol.

Transpl Immunol 2019 10 6;56:101207. Epub 2019 May 6.

Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States.

Conversion to belatacept immunosuppression is a therapeutic option for renal-transplant recipients with calcineurin inhibitors (CNI) toxicity, but it associates with high risk of acute rejection. Gradual conversion and serial immune monitoring with urinary chemokine CXCL9 may allow increasing safety of this maneuver. We converted kidney transplant recipients with signs of toxicity to CNI or other immunosuppressive drugs to belatacept over a 2-month period. We monitored renal function, metabolic profile, and circulating lymphocyte subsets. We also quantified urinary CXCL9 over a 12-month follow-up period. Between September 2016 and March 2017, 35 patients were successfully switched to belatacept immunosuppression at 3.3 (1.3-7.2) years after transplant. Two patients had a reversible rise in serum creatinine, associated with acute rejection in one case. Urinary CXCL9 increased before serum creatinine. After conversion, blood pressure and HbA1c significantly declined while eGFR and proteinuria remained stable. The percentage of circulating effector T cells and memory B cells significantly declined. Conversion from CNI to belatacept, in this setting, was feasible and safe, provided it was performed over a 2-month time-period. Monitoring urinary CXCL9 may further increase safety through earlier identification of patients at risk for acute rejection. The procedure associates with improved blood pressure, metabolic profile, and reduced circulating effector T and B cells.
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http://dx.doi.org/10.1016/j.trim.2019.04.002DOI Listing
October 2019

Impact of bronchial colonization with Candida spp. on the risk of bacterial ventilator-associated pneumonia in the ICU: the FUNGIBACT prospective cohort study.

Intensive Care Med 2019 06 24;45(6):834-843. Epub 2019 Apr 24.

Medical ICU, Gabriel-Montpied University Hospital,, Clermont-Ferrand,, France.

Introduction: Respiratory tract Candida spp. colonization is associated with more frequent bacterial ventilator-associated pneumonia (VAP). However, this colonization could be causally related to VAP or simply reflect the immune paralysis associated with multiple organ failure.

Objective: To prospectively evaluate the relationship between Candida spp. colonization and bacterial VAP in mechanically ventilated patients with multiple organ failure.

Inclusion: Patients receiving mechanical ventilation for > 4 days and presenting multiple organ failure were included. Tracheal colonization with Candida spp. was evaluated at inclusion (day 0, D0) and every 4 days until extubation. Quantitative proximal and tracheal cultures were performed at each VAP episode. Monocyte human leukocyte antigen-DR isotype (mHLA-DR) expression and the ratio of polymononuclear leukocytes to lymphocytes were used to evaluate immunoparalysis at D0 and D7. The relationship between fungal colonization and VAP was modelled using cause-specific models for repeated events with adjustment for time-dependent confounders and immune factors.

Results: A total of 213 patients, with a median age of 64, simplified acute physiology score II (SAPS II) score 55 and sequential organ failure assessment (SOFA) score 10, mainly admitted for medical reasons (n = 197, 92%), were enrolled in 2012-2015. The median ICU stay was 24 days and the mortality rate was 32% (69 cases). Median mHLA-DR was 5916 Ab-bound/cell [3863-8934]; median lymphocyte count, 0.9Giga/L [0.6-1.3]; neutrophil-to-lymphocyte ratio, 10.9 [6.5-19.7]. Overall, 146 cases (68.5%) had tracheal colonization with Candida spp. An episode of VAP occurred (either for the first or only time) in 62 (29.1%) cases 5.5 days (median) after D0; a second episode occurred in 12 (5.6%) cases, 15.5 days (median) after D0. After adjustment, bronchial colonization with Candida was not associated with VAP [adjusted cause-specific hazard ratio = 0.98 (0.59-1.65), p = 0.95].

Conclusion: In patients with mechanical ventilation for more than 4 days and multiple organ failure, bronchial colonization with Candida spp. was not associated with VAP, even after adjustment for immune function.
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http://dx.doi.org/10.1007/s00134-019-05622-0DOI Listing
June 2019

Flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for ruling out myelodysplastic syndromes: a diagnostic accuracy study.

Haematologica 2019 12 19;104(12):2382-2390. Epub 2019 Apr 19.

Service d'Hématologie Biologique, Hopital Estaing, Centre Hospitalier Universitaire de Clermont-Ferrand, F-63003 Clermont-Ferrand.

Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% 30.9%; <0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.
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http://dx.doi.org/10.3324/haematol.2018.202275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959174PMC
December 2019

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.

J Vis Exp 2018 11 3(141). Epub 2018 Nov 3.

Department of Hematology, University Hospital of Saint-Etienne.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.
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http://dx.doi.org/10.3791/57867DOI Listing
November 2018

Dyserythropoiesis evaluated by the RED score and hepcidin:ferritin ratio predicts response to erythropoietin in lower-risk myelodysplastic syndromes.

Haematologica 2019 03 4;104(3):497-504. Epub 2018 Oct 4.

Department of Hematology, CHU Grenoble-Alpes, Grenoble.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level <10 g/dL. Patients could be red blood cell transfusion-dependent or not and were given epoetin zeta 40 000 IU/week. Serum erythropoietin level, iron parameters, hepcidin, flow cytometry Ogata and RED scores, and growth-differentiation factor-15 levels were determined at baseline, and molecular analysis by next-generation sequencing was also conducted. Erythroid response (defined according to the International Working Group 2006 criteria) was assessed at week 12. Seventy patients, with a median age of 78 years, were included in the study. There were 22 patients with refractory cytopenia with multilineage dysplasia, 19 with refractory cytopenia with unilineage dysplasia, 14 with refractory anemia with ring sideroblasts, four with refractory anemia with excess blasts-1, six with chronic myelomonocytic leukemia, two with del5q-and three with unclassifiable myelodysplastic syndrome. According to the revised International Prognostic Scoring System, 13 had very low risk, 47 had low risk, nine intermediate risk and one had high-risk disease. Twenty patients were transfusion dependent. Forty-eight percent had an erythroid response and the median duration of the response was 26 months. At baseline, non-responders had significantly higher RED scores and lower hepcidin:ferritin ratios. In multivariate analysis, only a RED score >4 (=0.05) and a hepcidin:ferritin ratio <9 (=0.02) were statistically significantly associated with worse erythroid response. The median response duration was shorter in patients with growth-differentiation factor-15 >2000 pg/mL and a hepcidin:ferritin ratio <9 (=0.0008 and =0.01, respectively). In multivariate analysis, both variables were associated with shorter response duration. Erythroid response to epoetin zeta was similar to that obtained with other erythropoiesis-stimulating agents and was correlated with higher baseline hepcidin:ferritin ratio and lower RED score. .
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http://dx.doi.org/10.3324/haematol.2018.203158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395339PMC
March 2019

Accurate quantification of fourteen normal bone marrow cell subsets in infants to the elderly by flow cytometry.

Cytometry B Clin Cytom 2018 09;94(5):627-636

Department of Immunology, Grenoble-Alpes University Hospital (CHUGA), Grenoble, F-38700, France.

Background: Studies of normal bone marrow (BM) cell composition by flow cytometry are scarce. Presently, we aimed to quantify 14 cell subsets from infants to elderly patients.

Methods: Cell subsets in BM samples from 180 individuals without morphologically abnormal leukocytes were analyzed using a single combination of eight antibodies: CD3/CD10/CD38/CD19/CD36/CD16/CD34/CD45.

Results: By comparison with the Holdrinet score, we first validated the immature granulocyte/neutrophil (IGRA/N) ratio as a readily obtainable criterion of BM sample purity in 145 cases. Then, the 115 highly pure samples were selected (IGRA/N ≥ 1.2) and analyzed according to age group. CD34+ myeloblasts became progressively more infrequent with age: median 1.4% in infancy to 0.5% in the elderly. Neutrophils increased: 10.7% to 22.8%; all other myeloid subsets, IGRA, eosinophils, basophils and monocytes remained stable: respectively 40.3% to 46.7%, 2.0% to 2.8%, 0.2% to 0.3%, and 4.4% to 5.0% throughout life. Erythroblasts were lower in children (8.4% to 10.3%) than in adults (12.5% to 15.1%). For lymphoid cells, hematogones and transitional B-cells decreased: 15.5% to 0.6% and 3.6% to 0.1%, respectively; mature lymphocytes remained stable: B-cells: 1.4% to 2.8%, T-cells: 5.8% to 8.7%, and NK-cells: 0.7% to 1.4%. Plasma cells varied slightly: 0.1% to 0.5%. Differences of about 40% were seen in moderately pure (IGRA/N: 0.5 to 1.2) BM samples.

Conclusion: We thus provide the first values for 14 myeloid and lymphoid subsets characterizing BM cell composition in 5 age ranges. They should provide important information when screening patients for hematological disorders or abnormal bone marrow development. © 2018 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21643DOI Listing
September 2018

Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry.

PLoS One 2016;11(9):e0162209. Epub 2016 Sep 22.

Laboratoire d'Immunologie, Département d'Hématologie, Oncogénétique et Immunologie, Pôle de Biologie, Grenoble University Hospital, Grenoble, France.

A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45) and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033467PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162209PLOS
September 2016

Remodeling of B-Cell Subsets in Blood during Pegylated IFNα-2a Therapy in Patients with Chronic Hepatitis B Infection.

PLoS One 2016 9;11(6):e0156200. Epub 2016 Jun 9.

CHU Grenoble, Michallon Hospital, Hepato-gastroenterology unit, Grenoble, F-38043, France.

The ultimate goal of pegylated interferon-alfa-2a (Peg-IFN-α) therapy in chronic hepatitis B (CHB) infection is HBsAg seroconversion. Even though B cells are major mediators of a positive clinical outcome, their modulation during Peg-IFN-α therapy has not yet been described. We investigated here the effects of Peg-IFN-α on eight circulating B-cell subsets thanks to an original multi-gating approach based on CD19, CD27, IgD, CD10, and CD38 markers in patients with CHB treated with nucleos(t)ide analog alone or in combination with Peg-IFN-α. These dynamic changes were analyzed during the 48-weeks of Peg-IFN-α therapy and up to 2 years after the cessation of treatment. The CD19+CD27-IgD+CD10+CD38high transitional B cells and the CD19+CD27+IgD-CD10-CD38high plasmablasts continuously increased, whereas the CD19+CD27-IgD+CD10-CD38low naive, CD19+CD27+IgD+ natural memory, and CD19+CD27+IgD-CD10-CD38low post-germinal center B cells decreased during the course of Peg-IFNα treatment. Such modulations correlated with a sustained increase in sCD30 levels and the decrease in plasma HBsAg. However, no seroconversion occurred and all parameters returned to baseline after the stop of the treatment. Peg-IFN-α therapy mediates a remodeling of B-cell compartmentalization, without clinical relevance. Our study provides new insights into the immunomodulatory effects of Peg-IFN-α on circulating B-cells, and questioned the benefit of the add-on Peg-IFN-α treatment in CHB.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156200PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900671PMC
July 2017

Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms.

Blood 2016 06 8;127(24):3040-53. Epub 2016 Apr 8.

INSERM U1209, CNRS UMR 5309, Faculté de Médecine, Université Grenoble Alpes, Institut Albert Bonniot, Grenoble, France;

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN.
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http://dx.doi.org/10.1182/blood-2015-09-671040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043425PMC
June 2016

One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry.

Cytometry B Clin Cytom 2017 07 6;92(4):299-309. Epub 2016 May 6.

Department of Immunology CHU Grenoble, La Tronche, F-38700, France.

Background: Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8-color tube for providing equivalent information, using flow cytometry.

Methods: 99 bone marrow samples were classified into 2 groups, (i) 51 samples, obtained from either healthy donors (n = 4) or patients with various diseases at diagnosis or during remission that did not present a hematological malignancy (n = 47), and (ii) 48 pathological samples with quantitative and/or qualitative abnormalities. A panel of eight antibodies-CD3-FITC/CD10-PE/CD38-PerCP-Cy5.5/CD19-PECy7/CD36-APC/CD16-APC-H7/CD34-BV421/CD45-V500-was tested to identify the main cell subsets at different stages of maturation using a FACSCanto-II analyzer.

Results: We first proposed a strategy of sequential gating leading to the identification of 14 leukocyte subsets, that is, erythroblasts, monocytes, B-lymphoid cells from hematogones to plasma-cells (5 subsets), T- and NK-cells, polymorphonuclear cells (neutrophils, eosinophils, and basophils), myeloblasts and other immature granular cells. This approach was validated by comparing flow cytometry and microscopic morphological examination, both in cases of normal and abnormal samples. Interestingly, cell identification, and numeration by flow cytometry was easy to perform and highly reproducible.

Conclusion: A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21369DOI Listing
July 2017

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

Cytometry B Clin Cytom 2015 Jan 1;88(1):21-9. Epub 2014 Nov 1.

Hematology Laboratory, Hôpital de la Timone, Marseille, France.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10(-4) . Here we report the MFC methodological aspects from this multi-center experience.

Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC, or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment.

Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10(-4) cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL.

Conclusions: Measurement of MRD by MFC at the 10(-4) cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL.
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http://dx.doi.org/10.1002/cyto.b.21195DOI Listing
January 2015

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

J Immunother 2013 Apr;36(3):181-9

Etablissement Français du Sang, 29 av du Maquis du Grésivaudan, BP35, 38701 La Tronche, France.

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.
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http://dx.doi.org/10.1097/CJI.0b013e318288f8c1DOI Listing
April 2013

Inter-laboratory assessment of flow cytometric monocyte HLA-DR expression in clinical samples.

Cytometry B Clin Cytom 2013 Jan-Feb;84(1):59-62. Epub 2012 Sep 14.

Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France.

Background: Diminished expression of human leukocyte antigen DR on circulating monocytes (mHLA-DR) is a reliable indicator of immunosuppression in critically ill patients, predictive of both adverse outcome and septic complications. The objective of the present work was to test, in an inter-laboratory clinical study, a standardized protocol for mHLA-DR measurement by flow cytometry.

Methods: mHLA-DR was assessed in fresh whole blood according to a standardized staining protocol. Cells were analyzed on different flow cytometers (FC500, Navios, FACS Canto II) in different laboratories (Lyon and Grenoble). Results were expressed as numbers of antibodies bound per cell (AB/C).

Results: Correlations between results were excellent (Pearson and interclass correlation coefficients > 0.98). Coefficients of variations for intra-assay precision ranged from 1.9 to 3.2%.

Conclusion: The present report highlights the robustness of this standardized flow cytometric protocol for mHLA-DR measurement in multicentric clinical studies.
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http://dx.doi.org/10.1002/cyto.b.21043DOI Listing
June 2013

Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire.

Mod Pathol 2012 Sep 25;25(9):1246-57. Epub 2012 May 25.

Université Joseph Fourier Grenoble 1, France.

Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vβ repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded Vß family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vβ expansions were commonly detected within CD4+ or CD8+ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vβ expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vβ expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vβ families, the occurrence of a Vβ17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vβ repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.
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http://dx.doi.org/10.1038/modpathol.2012.74DOI Listing
September 2012

Exploration of the lysis mechanisms of leukaemic blasts by chimaeric T-cells.

J Biomed Biotechnol 2010 13;2010:234540. Epub 2010 Jun 13.

Etablissement Français du Sang, 38701 La Tronche, France.

Adoptive transfer of specific cytotoxic T lymphocytes (CTL) and Cytokine Induced Killer Cells (CIK) following genetic engineering of T-cell receptor zeta hold promising perspective in immunotherapy. In the present work we focused on the mechanisms of anti-tumor action of effectors transduced with an anti-CD19 chimaeric receptor in the context of B-lineage acute lymphoblastic leukemia (B-ALL). Primary B-ALL blasts were efficiently killed by both z-CD19 CTL and z-CD19 CIK effectors. The use of death receptor mediated apoptosis of target cells was excluded since agonists molecules of Fas and TRAIL-receptors failed to induce cell death. Perforin/granzyme pathway was found to be the mechanism of chimaeric effectors mediated killing. Indeed, cytolytic effector molecules perforin as well as granzymes were highly expressed by CTL and CIK. CD19 specific stimulation of transduced effectors was associated with degranulation as attested by CD107 membrane expression and high IFN-gamma and TNF-alpha release. Moreover inhibitors of the perforin-based cytotoxic pathway, Ca(2+)-chelating agent EGTA and Concanamycin A, almost completely abrogated B-ALL blast killing. In conclusion we show that the cytolysis response of z-CD19 chimaeric effectors is predominantly mediated via perforin/granzyme pathway and is independent of death receptors signaling in primary B-ALL.
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http://dx.doi.org/10.1155/2010/234540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896659PMC
October 2010

Plasmacytoid dendritic cells and dermatological disorders: focus on their role in autoimmunity and cancer.

Eur J Dermatol 2010 Jan-Feb;20(1):16-23. Epub 2009 Oct 22.

Dermatologie, Pôle Pluridisciplinaire de Médecine, CHU Grenoble, Hôpital Michallon, F-38043 France.

Dendritic cells (DC), considered as immunological sentinels of the organism since they are antigen presenting cells, create the link between innate and adaptive immunity. DC include myeloid dendritic cells (MDC) and plasmacytoid dendritic cells (PDC). The presence of PDC, cells capable of producing large quantities of interferon alpha (IFN-alpha) in response to pathogenic agents or danger signals, seems to be closely related to pathological conditions. PDC have been observed in inflammatory immunoallergic dermatological disorders, in malignant cutaneous tumours and in cutaneous lesions of infectious origin. They seem to play a crucial role in the initiation of the pathological processes of autoimmune diseases such as lupus or psoriasis. Their function within a tumour context is not as well known and is controversial. They could have a tolerogenic role towards tumour cells in the absence of an activator but they also have the capacity to become activated in response to Toll-like receptor (TLR) ligands and could therefore be useful for therapeutic purposes.
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http://dx.doi.org/10.1684/ejd.2010.0816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881955PMC
April 2010

Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia.

Br J Haematol 2009 Jun 8;145(5):624-36. Epub 2009 Apr 8.

INSERM UMR645, Université of Franche-Comté, Etablissement Français du Sang Bourgogne Franche-Comté, 1 boulevard A. Fleming, Besançon, France.

The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4(+) CD56(+) lineage(neg) CD45RA(+)/RO(neg) CD11c(neg) CD116(low) CD123(+) CD34(neg) CD36(+) HLA-DR(+). Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL-specific markers for diagnosis by flow-cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non-pDC - lymphoid or myeloid - acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA-2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non-pDC acute leukaemia cases. BDCA-4 expression was found on 13/16 pDCL, but also in 12% of non-pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B-cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4(+) CD56(+/-) MPO(neg) cCD3(neg) cCD79a(neg) CD11c(neg) profile and then the CD123(high), BDCA-2 and BDCA-4 expression. Atypical pDCL can be also identified this way and non-pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07679.xDOI Listing
June 2009

The Aurora Kinase C c.144delC mutation causes meiosis I arrest in men and is frequent in the North African population.

Hum Mol Genet 2009 Apr 15;18(7):1301-9. Epub 2009 Jan 15.

Département de Génétique et Procréation, CHU de Grenoble, Grenoble Cedex 9, France.

Infertility concerns a minimum of 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. In a previous study, we demonstrated that a homozygous mutation (c.144delC) in the Aurora Kinase C (AURKC) gene led to the production of large-headed polyploid multi-flagellar spermatozoa, a primary infertility phenotype mainly observed in North Africans. We now want to estimate the prevalence of the defect, to improve our understanding of AURKC physiopathology in spermatogenesis and assess its implication in oogenesis. A carrier frequency of 1/50 was established from individuals from the Maghrebian general population, comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. A total of 62 patients were genotyped, all who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n = 32), whereas no AURKC mutations were detected in the others. Two homozygous females were identified; both were fertile indicating that AURKC is not indispensible in oogenesis. Previous FISH results had showed a great chromosomal heterogeneity in these patient's spermatozoa. We demonstrate here by flow cytometry that all spermatozoa have in fact a homogeneous 4C DNA content and are thus all blocked before the first meiotic division. Our data thus indicate that a functional AURKC protein is necessary for male meiotic cytokinesis while its absence does not impair oogenesis.
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http://dx.doi.org/10.1093/hmg/ddp029DOI Listing
April 2009
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