Publications by authors named "Carmen Aanei"

25 Publications

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Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Front Immunol 2021 27;12:665541. Epub 2021 Apr 27.

Hematology Laboratory, Universitary Hospital of Saint-Etienne, Saint-Etienne, France.

Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.
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http://dx.doi.org/10.3389/fimmu.2021.665541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112610PMC
October 2021

A fixed-duration, measurable residual disease-guided approach in CLL: follow-up data from the phase 2 ICLL-07 FILO trial.

Blood 2021 02;137(8):1019-1023

Department of Hematology, Avicenne Hospital, AP-HP, Bobigny, France.

Trials assessing first-line, fixed-duration approaches in chronic lymphocytic leukemia (CLL) are yielding promising activity, but few long-term data are available. We report follow-up data from a phase 2 trial (ICLL07 FILO) in previously untreated, medically fit patients (N = 135). Patients underwent obinutuzumab-ibrutinib induction for 9 months; then, following evaluation (N = 130 evaluable), those in complete remission and with bone marrow measurable residual disease (BM MRD) <0.01% (n = 10) received ibrutinib for 6 additional months; those in partial remission and/or with BM MRD ≥0.01%, the majority (n = 120), also received 4 cycles of immunochemotherapy (fludarabine/cyclophosphamide-obinutuzumab). Beyond end of treatment, responses were assessed every 3 month and peripheral blood MRD every 6 months. At median follow-up 36.7 months from treatment start, progression-free and overall survival rates (95% confidence interval) at 3 years were 95.7% (92.0% to 99.5%) and 98% (95.1% to 100%), respectively. Peripheral blood MRD <0.01% rates were 97%, 96%, 90%, 84%, and 89% at months 16, 22, 28, 34, and 40, respectively. No new treatment-related or serious adverse event occurred beyond end of treatment. Thus, in previously untreated, medically fit patients with CLL, a fixed-duration (15 months), MRD-guided approach achieved high survival rates, a persistent MRD benefit beyond the end of treatment, and low long-term toxicity. This trial was registered at www.clinicaltrials.gov as #NCT02666898.
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http://dx.doi.org/10.1182/blood.2020008164DOI Listing
February 2021

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

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

Obinutuzumab and ibrutinib induction therapy followed by a minimal residual disease-driven strategy in patients with chronic lymphocytic leukaemia (ICLL07 FILO): a single-arm, multicentre, phase 2 trial.

Lancet Haematol 2019 Sep 16;6(9):e470-e479. Epub 2019 Jul 16.

Department of Hematology Biology, Assistance Publique Hopitaux de Paris, Pitié Salpêtrière, Paris, France.

Background: In patients with chronic lymphocytic leukaemia, achievement of a complete response with minimal residual disease of less than 0·01% (ie, <1 chronic lymphocytic leukaemia cell per 10 000 leukocytes) in bone marrow has been associated with improved progression-free survival. We aimed to explore the activity of induction therapy for 9 months with obinutuzumab and ibrutinib, followed up with a minimal residual disease-driven therapeutic strategy for 6 additional months, in previously untreated patients.

Methods: We did a single-arm, phase 2 trial in 27 university hospitals, general hospitals, and specialist cancer centres in France. Eligible patients were at least 18 years old and previously untreated, and had immunophenotypically confirmed B-cell chronic lymphocytic leukaemia; an Eastern Cooperative Oncology Group (ECOG) performance status score of less than 2; a Binet stage C according to IWCLL 2008 criteria or Binet stage A and B with active disease; no 17p deletion or absence of p53 mutation; and were considered medically fit. In the first part of the study (induction phase), all participants received eight intravenous infusions of obinutuzumab 1000 mg over six 4-weekly cycles and oral ibrutinib 420 mg once per day for 9 months. In part 2, after assessment on day 1 of month 9, patients with a complete response and bone marrow minimal residual disease of less than 0·01% received only oral ibrutinib 420 mg once per day for 6 additional months. Patients with a partial response, or with a complete response and bone marrow minimal residual disease of 0·01% or more, received 6 months of four 4-weekly cycles of intravenous fludarabine, cyclophosphamide, and obinutuzumab 1000 mg, alongside continuing ibrutinib 420 mg once per day. The primary endpoint was the proportion of patients achieving a complete response with bone marrow minimal residual disease less than 0·01% on day 1 of month 16 assessed by intention to treat (ITT). This trial is registered with ClinicalTrials.gov (number NCT02666898) and is still open for follow-up.

Findings: Between Oct 27, 2015, and May 16, 2017, 135 patients were enrolled. After induction treatment (day 1 of month 9), 130 patients were evaluable, of which ten (8%) achieved a complete response with bone marrow minimal residual disease of less than 0·01% and were assigned to ibrutinib, and 120 (92%) were assigned to ibrutinib plus fludarabine, cyclophosphamide, and obinutuzumab. After minimal residual disease-guided treatment (day 1 of month 16), 84 (62%, 90% CI 55-69) of 135 patients (ITT population) achieved a complete response with bone marrow minimal residual disease of less than 0·01%. The most common haematological adverse event was thrombocytopenia (in 45 [34%] of 133 patients at grade 1-2 in months 1-9 and in 43 [33%] of 130 patients at grade 1-2 in months 9-15). The most common non-haematological adverse events were infusion-related reactions (in 83 [62%] patients at grade 1-2 in months 1-9) and gastrointestinal disorders (in 62 [48%] patients at grades 1 and 2 in months 9-15). 49 serious adverse events occurred, most frequently infections (ten), cardiac events (eight), and haematological events (eight). No treatment-related deaths occurred.

Interpretation: Obinutuzumab and ibrutinib induction therapy followed by a minimal residual disease driven strategy is safe and active in patients with previously untreated chronic lymphocytic leukaemia. With longer follow-up, including assessing the evolution of minimal residual disease, if response is maintained, this strategy could be an option in the first-line setting in patients with chronic lymphocytic leukaemia, although randomised evidence is needed.

Funding: Roche, Janssen.
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http://dx.doi.org/10.1016/S2352-3026(19)30113-9DOI Listing
September 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

Evaluation of bone marrow microenvironment could change how myelodysplastic syndromes are diagnosed and treated.

Cytometry A 2018 07 13;93(9):916-928. Epub 2018 Sep 13.

Laboratoire d'Hématologie, CHU de Saint-Etienne, 42055 Saint-Etienne Cedex 2, France.

Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic disorders. However, the therapies used against the hematopoietic stem cells clones have limited efficacy; they slow the evolution toward acute myeloid leukemia rather than stop clonal evolution and eradicate the disease. The progress made in recent years regarding the role of the bone marrow microenvironment in disease evolution may contribute to progress in this area. This review presents the recent updates on the role of the bone marrow microenvironment in myelodysplastic syndromes pathogenesis and tries to find answers regarding how this information could improve myelodysplastic syndromes diagnosis and therapy.
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http://dx.doi.org/10.1002/cyto.a.23506DOI Listing
July 2018

Editorial: A Revised Approach to the Pathogenesis and Diagnosis of Myelodysplastic Syndromes With Therapeutic Implications.

Front Oncol 2018 17;8:261. Epub 2018 Jul 17.

Département d'Hématologie et Thérapie Cellulaire, Institut de Cancérologie Lucien Neuwirth, Saint-Etienne, France.

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http://dx.doi.org/10.3389/fonc.2018.00261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063204PMC
July 2018

Minimal residual disease in chronic lymphocytic leukemia: A consensus paper that presents the clinical impact of the presently available laboratory approaches.

Crit Rev Clin Lab Sci 2018 08 25;55(5):329-345. Epub 2018 May 25.

e Research Center for Functional Genomics and Translational Medicine , Iuliu Hatieganu University of Medicine and Pharmacy , Cluj Napoca , Romania.

Chronic lymphocytic leukemia (CLL) is a malignancy defined by the accumulation of mature lymphocytes in the lymphoid tissues, bone marrow, and blood. Therapy for CLL is guided according to the Rai and Binet staging systems. Nevertheless, state-of-the-art protocols in disease monitoring, diagnostics, and prognostics for CLL are based on the assessment of minimal residual disease (MRD). MRD is internationally considered to be the level of disease that can be detected by sensitive techniques and represents incomplete treatment and a probability of disease relapse. MRD detection has been continuously improved by the quick development of both flow cytometry and molecular biology technology, as well as by next-generation sequencing. Considering that MRD detection is moving more and more from research to clinical practice, where it can be an independent prognostic marker, in this paper, we present the methodologies by which MRD is evaluated, from translational research to clinical practice.
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http://dx.doi.org/10.1080/10408363.2018.1463508DOI Listing
August 2018

Evaluation by Flow Cytometry of Mature Monocyte Subpopulations for the Diagnosis and Follow-Up of Chronic Myelomonocytic Leukemia.

Front Oncol 2018 12;8:109. Epub 2018 Apr 12.

Laboratoire d'Hématologie, CHU de Saint-Etienne, Saint-Etienne, France.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis and dysplasia in at least one myeloid cell lineage. This persistent monocytosis should be distinguished from the reactive monocytosis which is sometimes observed in a context of infections or solid tumors. In 2015, Selimoglu-Buet et al. observed an increased percentage of classical monocytes (CD14/CD16 >94%) in the peripheral blood (PB) of CMML patients. In this study, using multiparametric flow cytometry (MFC), we assessed the monocytic distribution in PB samples and in bone marrow aspirates from 63 patients with monocytosis or CMML suspicion, and in seven follow-up blood samples from CMML patients treated with hypomethylating agents (HMA). A control group of 12 healthy age-matched donors was evaluated in parallel in order to validate the analysis template. The CMML diagnosis was established in 15 cases in correlation with other clinical manifestations and biological tests. The MFC test for the evaluation of the repartition of monocyte subsets, as previously described by Selimoglu-Buet et al. showed a specificity of 97% in blood and 100% in marrow samples. Additional information regarding the expression of intermediate MO2 monocytes percentage improved the specificity to 100% in blood samples allowing the screening of abnormal monocytosis. The indicative thresholds of CMML monocytosis were different in PB compared to BM samples (classical monocytes >95% for PB and >93% for BM). A decrease of monocyte levels in PB and BM, along with a normalization of monocytes distribution, was observed after treatment in 4/7 CMML patients with favorable evolution. No significant changes were observed in 3/7 patients who did not respond to HMA therapy and also presented unfavorable molecular prognostic factors at diagnosis (, and mutations). Considering its simplicity and robustness, the monocyte subsets evaluation by MFC provides relevant information for CMML diagnosis.
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http://dx.doi.org/10.3389/fonc.2018.00109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906716PMC
April 2018

Epigallocatechin-3-O-gallate alleviates the malignant phenotype in A-431 epidermoid and SK-BR-3 breast cancer cell lines.

Int J Food Sci Nutr 2018 Aug 21;69(5):584-597. Epub 2017 Nov 21.

a Department of Biophysics , "Carol Davila" University of Medicine and Pharmacy , Bucharest , Romania.

In this study, we evaluated the effects of epigallocatechin-3-O-gallate (EGCG) in two cancer cell lines, A-431 overexpressing ErbB1 and SK-BR-3, overexpressing ErbB2. EGCG treatment showed dose-dependent collapse of mitochondrial membrane potential (Δψ), increase in reactive oxygen species (ROS) production, changes in nuclear morphology and reduced viability. Flow cytometry data indicated that EGCG partially decreases the phosphorylation of several proteins involved in cell proliferation and survival: pErbB1(Y1173, Y1068), pAkt(S473) and pERK(Y204). EGCG affected the clonogenic growth in both cell lines with an EC of 2.5 and 5.4 µM for A-431 and SK-BR-3, respectively. Wound scratch assay demonstrated that EGCG inhibited the healing in dose-dependent manner and the effect was correlated with partial reduction in phosphorylation of pFAK(S910). Our data suggest that EGCG administration might reduce the unfavourable traits, particularly associated with ErbB1/EGFR overexpression.
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http://dx.doi.org/10.1080/09637486.2017.1401980DOI Listing
August 2018

Potential Role of OCT4 in Leukemogenesis.

Stem Cells Dev 2017 11 16;26(22):1637-1647. Epub 2017 Oct 16.

1 Laboratoire d'Hématologie, Centre Hospitalier Universitaire de Saint-Etienne , Saint-Etienne, France .

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.
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http://dx.doi.org/10.1089/scd.2017.0134DOI Listing
November 2017

Impaired Expression of Focal Adhesion Kinase in Mesenchymal Stromal Cells from Low-Risk Myelodysplastic Syndrome Patients.

Front Oncol 2017 8;7:164. Epub 2017 Aug 8.

UMR 5239, Laboratoire de Biologie et Modélisation de la Cellule, Lyon, France.

The pathogenic role of mesenchymal stromal cells (MSCs) in myelodysplastic syndromes (MDS) development and progression has been investigated by numerous studies, yet, it remains controversial in some aspects (1, 2). In the present study, we found distinct features of MSCs from low-risk (LR)-MDS stromal microenvironment as compared to those from healthy subjects. At the molecular level, focal adhesion kinase, a key tyrosine kinase in control of cell proliferation, survival, and adhesion process, was found profoundly suppressed in expression and activation in LR-MDS MSC. At a functional level, LR-MDS MSCs showed impaired growth and clonogenic capacity, which were independent of cellular senescence and apoptosis. The pro-adipogenic differentiation and attenuated osteogenic capacity along with reduced SDF-1 expression could be involved in creating an unfavorable microenvironment for hematopoiesis. In conclusion, our experiments support the theory that the stromal microenvironment is fundamentally altered in LR-MDS, and these preliminary data offer a new perspective on LR-MDS pathophysiology.
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http://dx.doi.org/10.3389/fonc.2017.00164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551509PMC
August 2017

Expression of embryonic stem cell markers in acute myeloid leukemia.

Tumour Biol 2017 Jul;39(7):1010428317716629

1 Laboratoire d'Hématologie, CHU de Saint-Etienne, Saint-Etienne, France.

Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34CD38 and CD34CD38) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34CD38 than in CD34CD38 subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.
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http://dx.doi.org/10.1177/1010428317716629DOI Listing
July 2017

Revisiting the hallmarks of cancer.

Am J Cancer Res 2017 1;7(5):1016-1036. Epub 2017 May 1.

Hematology Laboratory, Pole De Biologie-Pathologie, University Hospital of St EtienneSt Etienne, France.

The hallmarks of cancer described by Hanahan and Weinberg have proved seminal in our understanding of cancer's common traits and in rational drug design. Not free of critique and with understanding of different aspects of tumorigenesis coming into clearer focus in the recent years, we attempt to draw a more organized and updated picture of the cancer hallmarks. We define seven hallmarks of cancer: selective growth and proliferative advantage, altered stress response favoring overall survival, vascularization, invasion and metastasis, metabolic rewiring, an abetting microenvironment, and immune modulation, while highlighting some considerations for the future of the field.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446472PMC
May 2017

Bone Marrow Immunity and Myelodysplasia.

Front Oncol 2016 20;6:172. Epub 2016 Jul 20.

Haematology Laboratory, Pole de Biologie-Pathologie, University Hospital of St Etienne , St Etienne , France.

Myelodysplastic syndrome (MDS) is characterized by an ineffective hematopoiesis with production of aberrant clones and a high cell apoptosis rate in bone marrow (BM). Macrophages are in charge of phagocytosis. Innate Immune cells and specific T cells are in charge of immunosurveillance. Little is known on BM cell recruitment and activity as BM aspirate is frequently contaminated with peripheral blood. But evidences suggest an active role of immune cells in protection against MDS and secondary leukemia. BM CD8(+) CD28(-) CD57(+) T cells are directly cytotoxic and have a distinct cytokine signature in MDS, producing TNF-α, IL-6, CCL3, CCL4, IL-1RA, TNFα, FAS-L, TRAIL, and so on. These tools promote apoptosis of aberrant cells. On the other hand, they also increase MDS-related cytopenia and myelofibrosis together with TGFβ. IL-32 produced by stromal cells amplifies NK cytotoxicity but also the vicious circle of TNFα production. Myeloid-derived suppressing cells (MDSC) are increased in MDS and have ambiguous role in protection/progression of the diseases. CD33 is expressed on hematopoietic stem cells on MDS and might be a potential target for biotherapy. MDS also has impact on immunity and can favor chronic inflammation and emergence of autoimmune disorders. BM is the site of hematopoiesis and thus contains a complex population of cells at different stages of differentiation from stem cells and early engaged precursors up to almost mature cells of each lineage including erythrocytes, megakaryocytes, myelo-monocytic cells (monocyte/macrophage and granulocytes), NK cells, and B cells. Monocytes and B cell finalize their maturation in peripheral tissues or lymph nodes after migration through the blood. On the other hand, T cells develop in thymus and are present in BM only as mature cells, just like other well vascularized tissues. BM precursors have a strong proliferative capacity, which is usually associated with a high risk for genetic errors, cell dysfunction, and consequent cell death. Abnormal cells are prone to destruction through spontaneous apoptosis or because of the immunosurveillance that needs to stay highly vigilant. High rates of proliferation or differentiation failures lead to a high rate of cell death and massive release of debris to be captured and destroyed (1). Numerous macrophages reside in BM in charge of home-keeping. They have a high capacity of phagocytosis required for clearing all these debris.
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http://dx.doi.org/10.3389/fonc.2016.00172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4953538PMC
August 2016

Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes.

Front Oncol 2016 27;6:161. Epub 2016 Jun 27.

CNRS UMR5239, Université de Lyon, Saint-Etienne, France; Laboratoire d'Hématologie, CHU de Saint-Etienne, Saint-Etienne, France.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that exhibit heterogeneous clinical presentation and morphological findings, which complicates diagnosis, especially in early stages. Recently, refined definitions and standards in the diagnosis and treatment of MDS were proposed, but numerous questions remain. Multiparameter flow cytometry (MFC) is a helpful tool for the diagnostic workup of patients with suspected MDS, and various scores using MFC data have been developed. However, none of these methods have achieved the sensitivity that is required for a reassuring diagnosis in the absence of morphological abnormalities. One reason may be that each score evaluates one or two lineages without offering a broad view of the dysplastic process. The combination of two scores (e.g., Ogata and Red Score) improved the sensitivity from 50-60 to 88%, but the positive (PPV) and negative predictive values (NPV) must be improved. There are prominent differences between study groups when these scores are tested. Further research is needed to maximize the sensitivity of flow cytometric analysis in MDS. This review focuses on the application of flow cytometry for MDS diagnosis and discusses the advantages and limitations of different approaches.
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http://dx.doi.org/10.3389/fonc.2016.00161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921489PMC
July 2016

[CXCR4: a new therapeutic target of the leukaemic cell? Role of the SDF-1/CXCR4 axis in acute myeloid leukaemia].

Bull Cancer 2014 Jun;101(6):593-604

Institut de cancérologie Lucien-Neuwirth, Service d'hématologie, 108 bis, avenue Albert-Raimond, 42270 Saint-Priest-en-Jarez, France, Université Jean-Monnet, UMR 5239, Laboratoire de biologie moléculaire de la cellule, 42000 Saint-Etienne, France.

CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. Recent flow cytometry analyses reported that CXCR4 expression levels have a major prognostic impact in acute myeloid leukaemia (AML). CXCR4 expression is associated with poor prognosis and can be useful to stratify patients, according to their phenotype, in order to establish risk-adapted strategies. Newly diagnosed AML are now routinely stratified according to molecular markers which guide prognosis and treatment. Many leukaemia are composed of multiples subclones with differential susceptibility to treatment and specific targeted therapies are missing. Despite therapeutic improvements for the treatment of AML, long term survival remains poor. Targeting CXCR4 is a novel promising approach of therapy. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies. This review summarises our current knowledge regarding the key role of CXCR4 in AML and discusses how targeting this pathway could provide an interesting approach to eradicate the LSC.
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http://dx.doi.org/10.1684/bdc.2014.1925DOI Listing
June 2014

Heat Shock Protein 90 is overexpressed in high-risk myelodysplastic syndromes and associated with higher expression and activation of Focal Adhesion Kinase.

Oncotarget 2012 Oct;3(10):1158-68

Laboratoire d'Hématologie, University Hospital of Saint-Etienne, University Hospital of Saint-Etienne, 42055 Saint-Etienne Cedex 2,

Myelodysplastic syndromes are characterized by a high risk of evolution into acute myeloid leukaemia which can involve activation of signalling pathways. As the chaperone heat shock protein 90 (HSP90) has a key role in signal transduction, we investigated its role in the pathogenesis and evolution of myelodysplastic syndromes. Expressions of HSP90 and signalling proteins clients (phosphorylated-AKT (pAKT), Focal Adhesion Kinase (FAK) and phosphorylated-FAK (pFAK)), were assessed in bone marrow mononuclear and CD34-positive (CD34+) cells from 177 patients with myelodysplasia. Effects of HSP90 inhibition were also evaluated in 39 samples. The levels of all proteins studied were significantly higher in patients with high grade disease, than those with low grade myelodysplastic syndrome or chronic myelomonocytic leukaemia. High levels of HSP90, FAK, pFAK and pAKT were associated with shorter survival and increased risk of progression into acute leukaemia. A down regulation of pFAK and pAKT and increased apoptosis was observed in mononuclear and CD34+ cells after 12 hours of incubation with 17-AAG. In conclusion, our data suggest the implication of HSP90 and FAK and AKT activation in the pathogenesis of myelodysplastic syndromes with excess of blasts and evolution to leukaemia. Moreover this signalling network could be a therapeutic target through HSP90 inhibition.
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http://dx.doi.org/10.18632/oncotarget.557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717957PMC
October 2012

Intrinsic growth deficiencies of mesenchymal stromal cells in myelodysplastic syndromes.

Stem Cells Dev 2012 Jul 27;21(10):1604-15. Epub 2011 Oct 27.

Laboratoire d'Hématologie, Hôpital Nord, CHU de Saint-Etienne, Saint-Etienne Cedex, France.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoietic stem cells (HSCs) characterized by ineffective hematopoiesis. MDSs are responsible for 1 or several peripheral cytopenias. The evidence accumulated in recent years demonstrates that in addition to HSC defects, a particular role is also played by stromal microenvironment dysfunctions, which mediate the direct contact with hematopoietic precursor cells (HPCs). These interactions help regulate different adhesion-related processes, such as progenitor cell proliferation, apoptosis, clonogenic growth, and maintenance in in vitro cultures. As previously reported, these interactions are responsible for altering the microenvironment in MDS. Herein, we present a novel selection protocol for obtaining a standards-compliant mesenchymal stromal cell (MSC) preparation. This method allowed us to comparatively analyze 2 subpopulations of bone marrow MSCs (BM-MSCs) in terms of their adhesion profiles and growth abilities: BM-MSCs selected from MDS settings and their normal counterparts. Functional assays revealed that the MSCs from MDS are intrinsically pathological, thus showing a continuous decline of proliferation and a reduced clonogenic capacity during 14 days of culture and in the absence of signals from hematopoietic cells. The MSC growth defects were significantly correlated with decreases in CD44 adhesion molecules and CD49e (α5-integrin).
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http://dx.doi.org/10.1089/scd.2011.0390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376465PMC
July 2012

Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells.

Exp Cell Res 2011 Nov 16;317(18):2616-29. Epub 2011 Aug 16.

Laboratoire Hématologie, CHU de Saint-Etienne, 42055 Saint-Etienne, France.

Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y(397)], and HSP90α/β and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y(397)], and HSP90α/β formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y(397)], and HSP90α/β and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90α/β client protein, these results suggest the utility of HSP90α/β inhibition as a target for adjuvant therapy for myelodysplasia.
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http://dx.doi.org/10.1016/j.yexcr.2011.08.007DOI Listing
November 2011

Congenital acute leukemia with initial indolent presentation--a case report.

Cytometry B Clin Cytom 2011 Mar 2;80(2):130-3. Epub 2010 Dec 2.

Laboratoire d'Hématologie, Hôpital Nord, Centre Hospitalier Universitaire, Saint Etienne, France.

Background: Congenital acute leukemia is a rare event, presenting usually as an aggressive disease with a poor prognosis. A differential diagnosis is the transient myeloproliferative disorder observed in Down syndrome. We describe the case of an apparently healthy newborn male child presenting with normal peripheral blood (PB) counts but with a blast population on differentials. The child's condition and the blast population remained unchanged during the first year of life.

Methods: Bone marrow and PB were morphologically analyzed. Multiparametric flow cytometry was performed at the time of diagnosis and repeated at 1 year. These studies were completed by cytogenetic and molecular analyses.

Results: Bone marrow contained 40% of undifferentiated blasts. Multiparametric flow cytometry showed low expression of CD38, HLA-DR, and CD33 markers. All other markers were negative. Constitutional and blast cell karyotypes were normal. Fluorescence in situ hybridization analysis showed no rearrangement. Molecular studies were negative. The blast percentage remained stable during several months. After 1 year, the PB counts showed thrombocytopenia, with an increase of blast cells exhibiting the same phenotype. Clinically, an enlarged spleen was found. The child did not respond to chemotherapy and only partially to gemtuzumab ozogamicin. A cord blood cell transplantation was finally performed. With a follow-up of 12 months, the child is doing well.

Conclusions: To our knowledge, this is the first case of congenital acute leukemia with initially indolent course in a newborn without Down syndrome. This observation emphasizes the importance of a careful follow-up.
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http://dx.doi.org/10.1002/cyto.b.20578DOI Listing
March 2011

Flow cytometry-based quantification of cytokine levels in AML patients plasma and cell culture supernatants.

Rev Med Chir Soc Med Nat Iasi 2008 Jan-Mar;112(1):196-202

Gr.T. Popa University of Medicine and Pharmacy Iaşi, School of Medicine, Department of Hematology.

Aim: Bone marrow stromal cells (BMSCs) have been found to support leukemic cell survival; however, the mechanisms responsible are far from being elucidated yet. The main aim of the current study is to identify particular cytokine/chemokine patterns of acute myeloid leukemia (AML) cells, and, on a longer term, to correlate them with the patient outcome and response to therapy. Therefore, the influence of BMSCs on in vitro modulation of cytokine secretion (IL-1beta, TNF-alpha, IL-10, IFN-gamma, IL-4, IL-5, and IL-2) by AML cells as well as the AML cells supportive capacity of BMSCs-derived soluble factors was investigated.

Material And Methods: With this purpose, we used an in vitro experimental model consisting in the evaluation of the effect of BMSC confluent layers-conditioning medium (BMSC-CM) on M4/5 AML cell cultures.

Results: Our results show that BMSC-CM from both AML patients and healthy subjects conferred a substantial beneficial effect on AML cells throughout the culture (p=0.0002 and 0.0020 respectively at 24 hours and p=0,0013 and 0,0030 respectively at 72 hours), with a temporary increase in AML cell viability conferred by BM plasma from AML patients. Significant differences were observed with respect to IL1 b secretion which was upregulated in AML cell cultures both after 24 and 72 hours following the addition of AML-BMSC-CM, in contrast to control-BMSC-CM.

Conclusion: Our results suggest the contribution of BMSCs from AML patients to the generation of particular factors which may be key players involved in the in vivo maintenance of the malignant clone.
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September 2008

[Relationship diabetes mellitus-periodontal disease: etiology and risk factors].

Rev Med Chir Soc Med Nat Iasi 2007 Jul-Sep;111(3):748-53

Universitatea de Medicină şi Farmacie Gr. T. Popa Iaşi, Departamentul de Biochimie.

The interrelation between diabetes mellitus and inflammatory periodontal disease has been intensively studied for more than 50 years, a real bidirectional influence existing between patient's glycemic level disorder and periodontal territories alteration. Several studies developed in this direction emerged to the evidences that reveal a general increase of prevalence, extent and severity of gingivitis and periodontitis. Inflammation plays an important role in this interrelation, orchestrating both the periodontal disease and diabetes mellitus pathogeny and complications. Conversely, periodontal disease--infectious disease characterized by a significant inflammatory component--can seriously impair metabolic control of some diabetic patient. Moreover, treatment of periodontal disease and reduction of oral signs of inflammation may have a beneficial result on the diabetic condition (1). Less clear are the mechanisms governing this interrelation (even the literature is abundant in this direction), and, very probably, periodontal diseases serve as initiators of insulin resistance (in a way similar to obesity), thereby aggravating glycemic control. Further research is so imposed in order to clarify this aspect of the relationship between diabetes and periodontal disease.
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June 2008
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