Publications by authors named "Anne-Sophie Alary"

16 Publications

  • Page 1 of 1

Improvement of Standardization of Molecular Analyses in Hematology: The 10-year GBMHM French Experience.

Hemasphere 2021 Dec 17;5(12):e658. Epub 2021 Nov 17.

Laboratoire d'hématologie, CHU de Saint-Étienne, Saint-Priest-en-Jarez, France.

Molecular tests have become an indispensable tool for the diagnosis and prognosis of hematological malignancies and are subject to accreditation according to the International Standard ISO 15189. National standardization of these techniques is essential to ensure that patients throughout France benefit from the same care. We report here on the experience of the GBMHM (). By organizing External Evaluation of Quality (EEQ) programs and training meetings, the GBMHM has contributed to improvement and standardization of molecular tests in 64 French laboratories. A retrospective analysis of the quality-control results of 11 national campaigns spanning 10 years was performed for the 3 most frequently prescribed tests: , V617F, and lymphoid clonality. For each test, particular attention was placed on comparing methodologies and their evolution throughout the period. The establishment of the , V617F, and lymphoid clonality EEQ programs and the associated training meetings have initiated a process of collective standardization concerning the methods of implementation ( V617F) and the interpretation and formulation of results (lymphoid clonality). In addition, it resulted in objective improvement in technical performance (). Our evaluation of the impact of these EEQ programs demonstrates that it is possible to obtain reproducible values across different laboratories in France by applying national recommendations. To our knowledge, this is the first publication that evaluates the impact of a national quality assurance program on improving molecular results in hematology.
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http://dx.doi.org/10.1097/HS9.0000000000000658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8598224PMC
December 2021

Myeloproliferative neoplasms and clonal hematopoiesis in patients with giant cell arteritis: a case-control and exploratory study.

Rheumatology (Oxford) 2021 Apr 9. Epub 2021 Apr 9.

Department of Internal Medicine, Cochin Hospital, National Referral Center for Rare Systemic Autoimmune Diseases, Paris University, Paris, France.

Objectives: Giant cell arteritis (GCA) is a large vessel vasculitis for which triggering factors remain unknown. Clonal hematopoiesis (CH) was associated with atherosclerosis through the induction of inflammation in myeloid cells, and data suggest that CH expansion and inflammation may support each other to induce a proinflammatory loop. Our objective was to describe the impact of JAK2p.V617F-mutated myeloproliferative neoplasms (MPN) on GCA and to screen MPN-free patients for CH mutations.

Methods: We performed a retrospective case-control study comparing characteristics of 21 GCA patients with MPN and 42 age and gender-matched GCA patients without MPN. Also, 18 GCA patients were screened for CH through Next Generation Sequencing.

Results: The most frequent associated MPN was essential thrombocythemia (ET) (n = 11). Compared to controls, GCA patients with MPN had less frequent cephalic symptoms (71.4 vs. 97.6%, p = 0.004) and higher platelets count at baseline [485 (346-586) vs. 346 [IQR 296-418] x 109/L, p = 0.02). There was no difference between groups for other clinical features. Overall survival was significantly shorter in patients with MPN compared to controls [HR 8.2 (95% CI 1.2-56.6), p = 0.03]. Finally, screening for CH using NGS in 15 GCA patients without MPN revealed CH in 33%.

Conclusion: GCA patients with MPN display higher platelets count and shorter overall survival than controls. This association could not be fortuitous given the possible pathophysiological relationship between the two diseases. CH was found in one third of GCA patients, which may be higher than the expected prevalence for similar age, what should be confirmed in a larger cohort.
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http://dx.doi.org/10.1093/rheumatology/keab337DOI Listing
April 2021

Battle of the clones: paroxysmal nocturnal hemoglobinuria vs myelodysplastic syndrome.

Ann Hematol 2020 Oct 13;99(10):2459-2461. Epub 2020 Jun 13.

Assistance Publique-Hôpitaux de Paris, Hôpitaux Universitaires de Paris, Hematology Laboratory, Georges Pompidou European Hospital, INSERM UMR-S1140, Paris, France.

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http://dx.doi.org/10.1007/s00277-020-04134-yDOI Listing
October 2020

[Accreditation strategy for rare somatic molecular abnormalities detected or quantified by polymerase chain reaction: GBMHM recommendations].

Ann Biol Clin (Paris) 2019 12;77(6):681-684

Laboratoire d'hématologie, Hôpital Nord, Centre hospitalier universitaire de Saint-Étienne, Saint-Étienne, France.

In 2020, accreditation of molecular tests according to ISO 15189 is a requirement for all French medical laboratories. For many years, the GBMHM group (French Group of Molecular Biologists in Hematology) supports this approach through organization of external quality evaluation campaigns, and by publishing recommendations that have allowed the accreditation of the most frequent molecular tests for most laboratories. However, some molecular abnormalities concerns very few patients (and sometimes a single patient), and therefore cannot be evaluated in the same way, because of the lack of external quality controls or inter-laboratory comparisons. In order to allow the accreditation of these rare analyzes, the GBMHM proposes recommendations, based on the fact that analyzes using the same methodology than those already accredited by an extensive validation process, may be accredited without the need for full analytical validation. In particular, assays based on quantitative PCR or endpoint PCR may be accredited after verification of primer specificity, repeatability and/or reproducibility, and the determination of detection or linearity limits. These recommendations, by defining the validation approach for rare molecular abnormalities, make it possible to extend the requirement of accreditation for rare tests, to provide the best patient care.
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http://dx.doi.org/10.1684/abc.2019.1498DOI Listing
December 2019

[Development of molecular analysis by digital PCR for clinical practice: positioning, current applications and perspectives].

Ann Biol Clin (Paris) 2019 12;77(6):619-637

ID Solutions, Grabels, France, Service d'anatomie pathologique et neuropathologie, Assistance Publique Hôpitaux de Marseille, AP-HM, Marseille, France.

This review is the second part of the workshop on digital PCR (dPCR) proposed by the working group of the French society of clinical biology. The first part of the paper discusses the advantages and limitations of dPCR for the search of different molecular abnormalities such as point mutations, copy number variants, DNA methylation, RNA analysis and a more innovative application, the single-cell dPCR. This synthesis makes it possible to propose a positioning of the dPCR compared to the other available technologies in a medical laboratory. In a second part, the main current applications of the dPCR will be addressed including the oncology of solid tumors and liquid biopsies, oncohematology and the follow-up of hemopathies treatments by hematopoietic stem cell transplantation. We will also detail non-invasive prenatal diagnosis and diagnosis of mosaic genetic disease, using the example of McCune-Albright syndrome. Several French specialists in the field who have implemented these techniques in their laboratory have written these different examples of applications jointly. In summary, this manuscript offers an up-to-date view of the positioning of dPCR in relation to other existing technologies in order to best meet the expectations of precision medicine.
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http://dx.doi.org/10.1684/abc.2019.1502DOI Listing
December 2019

Phenotypic landscape of granulocytes and monocytes by multiparametric flow cytometry: A prospective study of a 1-tube panel strategy for diagnosis and prognosis of patients with MDS.

Cytometry B Clin Cytom 2020 05 9;98(3):226-237. Epub 2019 Sep 9.

Assistance Publique-Hôpitaux de Paris, Hôpitaux Universitaires Paris Centre, Service d'Hématologie Biologique, Paris, France.

Background: Multiparametric flow cytometry (MFC) was recently reported to be a helpful additional tool in the diagnosis of myelodysplastic syndromes (MDS). However, numerous aberrancies have been reported that makes their evaluation difficult as part of a routine diagnosis.

Methods: Here, we validated a 1-tube panel for the evaluation of granulocytic and monocytic maturation by MFC and correlated our findings with diagnosis and prognosis of MDS. A total of 251 samples with MDS suspicion were prospectively analyzed and compared to an internal reference database leading to the calculation of the Diff score.

Results: The associated specificity and sensitivity values of this scoring system were 92.1% and 60.4% in a first learning cohort and 96.7% and 65.2% in a second independent validation cohort. The combination of the Diff score with the concomitantly calculated Ogata score increased the sensitivity to 74.2% and 78.3% in the learning and validation cohorts, respectively. Finally, a normal Diff score in MDS patients was associated with a significant prolonged progression-free survival.

Conclusions: Taken together, the present data indicate that our strategy is a sensitive and specific MFC tool for the diagnosis of MDS-related cytopenia(s) which could be also useful for predicting evolution of these diseases.
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http://dx.doi.org/10.1002/cyto.b.21843DOI Listing
May 2020

Paraneoplastic Hyperleukocytosis Mimicking Hematologic Malignancy Revealing a Localized Lung Cancer.

Ann Thorac Surg 2020 03 10;109(3):e203-e206. Epub 2019 Aug 10.

Université Paris Descartes, Faculté de Médecine, Sorbonne Paris Cité, Paris, France; Service d'Hématologie, Hôpital Cochin, Assistance Publique - Hôpitaux de Paris, Paris, France; INSERM U1016, Institut Cochin, Paris, France.

Paraneoplastic leukemoid reaction is a challenging differential diagnosis when it presents at the time of diagnosis of cancer. Severe hyperleukocytosis with elevation of blood neutrophils and monocytes counts can evoke myeloid hematologic malignancies. We report the case of a patient who presented with blood and bone marrow features highly suggestive of chronic myelomonocytic leukemia. The diagnosis of primary lung sarcomatoid carcinoma was performed. Surgical removal of this tumor which will always remain the priority led to full normalization of blood cell count.
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http://dx.doi.org/10.1016/j.athoracsur.2019.06.064DOI Listing
March 2020

A variant erythroferrone disrupts iron homeostasis in -mutated myelodysplastic syndrome.

Sci Transl Med 2019 07;11(500)

Université de Paris, Paris 75006, France.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the splicing factor gene. Patients with MDS with mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative transcript in patients with MDS with the mutation. Induction of this transcript in primary -mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an gene mutation than in patients with wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with -mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant transcript that was restricted to -mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.
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http://dx.doi.org/10.1126/scitranslmed.aav5467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005358PMC
July 2019

Impact of genotype in relapsed and refractory acute myeloid leukaemia patients treated with clofarabine and cytarabine: a retrospective study.

Br J Haematol 2019 10 18;187(1):65-72. Epub 2019 Jun 18.

Faculté de Médecine Sorbonne Paris Cité, Université Paris Descartes, Paris, France.

The treatment of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains a challenge. Among salvage chemotherapy regimens, the clofarabine and cytarabine (CLARA) combination has been widely evaluated and has a favourable safety/efficacy balance. Predictive factors of efficacy in patients with R/R AML are unclear, particularly the impact of AML-related gene mutations. We report our single-centre experience on 34 R/R AML patients treated with CLARA, with a focus on the genetic characterization of our cohort. CLARA yielded a 47% response rate among this poor-prognosis AML population, while two patients (5·8%) died due to treatment-related toxicity. The two-year progression-free survival and overall survival rates were 29·4% and 35·3%, respectively. Nine patients (26%) had long-term response with a median follow-up of 39·5 months among the responders, of whom six underwent haematopoietic stem cell transplantation. Adverse karyotype did not correlate with response or survival, and secondary AML were more frequent among responders to CLARA, suggesting that this combination may successfully salvage R/R AML patients regardless of adverse prognostic markers. We also observed that a low mutational burden and absence of splice mutations correlated with prolonged survival after CLARA, suggesting that extensive genotyping may have prognostic implications in R/R AML.
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http://dx.doi.org/10.1111/bjh.16045DOI Listing
October 2019

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

[Development of digital PCR molecular tests for clinical practice: principles, practical implementation and recommendations].

Ann Biol Clin (Paris) 2018 10;76(5):505-523

Assistance publique - Hôpitaux de Marseille, Service d'anatomie pathologique et neuropathologie, Marseille, France, ID Solutions, Grabels, France.

Digital PCR (dPCR) is a 3rd generation technology that complements traditional end-point PCR and real-time PCR. It was developed to overcome certain limitations of conventional amplification techniques, in particular for the detection of small amounts of nucleic acids and/or rare variants. This technology is in a full swing because of its high sensitivity and major applications in various domains such as oncology, transplantation or non-invasive prenatal testing. Consequently, PCRd also has great interest in many areas of medical biology, particularly for clinical applications aiming at detecting and quantifying specific genetic or epigenetic alterations of nucleic acids, even with specimens containing very low concentration of the nucleic acids of interest (e.g. liquid biopsies). However, this technique requires a good training of users and compliance with certain precautions. A lack in such a knowledge can lead to many errors in the conduct of the experiment and the interpretation of the results. In this review, we present the context in which this technology has emerged by describing in particular its principle and the main factors that can influence the quality of the analysis. Then, we propose a number of practical recommendations for the implementation of a test based on dPCR in clinical laboratories with an eye on quality requirements.
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http://dx.doi.org/10.1684/abc.2018.1372DOI Listing
October 2018

CD13 expression in B cell malignancies is a hallmark of plasmacytic differentiation.

Br J Haematol 2019 02 10;184(4):625-633. Epub 2018 Sep 10.

Service d'Hématologie-Immunologie-Transfusion, Hôpitaux Universitaires Paris Ile De France Ouest, Université Versailles Saint Quentin, Boulogne, France.

The diagnosis of Waldenström Macroglobulinaemia (WM)/lymphoplasmacytic lymphoma (LPL) remains one of exclusion because other B-cell lymphoproliferative disorders (B-LPD), such as marginal zone lymphoma (MZL), can fulfil similar criteria, including MYD88 L265P mutation. It has been suggested that expression of the myeloid marker CD13 (also termed ANPEP) is more frequent in LPL than in other B-LPD and has also been described on normal and malignant plasma cells. Here, CD13 expression was tested in a cohort of 1037 B-LPD patients from 3 centres by flow cytometry. The percentage of CD13-expressing cells was found to be variable among B-LPD but significantly higher in WM/LPL (median 31% vs. 0% in non-WM/LPL, P < 0·001). In multivariate linear regression, CD13 expression remained significantly associated with a diagnosis of WM/LPL (P < 0·001). A cut-off value of 2% of CD19 cells co-expressing CD13 yielded the best diagnostic performance for WM/LPL assertion. This was further improved by association with the presence or absence of IgM paraprotein. Finally, given that previously published transcriptomic data revealed no difference in CD13 (also termed ANPEP) mRNA between normal and pathological B-cells, the hypothesis of some post-transcriptional regulation must be favoured. These results suggest that testing for CD13 expression in routine flow cytometry panels could help to discriminate WM/LPL from other B-LPD.
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http://dx.doi.org/10.1111/bjh.15584DOI Listing
February 2019

Molecular dissection of engraftment in a xenograft model of myelodysplastic syndromes.

Oncotarget 2018 Mar 20;9(19):14993-15000. Epub 2018 Feb 20.

University Clinic of Hematology, CHU Grenoble Alpes, Grenoble, France.

Myelodysplastic syndromes (MDS) are oligoclonal disorders of the hematopoietic stem cells (HSC). Recurrent gene mutations are involved in the MDS physiopathology along with the medullar microenvironment. To better study the heterogeneity of MDS, it is necessary to create patient derived xenograft (PDX). We have reproduced a PDX model by xenografting HSC (CD34) and mesenchymal stromal cells (MSC) in NOD/SCID/IL2rγ mice with primary samples from one RAEB2, two RAEB1 and one RARS patients harboring karyotype abnormalities and gene mutations. The average human chimerisms ranged from 59.7% to 0.0175% for the 4 patients. Secondary grafts (G2) were only performed for mice derived from the RAEB2 patient and the average human chimerism was 53.33%. G1 mice 1 and 2, and their derived G2 mice showed less than 20% of medullar blasts whereas mouse 3 and the resulting G2 mice transformed to AML. Clonal architecture was dissected in the different hematopoietic progenitors (HP) harvested from G1 and G2 mice. By direct Sanger sequencing, we found the 4 initial mutations in each HP subpopulation and those mutations had the same variant allele frequency in the CD34 CD38 HSC from G1 and G2 mice by next generation sequencing (NGS). Targeted NGS analysis done in HSC of mouse 3 did not show any additional driver gene mutations explaining the transformation to AML. To conclude, we have generated a PDX mouse model that perfectly reproduces the MDS founder clone which is stable over time, allowing us to consider this system as a powerful tool to test therapeutic approaches.
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http://dx.doi.org/10.18632/oncotarget.24538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871091PMC
March 2018

The role of genetic factors in patients with hepatocellular carcinoma and iron overload - a prospective series of 234 patients.

Liver Int 2016 05 7;36(5):746-54. Epub 2015 Nov 7.

Laboratory of Hematology, CHRU de Montpellier, Hôpital Saint Eloi, France.

Background & Aims: Iron overload (IO) in HFE-related hereditary haemochromatosis is associated with increased risk of liver cancer. This study aimed to investigate the role of other genes involved in hereditary IO among patients with hepatocellular carcinoma (HCC).

Methods: Patients with HCC diagnosed in our institution were included in this prospective study. Those with ferritin levels ≥300 μg/L (males) or ≥200 μg/L (females) and/or transferrin saturation ≥50% (males) or ≥45% (females) had liver iron concentration (LIC) evaluated by MRI. HFE C282Y and H63D mutations were screened. Genetic analyses of genes involved in hereditary IO (HFE, HJV/HFE2, HAMP, TFR2, SLC40A1, GNPAT) were performed in patients with increased LIC.

Results: A total of 234 patients were included; 215 (92%) had common acquired risk factors of HCC (mainly alcoholism or chronic viral hepatitis). 119 patients had abnormal iron parameters. Twelve (5.1%) were C282Y homozygotes, three were compound C282Y/H63D heterozygotes. LIC was measured by MRI in 100 patients. Thirteen patients with a LIC>70 μmol/g were enrolled in further genetic analyses: two unrelated patients bore the HAMP:c.-153C>T mutation at the heterozygous state, which is associated with increased risk of IO and severe haemochromatosis. Specific haplotypes of SLC40A1 were also studied.

Conclusions: Additional genetic risk factors of IO were found in 18 patients (7.7%) among a large series of 234 HCC patients. Screening for IO and the associated at-risk genotypes in patients who have developed HCC, is useful for both determining etiologic diagnosis and enabling family screening and possibly primary prevention in relatives.
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http://dx.doi.org/10.1111/liv.12984DOI Listing
May 2016
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