SOD Laboratorio Centrale

Firenze, Italy

SOD Laboratorio Centrale

Firenze, Italy
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Mannelli F.,University of Florence | Mannelli F.,Instituto Toscano Tumori | Ponziani V.,University of Florence | Ponziani V.,Instituto Toscano Tumori | And 30 more authors.
Haematologica | Year: 2017

Mutations in CCAAT/enhancer binding protein α (CEBPA) occur in 5-10% of cases of acute myeloid leukemia. CEBPA-doublemutated cases usually bear bi-allelic N- and C-terminal mutations and are associated with a favorable clinical outcome. Identification of CEBPA mutants is challenging because of the variety of mutations, intrinsic characteristics of the gene and technical issues. Several screening methods (fragment-length analysis, gene expression array) have been proposed especially for large-scale clinical use; although efficient, they are limited by specific concerns. We investigated the phenotypic profile of blast and maturing bone marrow cell compartments at diagnosis in 251 cases of acute myeloid leukemia. In this cohort, 16 (6.4%) patients had two CEBPA mutations, whereas ten (4.0%) had a single mutation. First, we highlighted that the CEBPA-double-mutated subset displays recurrent phenotypic abnormalities in all cell compartments. By mutational analysis after cell sorting, we demonstrated that this common phenotypic signature depends on CEBPA-double-mutated multi-lineage involvement. From a multi-dimensional study of phenotypic data, we developed a classifier including ten core and widely available parameters. The selected markers on blasts (CD34, CD117, CD7, CD15, CD65), neutrophil (SSC, CD64), monocytic (CD14, CD64) and erythroid (CD117) compartments were able to cluster CEBPA-double-mutated cases. In a validation set of 259 AML cases from three independent centers, our classifier showed excellent performance with 100% specificity and 100% sensitivity. We have, therefore, established a reliable screening method, based upon multi-dimensional analysis of widely available phenotypic parameters. This method provides early results and is suitable for largescale detection of CEBPA-double-mutated status, allowing gene sequencing to be focused in selected cases. © 2017 Ferrata Storti Foundation.


Mannelli F.,University of Florence | Mannelli F.,Instituto Toscano Tumori | Bencini S.,University of Florence | Bencini S.,Instituto Toscano Tumori | And 21 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2013

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a unique disorder caused by a PIG-A gene mutation in a stem cell clone. Its clinical picture can sometimes make challenging the distinction from other disorders, and especially from myelodysplastic syndromes (MDS), since both diseases correlate with cytopenias and morphological abnormalities of bone marrow (BM) cells. Recently, flow cytometry (FC) has been proposed to integrate the morphologic assessment of BM dysplasia, and thus to improve the diagnostics of MDS. Methods. In the present study, we have analyzed systematically FC data resulting from the study of BM cells from patients with PNH and MDS. Results. Our data demonstrated abnormalities in PNH beyond the deficiency of glycosylphosphatidylinositol- linked proteins and the application of a systematic approach allowed us to separate effectively MDS and PNH in a cluster analysis and to highlight disease-specific abnormalities. Indeed, the parallel evaluation of some key parameters, i.e. patterns of expression of CD45 and CD10, provided information with practical diagnostic usefulness in the distinction between PNH and MDS. Moreover, the hypo-expression of CD36 that we observed on monocytes might be related to the thrombotic tendency in PNH. Conclusions. We investigated systematically the phenotypic profile of BM cells from patients with PNH; our data provide useful antigenic patterns to solve between PNH and MDS, sometimes morphologically overlapping. Moreover, some PNH-related phenotypic changes might be involved in the physiopathology of the disease and further studies addressing this issue are warranted. © 2013 International Clinical Cytometry Society.


Mannelli F.,University of Florence | Mannelli F.,Instituto Toscano Tumori | Ponziani V.,University of Florence | Ponziani V.,Instituto Toscano Tumori | And 21 more authors.
Experimental Hematology | Year: 2015

The presence of multilineage dysplasia (MLD) by morphology at diagnosis in acute myeloid leukemia (AML) defines a separate subset in the World Health Organization classification with still-debated prognostic value. A major controversy concerns MLD's role in NPM1-mutated (NPM1+) AML, which correlates with good prognosis. We used flow cytometry (FC), an emerging technique for assessing dysplasia, to investigate MLD in NPM1+ AML by an immunophenotypic score (IPS), a technique previously adopted in myelodysplastic syndrome. Eighty-five intensively treated NPM1+ AML cases were studied. Patients were grouped according to the combination of data in maturing cell compartments. FC-assessed dysplasia showed a significant correlation with morphology-assessed dysplasia, showing the efficacy of this method in highlighting dysplasia in AML. Except for MLD, IPS did not influence any patient- or disease-related characteristics at diagnosis. Furthermore, IPS did not influence complete remission rate, disease-free survival, or overall survival. By investigating NPM1 status on separated cell compartments, we established a correlation between FC-assessed MLD and belonging to AML clone. This study shows that dysplasia evaluated by immunophenotype has no impact on clinical-biological characteristics or on outcome of NPM1+ AML. Dysplasia is part of the spectrum of NPM1+ AML, and the prognostic stratification of this category of patients should not be based upon it. © 2015 ISEH - International Society for Experimental Hematology.


PubMed | SOD Laboratorio Centrale, University of Florence and SOD Diagnostica Genetica
Type: Journal Article | Journal: Experimental hematology | Year: 2015

The presence of multilineage dysplasia (MLD) by morphology at diagnosis in acute myeloid leukemia (AML) defines a separate subset in the World Health Organization classification with still-debated prognostic value. A major controversy concerns MLDs role in NPM1-mutated (NPM1) AML, which correlates with good prognosis. We used flow cytometry (FC), an emerging technique for assessing dysplasia, to investigate MLD in NPM1 AML by an immunophenotypic score (IPS), a technique previously adopted in myelodysplastic syndrome. Eighty-five intensively treated NPM1 AML cases were studied. Patients were grouped according to the combination of data in maturing cell compartments. FC-assessed dysplasia showed a significant correlation with morphology-assessed dysplasia, showing the efficacy of this method in highlighting dysplasia in AML. Except for MLD, IPS did not influence any patient- or disease-related characteristics at diagnosis. Furthermore, IPS did not influence complete remission rate, disease-free survival, or overall survival. By investigating NPM1 status on separated cell compartments, we established a correlation between FC-assessed MLD and belonging to AML clone. This study shows that dysplasia evaluated by immunophenotype has no impact on clinical-biological characteristics or on outcome of NPM1 AML. Dysplasia is part of the spectrum of NPM1 AML, and the prognostic stratification of this category of patients should not be based upon it.

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