title: Extensive next-generation sequencing analysis in chronic lymphocytic leukemia at diagnosis: clinical and biological correlations.
abstract: In chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations.
  
  
  
  We performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens.
  
  
  
  Mutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p < 0.001), and the complex karyotype (p = 0.003). A high risk as assessed by FISH analysis was associated with mutations affecting TP53 (p = 0.012), BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations (p = 0.003, 0.018, and 0.001, respectively). By multivariate analysis, the multi-hit profile (≥2 mutations by NGS) was independently associated with a shorter time to first treatment (p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001). Advanced stage (p = 0.010), TP53 disruption (p < 0.001), IGHV unmutated status (p = 0.020), and the complex karyotype (p = 0.007) were independently associated with a shorter overall survival.
  
  
  
  At diagnosis, an extensive biologic characterization including NGS and karyotype analyses using novel mitogens may offer new perspectives for a better refinement of risk stratification that could be of help in the clinical management of CLL patients.
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy. rglgmt@unife.it.
authors.forename: Gian Matteo
authors.identifier: 0000-0002-8370-5190
authors.initials: GM
authors.lastname: Rigolin
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Saccenti
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, and "Laboratorio per le Tecnologie delle Terapie Avanzate" (LTTA), University of Ferrara, Ferrara, Italy.
authors.forename: Cristian
authors.initials: C
authors.lastname: Bassi
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, and "Laboratorio per le Tecnologie delle Terapie Avanzate" (LTTA), University of Ferrara, Ferrara, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Lupini
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Francesca Maria
authors.initials: FM
authors.lastname: Quaglia
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cavallari
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Martinelli
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Formigaro
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Lista
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Maria Antonella
authors.initials: MA
authors.lastname: Bardi
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Volta
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Tammiso
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Aurora
authors.initials: A
authors.lastname: Melandri
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Urso
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cavazzini
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, and "Laboratorio per le Tecnologie delle Terapie Avanzate" (LTTA), University of Ferrara, Ferrara, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Negrini
authors.affiliation: Hematology Section, Department of Medical Sciences, Azienda Ospedaliero-Universitaria, Arcispedale S. Anna, University of Ferrara, Via Aldo Moro, 8, 44124, Ferrara, Cona, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Cuneo
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics; D000075747:Baculoviral IAP Repeat-Containing 3 Protein / Q000235:genetics; D004252:DNA Mutational Analysis; D000073979:F-Box-WD Repeat-Containing Protein 7 / Q000235:genetics; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics; D018570:Risk Assessment / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016159:Tumor Suppressor Protein p53; C494832:BIRC3 protein, human; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins
keywords: Chronic lymphocytic leukemia; Complex karyotype; Gene mutation analysis; Next-generation sequencing; Prognosis