title: Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia.
abstract: Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.
  
  
  
  Amplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19- early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.
  
  
  
  NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19- early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.
  
  
  
  Our study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Quijada-Álamo
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Hernández-Sánchez
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Robledo
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Jesús-María
authors.initials: JM
authors.lastname: Hernández-Sánchez
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Rocío
authors.initials: R
authors.lastname: Benito
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Adrián
authors.initials: A
authors.lastname: Montaño
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.|Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK.
authors.forename: Ana E
authors.initials: AE
authors.lastname: Rodríguez-Vicente
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Dalia
authors.initials: D
authors.lastname: Quwaider
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Ana-África
authors.initials: AÁ
authors.lastname: Martín
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: María
authors.initials: M
authors.lastname: García-Álvarez
authors.affiliation: Servicio de Hematología, Hospital Virgen Blanca, León, Spain.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Vidal-Manceñido
authors.affiliation: Servicio de Hematología, Hospital Miguel Servet, Zaragoza, Spain.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Ferrer-Garrido
authors.affiliation: Servicio de Hematología, Hospital Miguel Servet, Zaragoza, Spain.
authors.forename: María-Pilar
authors.initials: MP
authors.lastname: Delgado-Beltrán
authors.affiliation: Servicio de Hematología, Hospital del Bierzo, Ponferrada, León, Spain.
authors.forename: Josefina
authors.initials: J
authors.lastname: Galende
authors.affiliation: Servicio de Hematología, Hospital Juan Ramón Jiménez, Huelva, Spain.
authors.forename: Juan-Nicolás
authors.initials: JN
authors.lastname: Rodríguez
authors.affiliation: Servicio de Hematología, Hospital Virgen del Puerto, Plasencia, Cáceres, Spain.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Martín-Núñez
authors.affiliation: Servicio de Hematología, Hospital Río Carrión, Palencia, Spain.
authors.forename: José-María
authors.initials: JM
authors.lastname: Alonso
authors.affiliation: Servicio de Hematología, Hospital Clínico, Valladolid, Spain.
authors.forename: Alfonso
authors.initials: A
authors.lastname: García de Coca
authors.affiliation: Servicio de Hematología, Hospital General de Segovia, Segovia, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Queizán
authors.affiliation: Servicio de Hematología, Hospital Virgen de la Concha, Zamora, Spain.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Sierra
authors.affiliation: Servicio de Hematología, Hospital Santa Bárbara, Soria, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Aguilar
authors.affiliation: MLL Munich, Munich, Germany.|AstraZeneca, Personalized Healthcare and Biomarkers, Innovative Medicines, Cambridge, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kohlmann
authors.affiliation: Servicio de Hematología, Hospital Universitario Infanta Leonor, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: José-Ángel
authors.initials: JÁ
authors.lastname: Hernández
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain.
authors.forename: Marcos
authors.initials: M
authors.lastname: González
authors.affiliation: Servicio de Hematología & IBSAL, IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario, Salamanca, Spain. jmhr@usal.es.|IBMCC, CIC Universidad de Salamanca-CSIC, Hospital Universitario de Salamanca, Paseo de San Vicente s/n, 37007, Salamanca, Spain. jmhr@usal.es.
authors.forename: Jesús-María
authors.initials: JM
authors.lastname: Hernández-Rivas
mesh_terms: D018941:Antigens, CD19; D018952:Antigens, CD34; D002869:Chromosome Aberrations; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000473:pathology*; D009154:Mutation
chemical_list: D018941:Antigens, CD19; D018952:Antigens, CD34; C000604811:CD19 molecule, human
keywords: Chromosomal abnormality; Chronic lymphocytic leukemia; FISH; Hematopoietic progenitors; Mutation; Next-generation sequencing