title: Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.
abstract: OBJECTIVE
  Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method.
  
  
  METHODS
  With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain.
  
  
  RESULTS
  Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results.
  
  
  CONCLUSIONS
  This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance.
authors.affiliation: Department of Hematology, University Hospital of Son Espases, Carretera Valldemossa no. 79, Palma de Mallorca, Spain. jorgej.martinez@ssib.es
authors.forename: Jordi
authors.initials: J
authors.lastname: Martinez-Serra
authors.forename: Antonio
authors.initials: A
authors.lastname: Gutiérrez
authors.forename: Toni F
authors.initials: TF
authors.lastname: Marcús
authors.forename: Simona
authors.initials: S
authors.lastname: Soverini
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Amat
authors.forename: María
authors.initials: M
authors.lastname: Navarro-Palou
authors.forename: Teresa
authors.initials: T
authors.lastname: Ros
authors.forename: Teresa
authors.initials: T
authors.lastname: Bex
authors.forename: Carmen
authors.initials: C
authors.lastname: Ballester
authors.forename: Josep Miquel
authors.initials: JM
authors.lastname: Bauça
authors.forename: Sara
authors.initials: S
authors.lastname: SanFelix
authors.forename: Andrés
authors.initials: A
authors.lastname: Novo
authors.forename: Carmen
authors.initials: C
authors.lastname: Vidal
authors.forename: Carmen
authors.initials: C
authors.lastname: Santos
authors.forename: Joan
authors.initials: J
authors.lastname: Besalduch
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001483:Base Sequence; D001549:Benzamides; D001853:Bone Marrow / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D016044:Fusion Proteins, bcr-abl / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000188:drug therapy* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010879:Piperazines / Q000627:therapeutic use*; D011743:Pyrimidines / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction; D012189:Retrospective Studies
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D015342:DNA Probes; D010879:Piperazines; D011743:Pyrimidines; C406060:abl-bcr fusion protein, human; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl