title: Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH.
abstract: BACKGROUND
  Mutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABL (T315I) mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation.
  
  
  RESULTS
  The PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors.
  
  
  CONCLUSIONS
  We present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Rosso
authors.affiliation: Department of Oncology, University of Turin, Turin, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Bracco
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Pedrola
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Carturan
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Signorino
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Jessica
authors.initials: J
authors.lastname: Petiti
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Calabrese
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Nicoli
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: De Gobbi
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Gaidano
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Gallo
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ulisciani
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Fava
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Rege-Cambrin
authors.affiliation: Department of Pediatric Hemato-Oncology and Stem Cell and Cellular Therapy Laboratory, Institute G. Gaslini, Largo G Gaslini, Genoa, 16147 Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Frassoni
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Saglio
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Cilloni
keywords: BCR-ABL1; Chronic Myeloid Leukemia; PNA; T315I mutation