title: Mutational analysis of gastrointestinal stromal tumors (GISTs): procedural approach for diagnostic purposes.
abstract: BACKGROUND
  Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract characterized, in the majority of cases, by activating mutations in the KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) or PDGFRA (platelet-derived growth factor receptor, alpha polypeptide) genes. Mutations affecting these tyrosine kinase receptors are also responsible for the mechanisms of primary and secondary drug resistance during the treatment with tyrosine kinase inhibitors. We performed mutational analysis to evaluate the pharmacotherapy susceptibility of GISTs, adopting a comprehensive procedural approach, in order to optimize the identification of mutations that may result in cellular resistance to conventional therapy.
  
  
  METHODS
  DNA from paraffin-embedded tumor sections from 40 GISTs were analyzed using microdissection, direct sequencing analysis and allelic separation by cloning.
  
  
  RESULTS
  KIT mutations were found in 55.0% of the tumor samples. PDGFRA mutations were present in 5.0% of cases. Allelic cloning assay allowed for better definition of the extent of the mutations and clarification of the exact nucleotidic position of complex mutations.
  
  
  CONCLUSIONS
  Our experience suggests that sequential microdissection, direct sequencing and allelic separation by PCR cloning of large variants may improve the approach to mutational analysis and interpretation of sequence data of KIT and PDGFRA in patients with GIST.
authors.affiliation: Department of Advanced Biotechnologies and Bioimaging, Institute of Care and Scientific Research (IRCCS) San Raffaele Pisana, Rome, Italy. raffaele.palmirotta@sanraffaele.it
authors.forename: Raffaele
authors.initials: R
authors.lastname: Palmirotta
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: De Marchis
authors.forename: Giorgia
authors.initials: G
authors.lastname: Ludovici
authors.forename: Barbara
authors.initials: B
authors.lastname: Leone
authors.forename: Renato
authors.initials: R
authors.lastname: Covello
authors.forename: Salvatore
authors.initials: S
authors.lastname: Conti
authors.forename: Leopoldo
authors.initials: L
authors.lastname: Costarelli
authors.forename: David
authors.initials: D
authors.lastname: Della-Morte
authors.forename: Patrizia
authors.initials: P
authors.lastname: Ferroni
authors.forename: Mario
authors.initials: M
authors.lastname: Roselli
authors.forename: Fiorella
authors.initials: F
authors.lastname: Guadagni
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D046152:Gastrointestinal Stromal Tumors / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics
chemical_list: D019009:Proto-Oncogene Proteins c-kit; D020796:Receptor, Platelet-Derived Growth Factor alpha
keywords: Gastrointestinal stromal tumors; KIT; PDGFRA; mutational analysis; pharmacogenomics