title: Microfluidic deletion/insertion analysis for rapid screening of KIT and PDGFRA mutations in CD117-positive gastrointestinal stromal tumors: diagnostic applications and report of a new KIT mutation.
abstract: Gastrointestinal stromal tumors (GISTs) frequently harbor mutations in the KIT and PDGFRA genes, the presence and type of which correlate with the response to the kinase inhibitor imatinib mesylate. Because most GIST mutations are deletions/insertions, we used a microfluidic apparatus to detect these size variations in polymerase chain reaction-amplified DNA. This approach, termed microfluidic deletion/insertion analysis (MIDIA), identified mutations in 30 of 50 DNA samples from paraffin-embedded CD117-positive GISTs (60%), comprising 25 deletions and five insertions. Sequencing of 14 MIDIA-positive samples confirmed the deletions/insertions, including two 3-bp alterations. Sequencing of all 20 MIDIA-negative samples also showed highly consistent results with MIDIA because 10 cases were wild type and eight displayed a single base substitution in which detection by MIDIA was not expected. Sequencing also revealed a 3-bp deletion undetected by MIDIA, thus establishing the resolution limit of MIDIA at deletions/insertions >or=3 bp. Denaturing high-pressure liquid chromatography analysis confirmed all mutations detected by MIDIA and sequencing. We pro-pose MIDIA as the first step in mutational screening of GIST because it allowed the detection of 75% of mutated cases (94% of deletions/insertions) in less than 30 minutes after polymerase chain reaction amplification and at a lower cost compared with denaturing high-pressure liquid chromatography and sequencing, which might then be used only for MIDIA-negative cases.
authors: Department of Pathology, University of Verona, Verona, Italy.
authors: Alberto
authors: A
authors: Zamò
authors: Anna
authors: A
authors: Bertolaso
authors: Ilaria
authors: I
authors: Franceschetti
authors: Gregor
authors: G
authors: Weirich
authors: Paola
authors: P
authors: Capelli
authors: Sara
authors: S
authors: Pecori
authors: Marco
authors: M
authors: Chilosi
authors: Heinz
authors: H
authors: Hoefler
authors: Fabio
authors: F
authors: Menestrina
authors: Aldo
authors: A
authors: Scarpa
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003365:Costs and Cost Analysis; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons / Q000235:genetics; D005188:False Negative Reactions; D005189:False Positive Reactions; D046152:Gastrointestinal Stromal Tumors / Q000175:diagnosis* / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D044085:Microfluidics / Q000191:economics / Q000379:methods*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000235:genetics*; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; D019009:Proto-Oncogene Proteins c-kit; D020796:Receptor, Platelet-Derived Growth Factor alpha