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.

Figure 1

Example of MIDIA analysis. A: Pseudo-gel image, samples 3 and 7 harbor a KIT deletion. B: Electropherogram of the smallest KIT alteration detected by MIDIA, a 3-bp insertion of sample 25. Short arrows: 15-bp and 600-bp markers; long arrows: PCR product peaks.

Figure 2

A: Chromatogram view of the new combined deletion and point mutation at 5′ end of KIT exon 11. Wild-type sequence in red, mutated sequence in blue; G to T point mutation squared in red. B: Sequence alignment of wild-type (red) and mutated (blue) sequences.

Figure 3

Proposed flow chart for mutation detection in GISTs. The suggested flow chart differs according to the site of origin and the histological variant of the GIST. Green boxes refer to MIDIA, orange boxes refer to PCR sequencing or DHPLC analysis. Small squares below the indicated analysis describe the percentages of the resolved cases at each step. For example, among the spindle GIST originating from the stomach, 60% of mutations are detected at the first step by KIT exon 11 MIDIA analysis, a further 20% is detected by KIT exon 11 PCR sequencing/DHPLC, and an additional 20% by PDGFRA exon 12 and 18 DHPLC/sequencing. The remaining mutations are detected by KIT exon 9 MIDIA and by further DHPLC/sequencing analysis as indicated.