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Justification of direct Sanger sequencing application for detection of KIT and PDGFRα gene mutations in formalin-fixed, paraffin-embedded samples from gastrointestinal stromal tumours


Aims The knowledge concerning genetic background of gastrointestinal stromal tumours (GISTs) is well recognised, and the accurate detection of KIT and PDGFRα mutations is of great importance for the process of disease diagnosis and patient’s treatment. In this study, we compare the usefulness of real-time PCR-based techniques and Sanger sequencing to detect mutations of both genes in 41 formalin-fixed, paraffin-embedded GIST samples.

Methods The analysis encompassed most frequently mutated coding regions of KIT (exons 9, 11, 13 and 17) and PDGFRα (exons 12, 14 and 18) genes. The GIST Mutation Detection Kit (EntroGen), direct Sanger sequencing and high-resolution melting (HRM) analysis were applied to conduct the study.

Results With the application of EntroGen kit, we found alterations in 22/38 samples, with Sanger sequencing variants were found in 36/41 samples. The concordant results for both methods were observed in 19/38 samples. With subsequently applied HRM analysis, we have confirmed that all samples, except one, harboured alterations in the regions indicated by Sanger sequencing.

Conclusions Our results show that in GIST samples, carrying a broad spectrum of deletions, Sanger sequencing is a better, more sensitive method for mutational analysis of KIT and PDGFRα genes.

  • molecular oncology
  • molecular pathology
  • cancer genetics

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