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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
  1. Katarzyna Kiwerska1,2,
  2. Joanna Wroblewska3,
  3. Apolonia Kaluzna3,
  4. Andrzej Marszalek3
  1. 1 Department of Tumor Pathology, Greater Poland Cancer Centre, Poznan, Poland
  2. 2 Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
  3. 3 Department of Tumor Pathology and Prophylaxis, Poznan University of Medical Sciences & Greater Poland Cancer Centre, Poznan, Poland
  1. Correspondence to Dr Katarzyna Kiwerska, Department of Tumor Pathology, Greater Poland Cancer Centre, Poznan 61-866, Poland; katarzyna.kiwerska{at}


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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  • Handling editor Runjan Chetty.

  • Contributors KK designed the study, performed the analysis and drafted the manuscript. JW performed laboratory and data analysis. AK performed laboratory analysis. AM supervised the study and revised the manuscript critically for important intellectual content.

  • Funding This work was supported by the Greater Poland Cancer Centre, Poznan, Poland (grant no. 18/04/2017/WCO/0016).

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.