P-0002 - Detection of c-KIT and PDGFRa mutations in GIST by Next Generation Sequencing

Date 28 June 2014
Event World GI 2014
Session Poster Session
Topics GIST
Pathology/Molecular Biology
Personalised Medicine
Presenter Céline De Rop
Citation Annals of Oncology (2014) 25 (suppl_2): ii14-ii104. 10.1093/annonc/mdu165
Authors C. De Rop, G. Beniuga, P. Ngendahayo, P. Vannuffel
  • IPG, Gosselies/BE



Gastrointestinal stromal tumours (GIST) occur in the gastrointestinal track, most commonly in the stomach or small intestine. These sporadic tumours are associated with genetic changes. In GIST subsets, c-KIT or PDGFRa mutations correlate with distinct anatomical site, clinical phenotype and sensitivity to tyrosine kinase inhibitors. Here we depict a multiplexed evaluation strategy based on Next Generation Sequencing (NGS) to investigate for the most relevant regions of c-KIT and PDGFRa in a GIST context.


A custom panel, covering exons 9, 11, 13 and 17 of c-KIT and exons 12, 14 and 18 of PDGFRa, was designed using the Ion AmpliSeq Designer module. FFPE tissue sections were macro-dissected and DNA was extracted using the Maxwell FFPE Tissue LEV purification kit. Sequencing was performed according to the AmpliSeq protocol on the Ion Torrent PGM, from as less as 6 ng of input DNA. Data were analysed with both Torrent Suite v4 and NextGENe software v2.3 with a whole exons-targeting bed file. Sensitivity of the technique was assigned from 2.5% at 500 x coverage to 10% at 100 x coverage, assuming a forward to reverse reads ratio >0.25.


DNA from samples for which c-KIT and PDGFRa mutations have been previously identified by Sanger sequencing (10 providing from EQC schemes and 30 from our regular practice), as well as from 10 wild-type samples, were tested with the designed panel. For all samples, there is a perfect correlation between our multiplexed sequencing strategy and the « gold standard » Sanger sequencing. Mutations - point mutations, duplications, deletions, insertions or both, mainly located in exons 9 and 11 of c-KIT and in exon 18 of PDGFRa – were found in all 40 mutated samples by both techniques, while no critical variant was detected in the 10 normal samples.


This multiplexed amplification of 7 exons in GIST samples demonstrates that custom NGS panels can be designed to identify mutations in genes or regions more relevant to diagnosis, prognosis or therapeutic choices. For any tumour setting or pathway, NGS panels allow more comprehensive sequence coverage than standard techniques and are scalable in term of regions of interest and number of samples.