Abstract 3107: A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors
Authors: Boonstra Pieter A. , Tibbesma Marco , Bosman Lisette J. , Mathijssen Ron H.J. , van Coevorden Frits , Steeghs Neeltje , Suurmeijer Albert J.H. , ter Elst Arja , Gietema Jourik A. , Reyners Anna K.L. , Schuuring Ed M.D. ,   Dutch GIST Consortium
Published: July 2016

Abstract
.
Introduction
Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract. These tumors are characterized by genomic mutations in the c-KIT gene that drive tumor growth and are target for specific inhibitors. Mutations in exon 11 (single nucleotide variations and deletions) are detected in approximately 70% of GIST. Although unique for each patient, most variations cluster in two different mutational hotspots each 25bp in size.
The aim of this study was to develop and test a single quantitative digital droplet PCR (ddPCR) assay to detect exon 11 c-KIT mutations in both formalin fixed paraffin embedded (FFPE) tissue and serial collected cell-free plasma DNA (cfDNA) of GIST patients for diagnostic and treatment response monitoring.
Materials and methods
The drop-off ddPCR assay for detection of exon 11 mutations consists of two fluorescent (FAM and HEX) labeled probes that each cover one of the 2 mutation hotspots. Since double exon 11 mutations are very rare in GIST, one probe will anneal to the non-mutated sequence (reference probe), while the other probe will not anneal. A decrease of one of the 2 fluorescent signals is considered as the presence of a mutation (“drop-off”). For validation, ddPCR results from tumor biopsies were compared with data from next generation sequencing (NGS) using the IonTorrent platform. Pretreatment FFPE tumor material was obtained from 29 (18 exon 11 mutated, 11 non-exon 11 mutated) GIST patients treated in 2014 and 2015. Plasma samples were collected at baseline (before start of treatment with imatinib) and at consecutive time points during treatment.
Results
Using the drop-off ddPCR assay on the same DNA used for NGS, mutations in 17 of the 18 samples were detected. Comparison of the allelic fraction of the mutation shows that ddPCR analysis is very similar to NGS. In 11 control samples, who were wild-type (4 samples) or had mutations other than c-KIT exon 11 (7 samples) no loss of signal was detected.
CfDNA was analyzed of a 72-year old representative patient with metastatic GIST carrying a c-KIT deletion in exon 11 with a fractional abundance (FA) in tissue of 91%. We detected the mutation using the drop-off ddPCR assay in the plasma sample collected before start of imatinib treatment (FA 16%).
Two weeks after initiation of imatinib a rise in mutant allelic frequency was observed (FA 61%) and a decrease after 4 weeks (FA 3%) of therapy.
Conclusion
The detection of multiple exon 11 mutations/deletions using a single ddPCR assay could be used as a rapid prescreening method for the detection of c-KIT mutations in FFPE tissue in GIST patients. Because of the quantitative nature of this assay, ddPCR analysis might be used to monitor response to treatment. The relevance of this drop-off assay for treatment decision making seems promising and is currently validated on a large series of cfDNA samples and a prospective study in GIST patients (NCT02331914).
Citation Format: Pieter A. Boonstra, Marco Tibbesma, Lisette J. Bosman, Ron H.J. Mathijssen, Frits van Coevorden, Neeltje Steeghs, Albert J.H. Suurmeijer, Arja ter Elst, Jourik A. Gietema, Anna K.L. Reyners, Ed M.D. Schuuring, Dutch GIST Consortium. A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3107.