A single digital droplet PCR assay to detect multiple KIT exon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors
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Pieter A. Boonstra1, Arja ter Elst2, Marco Tibbesma1,2, Lisette J. Bosman2, Ron Mathijssen3, Florence Atrafi3, Frits van Coevorden4, Neeltje Steeghs5, Sheima Farag5, Hans Gelderblom6, Winette T.A. van der Graaf7, Ingrid M.E. Desar7, Jacqueline Maier8, Jelle Overbosch9, Albert J.H. Suurmeijer2, Jourik Gietema1, Ed Schuuring2,* and Anna K.L. Reyners1,*
1University of Groningen, University Medical Center Groningen, Department of Medical Oncology, Groningen 9713 GZ, The Netherlands
2University of Groningen, University Medical Center Groningen, Department of Pathology, Groningen 9713 GZ, The Netherlands
3Department of Medical Oncology, Erasmus University Medical Center Rotterdam, Rotterdam 3015 CE, The Netherlands
4Antoni van Leeuwenhoek, Netherlands Cancer Institute, Department of Surgery, Amsterdam 1066 CX, The Netherlands
5Antoni van Leeuwenhoek, Netherlands Cancer Institute, Department of Medical Oncology, Amsterdam 1066 CX, The Netherlands
6Leiden University Medical Center, Department of Medical Oncology, Leiden 2300 RC, The Netherlands
7Radboud University Medical Center, Department of Medical Oncology, Nijmegen 6500 HB, The Netherlands
8University of Leipzig, Center for Internal Medicine, Department of Hematology/Oncology, Leipzig 04103, Germany
9University of Groningen, University Medical Center Groningen, Department of Radiology, Groningen 9713 GZ, The Netherlands
*These authors share authorship
Anna K.L. Reyners, email: firstname.lastname@example.org
Keywords: GIST; cell-free circulating DNA; single assay; plasma; digital droplet PCR
Received: October 05, 2017 Accepted: January 13, 2018 Published: February 14, 2018
Background: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients’ plasma.
Methods: A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample.
Results: The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease.
Conclusion: A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.
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