Background: The GRID study demonstrated that regorafenib provides a significant improvement in progression-free survival (HR 0.27; p<0.0001) compared with placebo in patients with advanced gastrointestinal stromal tumors (GIST) following failure of at least imatinib and sunitinib. Mutations in KIT play an important role in GIST and identification of the specific KIT mutations that each GIST patient harbors could have important therapeutic implications. However, determining tumor genotype in TKI-refractory GIST has proven challenging due to intra and inter-tumoral genetic heterogeneity and the difficulty associated with obtaining fresh tumor biopsies. To overcome this limitation, tumor genotyping in GRID was performed using baseline plasma as a source of circulating tumor-derived DNA. We previously performed genotyping using BEAMing (a mutation-specific technology) and have now extended this analysis using Safe-SeqS (Kinde et al. 2011 PNAS 108:9530) a targeted NGS-based platform aiming to detect previously undetectable cKIT mutations.

Methods: 91 plasma samples from patients enrolled in the Ph 3 study (GRID) for which BEAMing data (Jeffers et al 2013 JCO 31:10503) were subjected to Safe-SeqS covering cKit Exon 8 to Exon 18.

Results: In 6 of 32 samples reported to be cKIT wildtype by BEAMing, mutations were identified by SafeSeqS. The detection of primary KIT exon 9 mutations showed a high degree of concordance among the two mutation-detection methods evaluated. Secondary / resistance hotspot mutations were also readily detected by both methods, although a greater number of such mutations were detected by Safe-SeqS than by BEAMing. The localization of the additional mutations detected by Safe-SeqS in known mutational hotspots supports their legitimacy. Safe-SeqS also detected KIT mutations for which BEAMing assays had not been developed, whereas in 17 samples a mutation for which a BEAMing assay was available was not detectable by Safe SeqS. In 58% (10/17) of samples, the mutant allele frequency found by BEAMing was close to the detection limit of this platform (<0.05). The cKITM541L mutation in Exon10 was found in 25% (23/91) of the samples.

Conclusion: Our data support the use of Safe-SeqS as a sensitive and specific “liquid biopsy” method for non-invasive tumor genotyping of patients with GIST, enabling the identification of known and novel tumor-associated mutations using circulating DNA. These results confirm and extend the genotypic heterogeneity that had previously been identified in GRID circulating DNA samples by BEAMing. The comprehensive tumor mutational profiles generated by Safe-SeqS will be used to evaluate potential correlations between tumor genotype and clinical outcome.

Citation Format: Michael Jeffers, Henrik Seidel, Susanne Schwenke, Joachim Reischl, Mark Rutstein, Christian Kappeler, Iris Kuss, Michael Teufel. Tumor genotyping in the phase III GRID study of regorafenib vs placebo in tyrosine kinase inhibitor (TKI)-refractory GIST: Detection of KIT mutations in circulating tumor DNA comparing digital PCR and massive parallel sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 929. doi:10.1158/1538-7445.AM2015-929