Abstract 4578: Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST)

Abstract

Abstract Introduction: Genotype-directed therapies are revolutionizing care for gastrointestinal stromal tumors (GIST). Somatic mutations in KIT exons (ex) 11 and 9 induce constitutive kinase activity and account for ~95% of the primary oncogenic events in KIT-driven GIST; KIT mutations correlate with prognosis and predict clinical activity of tyrosine kinase inhibitors (TKI), such as imatinib. After a median of 24 months on imatinib, 85% of patients (pts) develop resistance and progress multifocally, mostly due to secondary KIT mutations in ex 13 and/or ex 17. We describe here a rapid, clinical grade droplet digital PCR (ddPCR) assay to detect and quantify KIT mutations associated with TKI resistance in cfDNA from plasma of metastatic (met) GIST pts. Unlike allele-specific ddPCR that requires splitting input DNA into many reactions, we designed pan-ex 17 KIT assay to capture the polyclonal mutational landscape of KIT in a single reaction. Methods: Tumor and plasma samples and clinical data were collected from pts with met GIST. ddPCR assays were developed for the detection in cfDNA of all common secondary KIT mutations associated with imatinib resistance (V654A, T670I, A829P and multiple ex 17 mutations). The pan-ex 17 mutation drop-off assay uses a FAM-labeled mutant (mut) probe that spans sequences encoding amino acids 819~824, and a VIC-labeled reference probe that is shared by both wild-type (wt) and mut alleles. Common ex 17 mutations were detected via cluster separation. V654A, T670I, A829P are detected with allele-specific assays that use a FAM-labeled mut probe and a VIC-labeled wt probe. To demonstrate analytical sensitivity/specificity of each assay, ddPCR cycling conditions were optimized to yield the maximum fluorescence signal with minimal noise signal. Results: The KIT ddPCR assays detect a mutation prevalence of 0.01 - 0.05%, with a sensitivity of 5 - 50 KIT mut copies in a background of 10,000 KIT wt copies, depending on the mutation assayed, with absolute specificity. Results were validated in a pilot observational cohort of pts who were treated with imatinib and progressed, for which matching plasma (1 ml) and tumor samples were analyzed to reveal identical mutations. In a separate cohort, ddPCR results were validated by sensitive plasma NGS. Conclusions: We have developed rapid, highly sensitive, and 100% specific quantitative assays to enable plasma-based tumor genotyping for secondary KIT resistance mutations, which has been technically optimized for translation into clinical practice. Analysis of cfDNA by ddPCR can successfully detect KIT secondary mutations in met GIST pts. In an ongoing study, serial monitoring of KIT mutations in cfDNA from pts with advanced GIST should allow for early detection of secondary mutations and optimization of genotype-targeted therapeutic management. Citation Format: Adrian Marino-Enriquez, Suzanne George, Julianna Supplee, Grace Heavey, Pasi A. Janne, Chandrajit Raut, Jonathan Fletcher, George D. Demetri, Cloud P. Paweletz, Yanan Kuang. Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4578.