Liquid biopsy in gastrointestinal stromal tumors -- Simultaneous detection of primary and secondary mutations with an NGS based circulating tumor DNA assay

PURPOSE: Gastrointestinal stromal tumors (GISTs) are characterized by primary mutations (PM) in KIT or PDGFRA genes. Under pharmacological pressure secondary mutations (SM) develop resulting in resistance to treatment. Our group developed a multiplex-amplicon sequencing assay that can detect PM and SM simultaneously in the plasma samples of patients with GIST, at a variant allele frequency of 0.1%. This assay was also designed to optimize detection of exon 11 mutations. PATIENTS AND METHODS: The assay was applied on 238 plasma samples obtained from 65 GIST patients. Clinical data and plasma samples were obtained from a prospective institutional database. Among the 65 patients, 34 had metastatic disease, 4 were on neoadjuvant treatment and 27 had curative surgery followed by adjuvant treatment or surveillance. There were no prespecified timepoints of plasma collection in this study. CtDNA results were correlated with clinical and radiological data. RESULTS: Overall, there was 89% concordance in detection of exon 11 mutations in tissue and plasma. 100% concordance was demonstrated for exons 9,13 and 17. The assay had a sensitivity of 75% and specificity of 94% in detecting mutations in the appropriate clinical context in the metastatic setting. Secondary mutations were detected in the appropriate clinical settings as well. In the metastatic cohort, ctDNA levels were able to correlate with disease trajectory i.e. levels decrease with response to treatment and increase with disease progression. CONCLUSION: This assay is a promising tool that can reliably and accurately detect ctDNA in GIST patients, especially the traditionally difficult exon 11 mutations. Once validated in a larger, prospective setting, this can potentially establish ctDNA as a non invasive biomarker in monitoring disease trajectory in GIST patients.