LAUSR

Simple Summary At some point during long-term treatment, gastrointestinal stromal tumors (GIST) stop responding to treatment due to new genetic changes (mutations). These mutations can be found in the blood of patients using very expensive methods not covered by insurance. We discovered that some mutations can be found in the blood when using cheaper methods that are available in most university hospitals. If the blood was processed immediately, regular blood tubes could be used, and a robot would be better in isolating the genetic information (DNA). Mutations were easier to find in patients with bigger tumors. We compared two different methods to analyze the data. Mutations that were found by both methods were mostly important resistance mutations, but many known mutations from tumor tissue were missed. Taken together, we think that our method cannot replace tumor biopsy or radiologic imaging in GIST, but more precise methods have to be investigated in clinical trials. Abstract Circulating tumor DNA (ctDNA) from circulating free DNA (cfDNA) in GIST is of interest for the detection of heterogeneous resistance mutations and treatment monitoring. However, methodologies for use in a local setting are not standardized and are error-prone and difficult to interpret. We established a workflow to evaluate routine tumor tissue NGS (Illumina-based next generation sequencing) panels and pipelines for ctDNA sequencing in an academic setting. Regular blood collection (Sarstedt) EDTA tubes were sufficient for direct processing whereas specialized tubes (STRECK) were better for transportation. Mutation detection rate was higher in automatically extracted (AE) than manually extracted (ME) samples. Sensitivity and specificity for specific mutation detection was higher using digital droplet (dd)PCR compared to NGS. In a retrospective analysis of NGS and clinical data (133 samples from 38 patients), cfDNA concentration correlated with tumor load and mutation detection. A clinical routine pipeline and a novel research pipeline yielded different results, but known and resistance-mediating mutations were detected by both and correlated with the resistance spectrum of TKIs used. In conclusion, NGS routine panel analysis was not sensitive and specific enough to replace solid biopsies in GIST. However, more precise methods (hybridization capture NGS, ddPCR) may comprise important research tools to investigate resistance. Future clinical trials need to compare methodology and protocols.