Broad spectrum of regorafenib activity on mutant KIT and absence of clonal selection in gastrointestinal stromal tumor (GIST): correlative analysis from the GRID trial.

BACKGROUND: In the phase 3 GRID trial, regorafenib improved progression-free survival (PFS) independent of KIT mutations in exons 9 and 11. In this retrospective, exploratory analysis of the GRID trial, we investigated whether a more comprehensive KIT mutation analysis could identify mutations that impact treatment outcome with regorafenib and a regorafenib-induced mutation pattern. METHODS: Archived tumor samples, collected at any time prior to enrollment in GRID, were analyzed by Sanger sequencing (n = 102) and next-generation sequencing (FoundationONE; n = 47). Plasma samples collected at baseline were analyzed by BEAMing (n = 163) and SafeSEQ (n = 96). RESULTS: In archived tumor samples, 67% (68/102) had a KIT mutation; 61% (62/102) had primary KIT mutations (exons 9 and 11) and 12% (12/102) had secondary mutations (exons 13, 14, 17, and 18). At baseline, 81% of samples (78/96) had KIT mutations by SafeSEQ, including the M541L polymorphism (sole event in 6 patients). Coexisting mutations in other oncogenes were rare, as were mutations in PDGFR, KRAS, and BRAF. Regorafenib showed PFS benefit across all primary and secondary KIT mutational subgroups examined. Available patient-matched samples taken at baseline and end of treatment (n = 41; SafeSEQ), revealed heterogeneous KIT mutational changes with no specific mutation pattern emerging upon regorafenib treatment. CONCLUSION: These data support the results of the GRID trial, and suggest that patients may benefit from regorafenib in the presence of KIT mutations and without the selection of particular mutation patterns that confer resistance. The study was not powered to address biomarker-related questions, and the results are exploratory and hypothesis-generating.

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing.

OBJECTIVES: Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies. METHODS: Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS). RESULTS: Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes. CONCLUSION: Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.

Detection and Quantification of KIT Mutations in ctDNA by Plasma Safe-SeqS.

We have designed a highly sensitive assay based on the Safe-SeqS technology to detect de novo mutations in the KIT gene and tested its performance. This assay was applied to plasma samples of GIST patients before and after treatment with regorafenib (GRID III trial) and mutations at known and novel sites of potential secondary resistance were identified.