Serial profiling of circulating tumor DNA identifies biomarkers for anti-VEGF chemotherapy in mCRC patients

Abstract

Background: Understanding the molecular changes in tumors in response to anti-VEGF chemotherapy is crucial to optimize the treatment strategy for metastatic colorectal cancer. We investigated changes in the amount and constitution of circulating tumor DNA (ctDNA) with serial peripheral blood sampling during chemotherapy. Methods: Forty plasma samples taken at different time-points (pre-treatment, remission, after progression) and pre-treatment tumor formalin-fixed paraffin-embedded (FFPE) samples were collected from 11 patients who received bevacizumab-containing first-line chemotherapy (backbone cytotoxic regimen; mFOLFOX6 or CapeOx). DNA extracted from plasma and tumor was sequenced by next-generation sequencing using a panel of 90 cancer-related genes to identify gene alterations. Candidate ctDNA in plasma were validated by mutational data from matching tumors. Results: One to five trunk mutations were found as ctDNA in all cases, and the mutant allele frequency (MAF) was distributed over a wide range (1-80%). Significant decreases at remission and increases after progression were observed in MAF (p<0.001). Reduction in MAF to below 3% in the remission period was strongly associated with better survival (19.9 vs. 37.6 months, p=0.007). In two cases, newly detected mutations (FBXW7 and CREBBP) at a low frequency of around 1% were found in the after-progression period. Compared to other cases, these two patients were less sensitive to subsequent cetuximab treatment. Conclusion: Use of ctDNA allows elucidation of the clonal repertoire and evolution during anti-VEGF chemotherapy. Changes in ctDNA level could be useful as predictive biomarkers for survival. Newly detected mutations in the late treatment period might reveal the rise of a minor clone that may show resistance to subsequent EGFR blockade therapy.