Circulating Methylated DNA to Monitor the Dynamics of RAS Mutation Clearance in Plasma from Metastatic Colorectal Cancer Patients

SIMPLE SUMMARY: Ongoing clinical trials are recently investigating the efficacy of second-line EGFR inhibitors in initially RAS mutant metastatic colorectal cancer patients who convert to RAS wild-type in plasma, as assessed by circulating tumor DNA (ctDNA) analysis. To this purpose, discriminating between patients with a real clearance of RAS mutations in plasma (real wild-type) from those who do not shed ctDNA is mandatory. The aim of the present study was to confirm the presence of ctDNA in patients with RAS mutation clearance in plasma at different time points, using a colon-cancer-specific five-gene methylation panel. The methylation test confirmed the presence of ctDNA in most RAS wild-type samples at the time of disease progression, thus confirming that the negative selection of RAS mutant clones during the clonal evolution of mutant RAS colorectal cancer is not an infrequent event. ABSTRACT: The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who “convert” to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples.