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

Chiara Nicolazzo,Federica Di Nicolantonio,Marco Macagno,Ludovic Barault,Salvatore Caponnetto,Gianluigi De Renzi,Enrico Cortesi,Francesca Belardinilli,Angela Gradilone,Paola Gazzaniga

doi:10.3390/cancers12123633

Abstract

Simple SummaryOngoing 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.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.

Highlights

The use of cell-free DNA to monitor cancer clonal evolution allows scientists to rapidly identify the occurrence of drug resistance to targeted therapies [1,2,3]
RAS mutant metastatic colorectal cancer (mCRC), suggesting for the first time an unpredicted negative selection of RAS mutant clones during the clonal evolution of this cancer type [5]. This observation led to the design of the first clinical trials aimed to investigate the efficacy and safety of second-line epidermal growth factor receptor (EGFR) inhibitors plus chemotherapy in initially RAS mutant mCRC patients who convert to RAS wt in plasma at the time of first disease progression (PD) [6,7]
Characteristics of patient population are shown in Table 1. circulating tumor DNA (ctDNA) samples were analyzed for specific KRAS and NRAS variants with real-time PCR

Summary

Introduction

The use of cell-free DNA (cfDNA) to monitor cancer clonal evolution allows scientists to rapidly identify the occurrence of drug resistance to targeted therapies [1,2,3]. In wild-type (wt) RAS metastatic colorectal cancer (mCRC), the emergence of RAS mutant clones under the selective pressure of epidermal growth factor receptor (EGFR) inhibitors has been widely described, providing a molecular rationale for liquid biopsy-based adaptive therapies [4]. RAS mutant mCRC, suggesting for the first time an unpredicted negative selection of RAS mutant clones during the clonal evolution of this cancer type [5]. This observation led to the design of the first clinical trials aimed to investigate the efficacy and safety of second-line EGFR inhibitors plus chemotherapy in initially RAS mutant mCRC patients who convert to RAS wt in plasma at the time of first disease progression (PD) [6,7]. Circulating methylated DNA (cmDNA) defined by a five-gene panel was previously used as a non-invasive treatment-monitoring assay and was associated with outcomes in mCRC patients [9]

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