Circulating DNA Demonstrates Convergent Evolution and Common Resistance Mechanisms during Treatment of Colorectal Cancer

Abstract

Purpose: Liquid biopsies allow the tracking of clonal dynamics and detection of mutations during treatment.Experimental Design: We evaluated under blinded conditions the ability of cell-free DNA (cfDNA) to detect RAS/BRAF mutations in the plasma of 42 metastatic colorectal cancer patients treated on a phase Ib/II trial of FOLFOX and dasatinib, with or without cetuximab.Results: Prior to treatment, sequencing of archival tissue detected mutations in 25 of 42 patients (60%), while the cfDNA assay detected mutations in 37 of 42 patients (88%). Our cfDNA assay detected mutations with allele frequencies as low as 0.01%. After exposure to treatment, 41 of 42 patients (98%) had a cfDNA-detected RAS/BRAF mutation. Of 21 patients followed with serial measurements who were RAS/BRAF mutant at baseline, 11 (52%) showed additional point mutation following treatment and 3 (14%) no longer had detectable levels of another mutant allele. Of RAS/BRAF wild-type tumors at baseline, 4 of 5 (80%) showed additional point mutations. cfDNA quantitative measurements from this study closely mirrored changes in CEA and CT scan results, highlighting the importance of obtaining quantitative data beyond the mere presence of a mutation.Conclusions: Our findings demonstrate the development of new RAS/BRAF mutations in patients regardless of whether they had preexisting mutations in the pathway, demonstrating a convergent evolutionary pattern. Clin Cancer Res; 23(16); 4578-91. ©2017 AACR.

Conflict of interest statement

Conflict of interest: No conflict of interest.

©2017 American Association for Cancer Research.

Figures

Figure 1. Comparison of mutational status of RAS/BRAF as determined by tumor tissue and plasma analysis at baseline (n=42)

A) Plasma analysis was carried out in cohort 1 and 2 before initiation of treatments (baseline) according with targeted genes. B) Comparison of the type of point mutation as determined by tumor tissue and plasma analysis before initiation of treatments and for cohort 1 and 2. Extended RAS testing was only performed on plasma for patients who were KRAS exon 2 and BRAFV600E wild type on all assays. ND, scored positive but the mutation is non-determined; WT, wild type for mutations tested.

Figure 2. RAS/BRAF mutational status

Comparison of RAS/BRAF mutational status as determined by tumor tissue and plasma analysis. A) Before initiation of treatment of cohorts 1 and 2 (baseline). B) Longitudinal plasma genotyping during treatment (n=26). C) Distribution of mutational status from cfDNA analysis of cohort 2 patients before and after EGFR directed therapy. Percentage of mutant patients as determined by tumor tissue analysis and plasma analysis before initiation and during FOLFOX/dasatinib + cetuximab treatment as determined by serial plasma analysis. Results are shown according the target genes. An, acquired n additional mutation; CO, co-occurring mutations from plasma analysis; WT, wild type for mutations tested; Single, one mutation was found in patient; Multiple, at least two mutations were found in patient.

Figure 3. Impact of treatment on the evolution of cfDNA concentration

A) RefA values during Folfox/dasatinib treatment (Cohort 1, n=11), and B) during Folfox/dasatinib/cetuximab treatment (Cohort 2, n=15). RefA are expressed in fold change of baseline value. C) cfDNA mutant allele frequency of each detected mutant samples. mA% was determined by Intplex as described in materials and methods before (blue, baseline) and after treatment (red, follow-up) in both cohorts. mA% varies from 0.009% to 60.28%. 21% and 64% of all samples showed an mutant allele frequency below 0.1% and 1% (dotted lines), respectively. RefA, concentration of total cfDNA (ng/mL of plasma); BL, baseline; mA%, cfDNA mutation load.

Figure 4. Quantitative cfDNA analysis in the course of treatments in comparison with serum CEA and CT scan measurements

Patients #8, #28, #31 and #36 illustrate impact of treatment on those biomarkers. A) Patients #8, #28 and #36 are patients who responded to treatment with stable disease. B) patient #31 was the single patient in both cohort showing initial response to therapy (cetuximab). CEA, carcinoembryonic antigen (expressed in ng/mL); CT-scan, computed tomography scan (expressed as percentage change of baseline value) ; RefA, concentration of total cfDNA (ng/mL of plasma); mA, concentration of mutant cfDNA (ng/mL of plasma); mA%, mutation load; PR, Partial response; SD, Stable disease (RECIST criteria); PD, progressive disease (RECIST criteria).