RAS and BRAF mutations in cell-free DNA are predictive for outcome of cetuximab monotherapy in patients with tissue-tested RAS wild-type advanced colorectal cancer

Abstract

In metastatic colorectal cancer, RAS and BRAF mutations cause resistance to anti-EGFR therapies, such as cetuximab. Heterogeneity in RAS and BRAF mutations might explain nonresponse in a subset of patients receiving cetuximab. Analyzing mutations in plasma-derived circulating tumor DNA (ctDNA) could provide a more comprehensive overview of the mutational landscape as compared to analyses of primary and/or metastatic tumor tissue. Therefore, this prospective multicenter study followed 34 patients with metastatic colorectal cancer who were tissue-tested as RAS wild-type (exons 2-4) during routine work-up and received third-line cetuximab monotherapy. BRAF mutation status was also tested but did not exclude patients from therapy. At baseline and upon disease progression, cell-free DNA (cfDNA) was isolated for targeted next-generation sequencing (NGS). At 8 weeks, we determined that patients had benefited from treatment. NGS of cfDNA identified three patients with RAS mutations not detected in tumor tissue during routine work-up. Another six patients had a BRAF or rare RAS mutation in ctDNA and/or tumor tissue. Relative to patients without mutations in RAS/BRAF, patients with mutations at baseline had shorter progression-free survival [1.8 versus 4.9 months (P < 0.001)] and overall survival [3.1 versus 9.4 months (P = 0.001)]. In patients with clinical benefit (progressive disease after 8 weeks), ctDNA testing revealed previously undetected mutations in RAS/BRAF (71%) and EGFR (47%), which often emerged polyclonally. Our results indicate that baseline NGS of ctDNA can identify additional RAS mutation carriers, which could improve patient selection for anti-EGFR therapies. Acquired resistance, in patients with initial treatment benefit, is mainly explained by polyclonal emergence of RAS, BRAF, and EGFR mutations in ctDNA.

Keywords: BRAF; RAS mutations; biomarkers; cell-free DNA; cetuximab; colorectal cancer.

Figure 1

Comparison of mutational status as determined by ctDNA analyses at baseline and PD in patients with and without clinical benefit. The number behind the ‘X’ indicates the number of hotspot mutations within a gene.

Figure 2

Progression‐free survival (A) and OS (B) for patients with RAS and/or BRAF mutations (mutant) versus patients without RAS/BRAF mutations (wild‐type) in tissue and ctDNA.

Figure 3

Paired baseline, 2‐week, and PD ctDNA‐sequencing results of patients with clinical benefit. Mutations were grouped per gene, and if patients harbored polyclonal mutations, the clones were numbered. For example, in patient 18 two TP53 mutations were detected at baseline, clones 1 and 2, which both decreased in MAF at 2 weeks.

Figure 4

Paired baseline and PD ctDNA mutational analyses in patients with initial clinical benefit (A) and patients without clinical benefit (B). Mutations are depicted per gene, each gene having a separate color. Higher bars indicate polyclonal mutations. For example, patient 2 gained seven different KRAS hotspot mutations at disease progression. Patient 20 with clinical benefit and patient 30 without clinical benefit were not included in the graph because of the absence of mutations at baseline as well as PD.