Acquired RAS or EGFR mutations and duration of response to EGFR blockade in colorectal cancer

Abstract

Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.

Figure 1. Clinical benefit of CRC patients treated with anti-EGFR therapy according to the emergence of EGFR ECD versus RAS mutations.

(a) Response measured by RECIST criteria 1.1 in 27 CRC patients, according to the emergence of EGFR ECD versus RAS mutations detected in tumour tissue and/or plasma. Most patients with emergence of EGFR ECD mutations achieved a partial response, whereas patients with emergence of RAS mutations mostly had stable disease as best response (P=0.128 by Fisher's exact test). Cases with co-existence of EGFR ECD and RAS mutations were excluded from the group of RAS mutant tumours (RAS group) and included in the group of EGFR ECD mutant tumours (EGFR group). (b) Kaplan–Meier estimates of PFS according to the emergence of RAS mutations versus EGFR ECD mutations detected in tumour tissue and/or plasma. The hazard ratio for the RAS group (grey line) as compared with the EGFR group (red line) was 2.56 (95% CI, 1.06–6.21; P=0.013 by a stratified log-rank test). Median PFS time in the RAS group was 25.6 weeks (95% CI, 24–27), as compared with 44.6 weeks (95% CI, 38–49) in the EGFR group.

Figure 2. Longitudinal analysis of plasma ctDNA collected during anti-EGFR therapy.

Shades of black and grey indicate frequency of circulating RAS mutant alleles; and shades of red and orange indicate frequency of circulating EGFR mutant alleles. All patients achieved partial response or disease stabilization under EGFR blockade administered alone or in combination with standard chemotherapy. RAS mutant clones are apparent in the circulation months earlier than EGFR clones in all of the three cases. CT, computed tomography; PD, progressive disease; Pmab, panitumumab; PR, partial response; SD, stable disease.

Figure 3. Selection of RAS and EGFR ECD mutations during development of resistance to EGFR blockade in CRC cells.

Mutational profiles were used to monitor clonal dynamics of LIM1215 CRC cells under cetuximab treatment. Frequency of the NRAS G12C and EGFR S492R mutations at each time point are shown as determined by NGS. The frequencies presented were adjusted for gene copy number as it was determined by FISH analysis that LIM1215 has a duplication of the EGFR locus (7p.11.2) in the p-arm of chromosome 7 (Supplementary Fig. 5).

Figure 4. Fitness of individual and double-mutated cells in the presence of cetuximab.

(a) Sensitivity to cetuximab of LIM1215 parental, resistant pool and single-cell clones containing the indicated mutations. Cell populations and individual clones were treated with increasing doses of cetuximab for 6 days. Cell viability was measured by ATP assay and the mean calculated as % of untreated control. Error bars are s.d. of three independent experiments with three technical replicates. ***P<0.001 by unpaired two-tailed Student's t-test (NRAS+EGFR clone versus NRAS clone). (b) Western blot analysis of the indicated cells treated with 50 μg ml⁻¹ of cetuximab and lysed after 2 h of treatment. Lysates were immunoblotted with the indicated antibodies. Vinculin was included as a loading control. Cetux, cetuximab.

Figure 5. Emergence of RAS and EGFR ECD mutations in CRC cell populations.

RAS and EGFR mutation frequencies were measured by ddPCR and are indicated for LIM1215 cells cultured in low-serum, chemotherapy, chemotherapy plus cetuximab and cetuximab-only conditions. R1 and R5 cetux indicate two resistant populations of LIM1215 generated independently. Cetux, cetuximab.