Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment

Abstract

Introduction: KRAS and EGFR ectodomain-acquired mutations in patients with metastatic colorectal cancer (mCRC) have been correlated with acquired resistance to anti-EGFR monoclonal antibodies (mAbs). We investigated the frequency, co-occurrence, and distribution of acquired KRAS and EGFR mutations in patients with mCRC refractory to anti-EGFR mAbs using circulating tumor DNA (ctDNA).

Patients and methods: Sixty-two post-treatment plasma and 20 matching pretreatment archival tissue samples from KRAS (wt) mCRC patients refractory to anti-EGFR mAbs were evaluated by high-sensitivity emulsion polymerase chain reaction for KRAS codon 12, 13, 61, and 146 and EGFR 492 mutations.

Results: Plasma analyses showed newly detectable EGFR and KRAS mutations in 5/62 [8%; 95% confidence interval (CI) 0.02-0.18] and 27/62 (44%; 95% CI 0.3-0.56) samples, respectively. KRAS codon 61 and 146 mutations were predominant (33% and 11%, respectively), and multiple EGFR and/or KRAS mutations were detected in 11/27 (41%) cases. The percentage of mutant allele reads was inversely correlated with time since last treatment with EGFR mAbs (P = 0.038). In the matching archival tissue, these mutations were detectable as low-allele-frequency clones in 35% of patients with plasma mutations after treatment with anti-EGFR mAbs and correlated with shorter progression-free survival (PFS) compared with the cases with no new mutations (3.0 versus 8.0 months, P = 0.0004).

Conclusion: Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

Keywords: EGFR acquired mutation; KRAS acquired mutation; anti-EGFR mAbs; ctDNA.

© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Figures

Figure 1.

KRAS codon mutation distribution in treatment-naïve patients versus patients with acquired EGFR-inhibitor resistance (EGFR-I resistant). Atypical KRAS codon 61 and codon 146 mutations were more frequent in metastatic colorectal cancer patients with acquired KRAS mutations after anti-EGFR mAb treatment than in treatment-naïve patients with KRAS mutations (*P < 0.05) .

Figure 2.

Concentration of ctDNA-detectable alleles by timing of mutation and tumor size. (A) Patients with tumors carrying de novo KRAS mutations had a significantly higher percentage of KRAS mutant alleles (defined as the number of mutant KRAS allele reads divided by the total number of wild-type or mutant KRAS allele reads) detected in plasma ctDNA than patients characterized as having wild-type KRAS initially but who had newly detected KRAS mutations after progression on EGFR mAb treatment (P < 0.001, Mann–Whitney U-test). (B) Tumor volumes were calculated by three independent readers by manually tracing the outer edge of the lesions on each image, and the whole-tumor burden was calculated by summing each lesion volumes. No correlation was found between percentage of acquired mutant alleles and tumor burden.

Figure 3.

Time from last treatment with EGFR mAbs correlates with quantification of KRAS mutations detected in ctDNA. Time from last dose of EGFR mAbs correlated with the percentage of KRAS mutation reads detected in ctDNA from the plasma of metastatic colorectal cancer patients who developed resistance to treatment (P = 0.038).

Figure 4.

Low-frequency KRAS mutation expression in patient archival tissue and patient plasma samples correlate with shorter PFS with anti-EGFR treatment. (A) In 7of 20 patients with newly detected KRAS mutations in the plasma and wild-type KRAS by standard-of-care testing in the primary tumor, there were detectable low-frequency mutations present in microdissected tumor analyzed by BEAMing assay. (B) Patients with low-frequency KRAS mutations detected in archival primary tumor tissues had shorter PFS than patients with no detectable mutations by BEAMing (P = 0.0004).