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

Ann Oncol. 2015 Apr;26(4):731-736. doi: 10.1093/annonc/mdv005. Epub 2015 Jan 26.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / therapeutic use*
  • Clone Cells
  • Colorectal Neoplasms / drug therapy
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / mortality
  • Colorectal Neoplasms / pathology*
  • DNA, Neoplasm / blood
  • DNA, Neoplasm / genetics
  • Drug Resistance, Neoplasm / genetics*
  • ErbB Receptors / antagonists & inhibitors*
  • ErbB Receptors / blood
  • ErbB Receptors / genetics
  • Female
  • Follow-Up Studies
  • Humans
  • Male
  • Middle Aged
  • Mutation / genetics*
  • Neoplasm Staging
  • Neoplastic Cells, Circulating / pathology*
  • Prognosis
  • Proto-Oncogene Proteins / blood
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins B-raf / blood
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins p21(ras)
  • Retrospective Studies
  • Survival Rate
  • ras Proteins / blood
  • ras Proteins / genetics


  • Antineoplastic Agents
  • DNA, Neoplasm
  • KRAS protein, human
  • Proto-Oncogene Proteins
  • EGFR protein, human
  • ErbB Receptors
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins