Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer

Abstract

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models.Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance.Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab.Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.

Broad Detection of Alterations Predicted to Confer Lack of Benefit From EGFR Antibodies or Sensitivity to Targeted Therapy in Advanced Colorectal Cancer

“…Potential cetuximab/panitumumab resistance alterations in RAS/RAF wild-type tumors included alterations that impact antibody binding, bypass track activation, and alteration of downstream signaling cascade nodes-all well-established resistance mechanisms in cancer. Specifically, we observed EGFR EC domain mutations, which have been shown to disrupt antibody binding, and amplification of MET and FLT3, all of which have been associated with resistance to anti-EGFR therapy in CRC [19,[29][30][31][32]. Concurrent or compensatory ERBB2 amplification is a poor prognostic indicator independent of KRAS and ERBB2 mutations, and mediates resistance in a portion of anti-EGFRtreated patients [33,34].…”

“…PI3K pathway alterations, including PIK3CA mutation and PTEN mutation and loss of expression, have been associated with poor overall response rate and shorter overall survival in patients with KRAS wild-type CRC treated with cetuximab and panitumumab [36][37][38]. However, PIK3CA mutation has been reported in both pre-and post-treatment biopsy specimens from CRC patients who have developed resistance to anti-EGFR therapies, and we and others observed high co-occurrence with other alterations, including RAS mutations, suggesting that the functional significance of PIK3CA mutation in CRC is less clear [21,29]. The frequencies of RTK and MEK1 alterations reported here were similar to those observed in a recent smaller study, which suggested a poor response rate to anti-EGFR antibodies in patients with RAS wild-type CRC with these alterations [39]; however, we did identify some co-occurrence of MEK1 and RAS/RAF mutations.…”

Broad detection of alterations predicted to confer lack of benefit from EGFR antibodies or sensitivity to targeted therapy in advanced colorectal cancer

“…Potential cetuximab/panitumumab resistance alterations in RAS/RAF wild-type tumors included alterations that impact antibody binding, bypass track activation, and alteration of downstream signaling cascade nodes-all well-established resistance mechanisms in cancer. Specifically, we observed EGFR EC domain mutations, which have been shown to disrupt antibody binding, and amplification of MET and FLT3, all of which have been associated with resistance to anti-EGFR therapy in CRC [19,[29][30][31][32]. Concurrent or compensatory ERBB2 amplification is a poor prognostic indicator independent of KRAS and ERBB2 mutations, and mediates resistance in a portion of anti-EGFRtreated patients [33,34].…”

“…PI3K pathway alterations, including PIK3CA mutation and PTEN mutation and loss of expression, have been associated with poor overall response rate and shorter overall survival in patients with KRAS wild-type CRC treated with cetuximab and panitumumab [36][37][38]. However, PIK3CA mutation has been reported in both pre-and post-treatment biopsy specimens from CRC patients who have developed resistance to anti-EGFR therapies, and we and others observed high co-occurrence with other alterations, including RAS mutations, suggesting that the functional significance of PIK3CA mutation in CRC is less clear [21,29]. The frequencies of RTK and MEK1 alterations reported here were similar to those observed in a recent smaller study, which suggested a poor response rate to anti-EGFR antibodies in patients with RAS wild-type CRC with these alterations [39]; however, we did identify some co-occurrence of MEK1 and RAS/RAF mutations.…”

Detection of KRAS Mutations in Plasma DNA Using a fully Automated Rapid Detection System in Colorectal Cancer Patients

“…Mechanisms of acquired resistance to anti-EGFR antibodies include activation of an alternative pathway, such as MET/HGF or EGFR, or activation of RAS/RAF/MAPK mediated through the appearance of KRAS mutations [12][13][14]. Because the emergence of KRAS mutations causes resistance to anti-EGFR antibodies, detection of these mutations is indispensable for deciding subsequent treatment.…”