Abstract
Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation1. Recent studies have identified alterations in KRAS2,3,4 and other genes5,6,7,8,9,10,11,12,13 as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
Primary accessions
EMBL/GenBank/DDBJ
Data deposits
Sequence data have been deposited at the European Genome-phenome Archive, which is hosted at the European Bioinformatics Institute, under study accession EGAS00001001305.
References
Van Cutsem, E., Cervantes, A., Nordlinger, B. & Arnold, D. on behalf of the ESMO Guidelines Working Group. Metastatic colorectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 25 (Suppl. 3), iii1–iii9 (2014)
Diaz, L. A., Jr et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486, 537–540 (2012)
Misale, S. et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 486, 532–536 (2012)
Amado, R. G. et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J. Clin. Oncol. 26, 1626–1634 (2008)
De Roock, W. et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 11, 753–762 (2010)
Tol, J. et al. Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab. Eur. J. Cancer 46, 1997–2009 (2010)
Sartore-Bianchi, A. et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res. 69, 1851–1857 (2009)
Bardelli, A. et al. Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer. Cancer Discov. 3, 658–673 (2013)
Bertotti, A. et al. A molecularly annotated platform of patient-derived xenografts (“xenopatients”) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer. Cancer Discov. 1, 508–523 (2011)
Yonesaka, K. et al. Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab. Sci. Transl. Med. 3, 99ra86 (2011)
Montagut, C. et al. Identification of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer. Nature Med. 18, 221–223 (2012)
Bettegowda, C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci. Transl. Med. 6, 224ra224 (2014)
Diaz, L. A., Jr, Sausen, M., Fisher, G. A. & Velculescu, V. E. Insights into therapeutic resistance from whole-genome analyses of circulating tumor DNA. Oncotarget 4, 1856–1857 (2013)
Leary, R. J. et al. Integrated analysis of homozygous deletions, focal amplifications, and sequence alterations in breast and colorectal cancers. Proc. Natl Acad. Sci. USA 105, 16224–16229 (2008)
Barber, T. D., Vogelstein, B., Kinzler, K. W. & Velculescu, V. E. Somatic mutations of EGFR in colorectal cancers and glioblastomas. N. Engl. J. Med. 351, 2883 (2004)
Moroni, M. et al. Somatic mutation of EGFR catalytic domain and treatment with gefitinib in colorectal cancer. Ann. Oncol. 16, 1848–1849 (2005)
Wesche, J., Haglund, K. & Haugsten, E. M. Fibroblast growth factors and their receptors in cancer. Biochem. J. 437, 199–213 (2011)
Heinrich, M. C. et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299, 708–710 (2003)
Dibb, N. J., Dilworth, S. M. & Mol, C. D. Switching on kinases: oncogenic activation of BRAF and the PDGFR family. Nature Rev. Cancer 4, 718–727 (2004)
Marks, J. L. et al. Novel MEK1 mutation identified by mutational analysis of epidermal growth factor receptor signaling pathway genes in lung adenocarcinoma. Cancer Res. 68, 5524–5528 (2008)
Algars, A., Lintunen, M., Carpen, O., Ristamaki, R. & Sundstrom, J. EGFR gene copy number assessment from areas with highest EGFR expression predicts response to anti-EGFR therapy in colorectal cancer. Br. J. Cancer 105, 255–262 (2011)
Moroni, M. et al. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study. Lancet Oncol. 6, 279–286 (2005)
Parsons, D. W. et al. Colorectal cancer: mutations in a signalling pathway. Nature 436, 792 (2005)
Misale, S. et al. Blockade of EGFR and MEK intercepts heterogeneous mechanisms of acquired resistance to anti-EGFR therapies in colorectal cancer. Sci. Transl. Med. 6, 224ra226 (2014)
Zanella, E. R. et al. IGF2 is an actionable target that identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-EGFR therapies. Sci. Transl. Med. 7, 272ra212 (2015)
Kavuri, S. M. et al. HER2 activating mutations are targets for colorectal cancer treatment. Cancer Discov. 5, 832–841 (2015)
Voigt, M. et al. Functional dissection of the epidermal growth factor receptor epitopes targeted by panitumumab and cetuximab. Neoplasia 14, 1023–1031 (2012)
Koefoed, K. et al. Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor. MAbs 3, 584–595 (2011)
Pavlicek, A. et al. Molecular predictors of sensitivity to the insulin-like growth factor 1 receptor inhibitor Figitumumab (CP-751,871). Mol. Cancer Ther. 12, 2929–2939 (2013)
Jones, S. et al. Comparative lesion sequencing provides insights into tumor evolution. Proc. Natl Acad. Sci. USA 105, 4283–4288 (2008)
Siena, S. et al. Phase II open-label study to assess efficacy and safety of lenalidomide in combination with cetuximab in KRAS-mutant metastatic colorectal cancer. PLoS One 8, e62264 (2013)
Galimi, F. et al. Genetic and expression analysis of MET, MACC1, and HGF in metastatic colorectal cancer: response to met inhibition in patient xenografts and pathologic correlations. Clin. Cancer Res. 17, 3146–3156 (2011)
Baralis, E., Bertotti, A., Fiori, A. & Grand, A. LAS: a software platform to support oncological data management. J. Med. Syst. 36 (Suppl. 1), S81–S90 (2012)
Jones, S. et al. Personalized genomic analyses for cancer mutation discovery and interpretation. Sci. Transl. Med. 7, 283ra253 (2015)
Needleman, S. B. & Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443–453 (1970)
Leary, R. J., Cummins, J., Wang, T. L. & Velculescu, V. E. Digital karyotyping. Nature Protocols 2, 1973–1986 (2007)
Jiao, Y. et al. Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nature Genet. 45, 1470–1473 (2013)
Sjoblom, T. et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006)
Kan, Z. et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature 466, 869–873 (2010)
Acknowledgements
We thank S. Angiuoli, D. Riley, L. Kann, M. Shukla, and C. L. McCord for their assistance with next-generation sequencing analyses, and F. Galimi and S. M. Leto for their help with Sanger sequencing analyses and functional studies. This work was supported by the John G. Ballenger Trust, FasterCures Research Acceleration Award, the European Community’s Seventh Framework Programme, the AIRC Italian Association for Cancer Research (Special Program Molecular Clinical Oncology 5×1000, project 9970, and Investigator Grants projects 14205 and 15571), American Association for Cancer Research (AACR) – Fight Colorectal Cancer Career Development Award in memory of Lisa Dubow (project 12-20-16-BERT), the Commonwealth Foundation, Swim Across America, US National Institutes of Health grant CA121113, Fondazione Piemontese per la Ricerca sul Cancro-ONLUS (5×1000 Italian Ministry of Health 2011), Oncologia Ca’ Granda ONLUS, and the SU2C-DCS International Translational Cancer Research Dream Team Grant (SU2C-AACR-DT1415). We acknowledge Merck for a gift of cetuximab. Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. A.B. and L.T. are members of the EurOPDX Consortium.
Author information
Authors and Affiliations
Contributions
A.B. and E.P. conceived the project, designed and performed experiments, interpreted results and co-wrote the manuscript. S.J., V.A., V.A., B.L., M.S., J.P., C.A.H., M.N., K.L., F.S., F.C., G.M., E.R.Z., D.R., N.R., A.M., A.M., G.P., M.S., S.M., and A.C. performed experiments, analysed data, prepared tables, or participated in discussion of the results. M.K. and J.L. contributed reagents. Q.K.L. undertook all pathological evaluations. C.T., N.N., R.K., and R.S. performed statistical analyses. A.S.-B., S.S., and L.A.D. provided clinically annotated samples and supervised experimental designs. L.T. and V.E.V. conceived the project, supervised experimental designs, interpreted results, and co-wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
L.A.D. and V.E.V. are co-founders of Personal Genome Diagnostics and are members of its Board of Directors. V.E.V. and L.A.D. own Personal Genome Diagnostics stock, which is subject to certain restrictions under Johns Hopkins University policy. The authors are entitled to a share of the royalties received by the University on sales of products related to genes described in this manuscript. The terms of these arrangements are managed by the Johns Hopkins University in accordance with its conflict-of-interest policies.
Extended data figures and tables
Extended Data Figure 1 EGFR signalling pathway genes involved in cetuximab resistance or sensitivity.
Altered cell-surface receptors or members of RAS or PI3K pathways identified in this study are indicated. Somatic alterations related to resistance or sensitivity are highlighted in red or green boxes, respectively. The percentages indicate the fraction of KRAS wild-type tumours containing the somatic alterations in the specified genes. For the following genes a subset of alterations are indicated: PDGFRA kinase domain mutations; EGFR ecto- and kinase domain mutations and amplifications.
Extended Data Figure 2 Pan-HER monoclonal antibody mixture binds epitopes different from those recognized by cetuximab.
a, The H383 (green) and the S484/G485 (light blue) residues in EGFR domain III are critical for the binding of Pan-HER anti-EGFR antibodies 1277 and 1565, respectively28. Antibodies 1277 and 1565 (ref. 28) bind to an epitope distinct from that of cetuximab, which may contribute to the superior tumour growth inhibition in the presence of mutations at residue 465. Mutations identified in this study affecting G465 (red) and the S492 amino acid (yellow) previously reported to confer cetuximab resistance11 are shown for reference. Similarly to mutations affecting S492, the alterations at 465 that we identified in this study (G465R and G465E) involve changes from a non-polar uncharged side chain to large electrically charged arginine or glutamic acid residues, respectively, and predict resistance to cetuximab. b, Critical EGFR amino acids selectively recognized by both cetuximab and panitumumab as determined by phage screening are shown in blue and include P373, K467, P411, K489, D379, F376 (ref. 27). Residue G465 is in close proximity to K467 and other residues that have been shown to influence the binding of both cetuximab and panitumumab27.
Extended Data Figure 3 Expression of IRS2 according to response categories in tumour graft models.
Results were obtained using Illumina-based oligonucleotide microarrays in 100 tumour grafts that had no mutations in the KRAS, NRAS, BRAF, or PIK3CA genes. Response categories are defined in the main text. OR, objective response; SD, stable disease; PD, progressive disease. P < 0.001 for OR compared with PD and SD compared with PD by one-way ANOVA and Bonferroni’s multiple comparison test. IRS2 expression values are shown in Supplementary Table 10.
Extended Data Figure 4 Functional studies of genetic alterations associated with cetuximab response.
a, b, Ectopic expression of mutations that correlated with resistance to EGFR blockade prevented responsiveness to cetuximab. NCI-H508 cells expressing EGFR G465E (a, left) or DDK-tagged MAP2K1 K57N (b, left) were refractory to cetuximab in dose-dependent viability assays after 6 days of treatment. Results are the means ± s.d. of two independent experiments performed in biological triplicates (n = 6) for EGFR G465E and three independent experiments performed in biological triplicates (n = 9) for MAP2K1 K57N compared with mock vector controls. Biochemical responses of NCI-H508 EGFR G465E (a, right) and NCI-H508 MAP2K1 K57N (b, right) treated with cetuximab for 24 h were documented by western blot analyses. c, Genetic silencing of IRS2 (IRS2 shRNA) in NCI-H508 cells reduced sensitivity to cetuximab in dose-dependent viability assays (left). Results are the means ± s.d. of two independent experiments performed in biological triplicates (n = 6). In biochemical studies using western blot analyses (right), IRS2 knockdown attenuated EGF-dependent activation of AKT (P-AKT) and ERK (P-ERK). Cells were treated for 10 min with the indicated concentrations of EGF. Tubulin was used as a loading control. Western blots for total EGFR, ERK, and AKT proteins were run with the same lysates as those used for anti-phosphoprotein detection but on different gels. All western blots are representative of two independent experiments.
Extended Data Figure 5 Signalling consequences of FGFR inhibition in FGFR1-amplified CRC477.
Immunohistochemistry with the indicated antibodies and morphometric quantitations of representative tumours at the end of treatment. Results are the means ± s.d. of five fields (×40) from two tumours for each experimental point (n = 10). Scale bar, 300 μm. P-ERK, phospho-ERK; P-S6, phospho-S6. NT, not treated (vehicle); CET, cetuximab; BGJ, BGJ398. *P < 0.05; **P < 0.01 by two-tailed Student’s t-test.
Extended Data Figure 6 Signalling consequences of EGFR inhibition in EGFR mutant (V843I) CRC334.
Immunohistochemistry with the indicated antibodies and morphometric quantitations of representative tumours at the end of treatment. Results are the means ± s.d. of five fields (×40) from two tumours for each experimental point (n = 10). Scale bar, 300 μm. AFA, afatinib. **P < 0.01; ***P < 0.001 by two-tailed Student’s t-test.
Extended Data Figure 7 Signalling consequences of PDGFR inhibition in PDGFRA mutant (R981H) CRC525.
Immunohistochemistry with the indicated antibodies and morphometric quantitations of representative tumours after acute treatment (4 h after imatinib and 24 h after cetuximab administration). Results are the means ± s.d. of five fields (×40) from two tumours for each experimental point (n = 10). Scale bar, 300 μm. **P < 0.01 by two-tailed Student’s t-test.
Extended Data Figure 8 Signalling consequences of MEK1 inhibition in MAP2K1 mutant (K57KN) CRC343.
Immunohistochemistry with the indicated antibodies and morphometric quantitations of representative tumours at the end of treatment. Results are the means ± s.d. of five fields (×40) from two tumours for each experimental point (n = 10). Scale bar, 300 μm. AZD, AZD6244; SCH, SCH772984. ***P < 0.001 by two-tailed Student’s t-test.
Extended Data Figure 9 Signalling consequences of EGFR inhibition in EGFR mutant (G465E) CRC104.
Immunohistochemistry with the indicated antibodies and morphometric quantitations of representative tumours at the end of treatment. Results are the means ± s.d. of five fields (×40) from two tumours for each experimental point (n = 10). Scale bar, 300 μm. PAN, panitumumab. NS, not significant; **P <0.01 by two-tailed Student’s t-test.
Extended Data Figure 10 Signalling consequences of EGFR inhibition in EGFR mutant (G465R) CRC177.
Immunohistochemistry with the indicated antibodies and morphometric quantitations of representative tumours at the end of treatment. Results are the means ± s.d. of five fields (×40) from two tumours for each experimental point (n = 10). Scale bar, 300 μm. *P < 0.05; ***P < 0.001 by two-tailed Student’s t-test.
Supplementary information
Supplementary Information
This file contains Supplementary Tables 1-10. (XLSX 6636 kb)
Rights and permissions
About this article
Cite this article
Bertotti, A., Papp, E., Jones, S. et al. The genomic landscape of response to EGFR blockade in colorectal cancer. Nature 526, 263–267 (2015). https://doi.org/10.1038/nature14969
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature14969
This article is cited by
-
The emergence of RAS mutations in patients with RAS wild-type mCRC receiving cetuximab as first-line treatment: a noninterventional, uncontrolled multicenter study
British Journal of Cancer (2023)
-
Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT)
Cell Death & Disease (2023)
-
Final results of DESTINY-CRC01 investigating trastuzumab deruxtecan in patients with HER2-expressing metastatic colorectal cancer
Nature Communications (2023)
-
Organoids and organs-on-chips: insights into predicting the efficacy of systemic treatment in colorectal cancer
Cell Death Discovery (2023)
-
Rational combinations of targeted cancer therapies: background, advances and challenges
Nature Reviews Drug Discovery (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.