Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

Journal name:
Nature
Volume:
486,
Pages:
532–536
Date published:
DOI:
doi:10.1038/nature11156
Received
Accepted
Published online

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers1. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug2. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood3, 4, 5, 6, 7, 8. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

At a glance

Figures

  1. KRAS amplification mediates acquired resistance to cetuximab in DiFi cells.
    Figure 1: KRAS amplification mediates acquired resistance to cetuximab in DiFi cells.

    a, Parental (wild-type, WT) and cetuximab-resistant (-R1, -R2) DiFi cells were treated for one week with increasing concentrations of cetuximab. Cell viability was assayed by the ATP assay. Data points represent the mean±s.d. of three independent experiments. b, Whole exome gene copy number analysis of parental and cetuximab-resistant DiFi cells. Individual chromosomes are indicated on the x-axis. The lines indicate the sequencing depth (y-axis) over exome windows of 100,000base pairs. c, Fluorescent in situ hybridization (FISH) analysis confirming KRAS amplification in resistant (DiFi-R) but not parental DiFi cells. KRAS locus bacterial artificial chromosome (BAC) DNA (probe RP11-707G18; green) and chromosome 12 paint (red) were hybridized to the metaphase spreads of DiFi cells. Original magnification, ×100. d, DiFi cells were treated with 35nM cetuximab for 24h, and whole-cell extracts were then subjected to western blot analysis and compared with untreated cells. DiFi-R1 and -R2 cells were plated in the absence of cetuximab for 7days or maintained in their normal growth medium (with 35nM cetuximab) before protein analysis. Active KRAS (GTP-KRAS) was assessed by glutathione S-transferase (GST)–RAF1 pull-down. Whole-cell extracts were blotted with phosphorylated EGFR (pEGFR; Tyr1068), total EGFR, total KRAS, phosphorylated AKT (Thr308), phosphorylated AKT (Ser473), total AKT, total MEK1/2 and phosphorylated MEK1/2, total ERK1/2 and phosphorylated ERK1/2 antibodies. Vinculin was included as a loading control. e, Western blot analysis of KRAS protein in DiFi cells infected with a KRAS lentivirus. Actin is shown as a loading control. f, Ectopic expression of wild-type KRAS in parental DiFi cells confers resistance to cetuximab. Data points represent the mean±s.d. of three independent experiments.

  2. KRAS mutations mediate acquired resistance to cetuximab in Lim1215 cells.
    Figure 2: KRAS mutations mediate acquired resistance to cetuximab in Lim1215 cells.

    a, Parental (Lim) and cetuximab-resistant (Lim-R1 and Lim-R2) Lim1215 cells were treated for one week with increasing concentrations of cetuximab. Cell viability was assayed by the ATP assay. Data points represent the mean±s.d. of three independent experiments. b, Sanger sequencing of KRAS exon 2 in parental and two representative cetuximab-resistant Lim1215 cells obtained in independent selection procedures. c, Western blot analysis of the EGFR signalling pathway in parental and cetuximab-resistant Lim1215 cells. d, Schematic representation of the vectors used to knock-in the G12R and G13D mutations into the genome of Lim1215 parental cell lines by AAV-mediated homologous recombination. Targeting was assessed by Sanger sequencing. ITR, inverted terminal repeat; P Neo, neomycin-resistance plasmid. e, Parental and isogenic Lim1215 cells carrying the indicated mutations were treated for one week with increasing doses of cetuximab. KI, knock-in. Data points represent the mean±s.d. of three independent experiments.

  3. Mutational analysis of the KRAS gene in patients.
    Figure 3: Mutational analysis of the KRAS gene in patients.

    a, Mutational analysis of KRAS in patients with chemotherapy-refractory CRC. b, Mutational analysis of the KRAS gene in patients who progressed on anti-EGFR antibodies. The results in a and b are based on assays performed by deep sequencing technologies, either 454 pyrosequencing (*) or BEAMing (). c, Dot plot of the percentage of mutated KRAS alleles in patients with chemotherapy-refractory and anti-EGFR-resistant CRC. P value was calculated using a two-tailed unpaired Mann–Whitney test.

  4. Detection of circulating KRAS mutant DNA in a patient with acquired resistance to cetuximab therapy.
    Figure 4: Detection of circulating KRAS mutant DNA in a patient with acquired resistance to cetuximab therapy.

    a, Size of liver metastasis (blue bars) and carcinoembryonic antigen (CEA) levels in blood (blue line) at the indicated time points, showing an initial response to cetuximab followed by progression (patient 8). PR, partial response; PD, progressive disease. b, Quantitative analysis of KRAS(Q61H) mutant DNA in plasma, as assessed by BEAMing. c, Two-dimensional dot plot showing quantitative analysis of the KRAS(Q61H) mutation in plasma using BEAMing at individual time points. d, Mutational analysis of KRAS on tumour samples collected before cetuximab treatment and at the time of disease progression.

References

  1. Ciardiello, F. & Tortora, G. EGFR antagonists in cancer treatment. N. Engl. J. Med. 358, 11601174 (2008)
  2. Karapetis, C. S. et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N. Engl. J. Med. 359, 17571765 (2008)
  3. Wheeler, D. L. et al. Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members. Oncogene 27, 39443956 (2008)
  4. Benavente, S. et al. Establishment and characterization of a model of acquired resistance to epidermal growth factor receptor targeting agents in human cancer cells. Clin. Cancer Res. 15, 15851592 (2009)
  5. Li, C., Iida, M., Dunn, E. F., Ghia, A. J. & Wheeler, D. L. Nuclear EGFR contributes to acquired resistance to cetuximab. Oncogene 28, 38013813 (2009)
  6. Hatakeyama, H. et al. Regulation of heparin-binding EGF-like growth factor by miR-212 and acquired cetuximab-resistance in head and neck squamous cell carcinoma. PLoS ONE 5, e12702 (2010)
  7. Yonesaka, K. et al. Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab. Sci. Transl. Med. 3, 99ra86 (2011)
  8. 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, 221223 (2012)
  9. 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, 279286 (2005)
  10. 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, 753762 (2010)
  11. Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nature Med. 14, 985990 (2008)
  12. Di Nicolantonio, F. et al. Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses. Proc. Natl Acad. Sci. USA 105, 2086420869 (2008)
  13. Bardelli, A. & Siena, S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. J. Clin. Oncol. 28, 12541261 (2010)
  14. Van Cutsem, E. et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N. Engl. J. Med. 360, 14081417 (2009)
  15. Amado, R. G. et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J. Clin. Oncol. 26, 16261634 (2008)
  16. Janne, P. A. Challenges of detecting EGFR T790M in gefitinib/erlotinib-resistant tumours. Lung Cancer 60, (suppl. 2)S3S9 (2008)
  17. Engelman, J. A. et al. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J. Clin. Invest. 116, 26952706 (2006)
  18. Arcila, M. E. et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin. Cancer Res. 17, 11691180 (2011)
  19. Molinari, F. et al. Increased detection sensitivity for KRAS mutations enhances the prediction of anti-EGFR monoclonal antibody resistance in metastatic colorectal cancer. Clin. Cancer Res. 17, 49014914 (2011)
  20. Whitehead, R. H., Macrae, F. A., St John, D. J. & Ma, J. A colon cancer cell line (LIM1215) derived from a patient with inherited nonpolyposis colorectal cancer. J. Natl. Cancer Inst. 74, 759765 (1985)
  21. Smith, G. et al. Activating K-Ras mutations outwith ‘hotspot’ codons in sporadic colorectal tumours - implications for personalised cancer medicine. Br. J. Cancer 102, 693703 (2010)

Download references

Author information

  1. These authors contributed equally to this work.

    • Sandra Misale,
    • Rona Yaeger,
    • Sebastijan Hobor,
    • Elisa Scala &
    • Manickam Janakiraman

Affiliations

  1. Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment (IRCC), 10060 Candiolo (Torino), Italy

    • Sandra Misale,
    • Sebastijan Hobor,
    • Elisa Scala,
    • Michela Buscarino,
    • Giulia Siravegna,
    • Carlo Zanon,
    • Federica Di Nicolantonio &
    • Alberto Bardelli
  2. Department of Oncological Sciences, University of Torino Medical School, 10060 Candiolo (Torino), Italy

    • Sandra Misale,
    • Elisa Scala,
    • Michela Buscarino,
    • Enzo Medico &
    • Alberto Bardelli
  3. Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Rona Yaeger,
    • Andrea Cercek &
    • David Solit
  4. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Manickam Janakiraman &
    • David Solit
  5. Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • David Liska,
    • Chin-Tung Chen &
    • Martin Weiser
  6. Division of Pathology, Ospedale Niguarda Ca’ Granda, 20162 Milano, Italy

    • Emanuele Valtorta,
    • Silvio Veronese &
    • Marcello Gambacorta
  7. Falck Division of Medical Oncology, Ospedale Niguarda Ca’ Granda, 20162 Milano, Italy

    • Roberta Schiavo,
    • Katia Bencardino,
    • Andrea Sartore-Bianchi &
    • Salvatore Siena
  8. Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, 10125 Torino, Italy

    • Margherita Gallicchio &
    • Valentina Boscaro
  9. Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Efsevia Vakiani
  10. Laboratory of Functional Genomics, Institute for Cancer Research and Treatment (IRCC), 10060 Candiolo (Torino), Italy

    • Enzo Medico
  11. FIRC Institute of Molecular Oncology (IFOM), 20139 Milano, Italy

    • Federica Di Nicolantonio &
    • Alberto Bardelli

Contributions

A.B., D.S., S.S. and F.D.N. planned the project and supervised all research. A.B., D.S. and F.D.N. wrote the manuscript. S.M., R.Y., S.H., E.S., M.W. and F.D.N. designed the experiments. A.B. conceived the molecular analysis of plasma samples. S.M., R.Y., S.H., E.S., M.J., D.L., E.V., R.S., M.B., G.S., C.-T.C., S.V., M.G. and V.B. performed the experiments. C.Z., A.S.-B., M.G. and E.M. analysed data. K.B., A.C. and E.V. provided samples for analysis. S.S., D.S. and A.B. devised dual biopsy clinical protocols for EGFR mAb resistant mCRC.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (3.5M)

    This file contains Supplementary Figures 1-7 and Supplementary Tables 1-4.

Additional data