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.

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We are particularly indebted to S. Lamba for generating the KRAS(G12R) knock-in in Lim1215 cells. We thank C. Cancelliere and S. Destefanis for technical assistance. We thank S. Arena, M. Russo and D. Zecchin for critically reading the manuscript. We also thank A. Heguy, A. Viale, N. Socci and M. Pirun for assistance with analysis of next generation sequencing data. This work was supported by European Union Seventh Framework Programme, grant 259015 COLTHERES (A.B. and S.S.); Associazione Italiana per la Ricerca sul Cancro (AIRC) 2010 Special Program Molecular Clinical Oncology 5 × 1000, project 9970 (A.B. and S.S.); Regione Piemonte (A.B. and F.D.N.); Fondazione Piemontese per la Ricerca sul Cancro (FPRC) Intramural Grant, 5xmille 2008, ONLUS (A.B. and F.D.N.); AIRC MFAG 11349 (F.D.N.); Oncologia Ca’ Granda ONLUS (OCGO) (S.S.); Mr William H. Goodwin and Mrs Alice Goodwin and the Commonwealth Foundation for Cancer Research; the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (D.S.); the Society of MSKCC (M.W.); the National Institutes of Health (D.S.); the Beene Foundation (D.S.) and Regione Lombardia and Ministerio Salute grant ‘Gene Mutation Monitoring in mCRC’ (S.S.).

Author information

Author notes

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

    These authors contributed equally to this work.


  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


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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 interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to David Solit or Alberto Bardelli.

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