A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/β-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.

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  1. 1.

    et al. Spatial and temporal diversity in genomic instability processes defines lung cancer evolution. Science 346, 251–256 (2014).

  2. 2.

    et al. Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing. Science 346, 256–259 (2014).

  3. 3.

    et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell 17, 77–88 (2010).

  4. 4.

    et al. Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat. Genet. 44, 852–860 (2012).

  5. 5.

    et al. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients. Nat. Commun. 7, 11815 (2016).

  6. 6.

    et al. Mutations in TP53, PIK3CA, PTEN and other genes in EGFR mutated lung cancers: correlation with clinical outcomes. Lung Cancer 106, 17–21 (2017).

  7. 7.

    et al. Impact of TP53 mutations on outcome in EGFR-mutated patients treated with first-line tyrosine kinase inhibitors. Clin. Cancer Res. 23, 2195–2202 (2017).

  8. 8.

    Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550 (2014).

  9. 9.

    et al. Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumor DNA. PLoS One 10, e0140712 (2015).

  10. 10.

    et al. Detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next-generation sequencing of cell-free circulating tumor DNA. Clin. Cancer Res. 22, 5772–5782 (2016).

  11. 11.

    et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin. Cancer Res. 19, 2240–2247 (2013).

  12. 12.

    et al. Poor response to erlotinib in patients with tumors containing baseline EGFR T790M mutations found by routine clinical molecular testing. Ann. Oncol. 25, 423–428 (2014).

  13. 13.

    , , , & Erlotinib resistance in mouse models of epidermal growth factor receptor-induced lung adenocarcinoma. Dis. Model. Mech. 3, 111–119 (2010).

  14. 14.

    et al. Acquired resistance to the mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models. Cancer Res. 75, 2489–2500 (2015).

  15. 15.

    et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat. Med. 21, 560–562 (2015).

  16. 16.

    , & High MET amplification level as a resistance mechanism to osimertinib (AZD9291) in a patient that symptomatically responded to crizotinib treatment post-osimertinib progression. Lung Cancer 98, 59–61 (2016).

  17. 17.

    et al. Acquired BRAF V600E mutation as resistant mechanism after treatment with osimertinib. J. Thorac. Oncol. 12, 567–572 (2017).

  18. 18.

    et al. Rociletinib in EGFR-mutated non-small-cell lung cancer. N. Engl. J. Med. 372, 1700–1709 (2015).

  19. 19.

    et al. Beta-catenin expression pattern in stage I and II ovarian carcinomas : relationship with beta-catenin gene mutations, clinicopathological features, and clinical outcome. Am. J. Pathol. 155, 527–536 (1999).

  20. 20.

    , & Antisense inhibition of protein kinase Cα reverses the transformed phenotype in human lung carcinoma cells. Exp. Cell Res. 250, 253–263 (1999).

  21. 21.

    , , , & Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α (PIK3CA). Proc. Natl. Acad. Sci. USA 109, 15259–15264 (2012).

  22. 22.

    et al. Mutationally activated PIK3CA(H1047R) cooperates with BRAF(V600E) to promote lung cancer progression. Cancer Res. 73, 6448–6461 (2013).

  23. 23.

    , & Histopathological transformation to small-cell lung carcinoma in non-small cell lung carcinoma tumors. Transl. Lung Cancer Res. 5, 401–412 (2016).

  24. 24.

    et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci. Transl. Med. 3, 75ra26 (2011).

  25. 25.

    et al. Heterogeneity underlies the emergence of EGFRT790 wild-type clones following treatment of T790M-positive cancers with a third-generation EGFR inhibitor. Cancer Discov. 5, 713–722 (2015).

  26. 26.

    et al. Effect of mutation order on myeloproliferative neoplasms. N. Engl. J. Med. 372, 601–612 (2015).

  27. 27.

    et al. Biopsy-free circulating tumor DNA assay identifies actionable mutations in lung cancer. Oncotarget 7, 66880–66891 (2016).

  28. 28.

    et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat. Med. 20, 548–554 (2014).

  29. 29.

    et al. Tracking the evolution of non-small-cell lung cancer. N. Engl. J. Med. 376, 2109–2121 (2017).

  30. 30.

    et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS One 4, e6529 (2009).

  31. 31.

    & Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).

  32. 32.

    et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9, 493–501 (2003).

  33. 33.

    et al. The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells. Proc. Natl. Acad. Sci. USA 102, 18443–18448 (2005).

  34. 34.

    , , & Neoplastic transformation of RK3E by mutant beta-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression. Mol. Cell. Biol. 19, 5696–5706 (1999).

  35. 35.

    et al. Inactivation of Capicua drives cancer metastasis. Nat. Genet. 49, 87–96 (2017).

  36. 36.

    et al. NF-κB-activating complex engaged in response to EGFR oncogene inhibition drives tumor cell survival and residual disease in lung cancer. Cell Rep. 11, 98–110 (2015).

  37. 37.

    et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

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The authors acknowledge funding support from the NIH (NCI-R01CA169338, NIH Director's New Innovator Award NCI-DP2CA174497), the Pew Charitable Trust, Stewart Foundation, and Searle Foundation (to T.G.B.), and the AACR and Lung Cancer Research Foundation (C.M.B.). The authors thank J. Blakely for artwork and A. Sabnis, R. Okimoto, A. Tulpule, and M. Hutchinson for critical review and input on the manuscript. The authors acknowledge the following researchers for providing plasmids through Addgene: E. Campeau (University of Massachusetts Medical School); H. Land and J. Morgenstern (Imperial Cancer Research Fund); B. Weinberg (Whitehead Institute for Biomedical Research); J. Zhao (Dana-Farber Cancer Institute, Harvard Medical School); and E. Fearon (University of Michigan School of Medicine).

Author information

Author notes

    • Collin M Blakely
    • , Thomas B K Watkins
    • , Wei Wu
    •  & Beatrice Gini

    These authors contributed equally to this work.


  1. Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

    • Collin M Blakely
    • , Wei Wu
    • , Beatrice Gini
    • , Victor R Olivas
    • , Julia Rotow
    • , Ashley Maynard
    • , Victoria Wang
    • , Matthew A Gubens
    •  & Trever G Bivona
  2. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.

    • Collin M Blakely
    • , Wei Wu
    • , Beatrice Gini
    • , Victor R Olivas
    • , Julia Rotow
    • , Ashley Maynard
    • , Victoria Wang
    • , Matthew A Gubens
    •  & Trever G Bivona
  3. The Francis Crick Institute, Cancer Research UK Lung Cancer Centre of Excellence, UCL Cancer Institute, London, UK.

    • Thomas B K Watkins
    • , Nicholas McGranahan
    • , Gareth A Wilson
    • , Nicolai J Birkbak
    •  & Charles Swanton
  4. Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, USA.

    • Jacob J Chabon
    •  & Maximilian Diehn
  5. Department of Medicine, Division of Medical Oncology, University of Colorado, Denver, Aurora, Colorado, USA.

    • Caroline E McCoach
    •  & Robert C Doebele
  6. Guardant Health, Inc., Redwood City, California, USA.

    • Kimberly C Banks
    •  & Richard B Lanman
  7. Driver Inc., San Francisco, California, USA.

    • Aleah F Caulin
    • , John St John
    • , Anibal R Cordero
    •  & Petros Giannikopoulos
  8. Clovis Oncology Inc., Boulder, Colorado, USA.

    • Andrew D Simmons
  9. University of California Davis Cancer Center, Sacramento, California, USA.

    • Philip C Mack
    • , David R Gandara
    •  & Jonathan W Riess
  10. Moores Cancer Center, University of California San Diego, San Diego, California, USA.

    • Hatim Husain


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C.M.B., T.B.K.W., C.S. and T.G.B. designed the study. C.M.B. performed medical-record review, analyzed data and prepared tables and figures. T.B.K.W. performed WES and clonality analysis and prepared tables and figures with assistance from N.M., G.A.W., and N.J.B. W.W. performed analysis of cfDNA-sequencing data on patient cohorts and prepared tables and figures. B.G. performed cell-line experiments and prepared figures with assistance from A.M. J.J.C. and M.D. performed cancer personalized profiling by deep sequencing (CAPP-seq) analysis. V.R.O. and J.R. performed immunohistochemistry analysis. C.E.M., M.A.G., V.W., A.D.S., P.C.M., D.R.G., H.H., R.C.D., and J.W.R. performed medical-record review and provided clinical data. K.C.B. and R.B.L. compiled and annotated cfDNA data from 1,150 patients with EGFR-mutant-positive NSCLC and 1,008 patients with EGFR-mutant-negative NSCLC. A.R.C. extracted DNA and prepared exome libraries from patient tumor samples. A.F.C. and J.S.J. performed exome sequencing alignment and quality analysis. P.G. harvested autopsy tissue and performed pathological assessments. C.M.B. and T.G.B. wrote the manuscript, to which all authors contributed.

Competing interests

K.C.B. and R.B.L. are employees of Guardant Health Inc.; A.F.C., J.S.J., A.R.C., and P.G. are employees of Driver Inc.; A.D.S. is an employee of Clovis Oncology Inc. T.G.B. is an advisor to Novartis, Astrazeneca, Takeda, Array Biopharma, and Revolution Medicines, and has received research funding from Ignyta and Revolution Medicines.

Corresponding authors

Correspondence to Charles Swanton or Trever G Bivona.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Tables 1–6 and Supplementary Note

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Data Set 1

    cfDNA alterations in EGFR-mutant positive lung cancer patientsSomatic variants, copy number gains and clonality of alterations detected in cfDNA of EGFR-mutant positive patients.

  2. 2.

    Supplementary Data Set 2

    cfDNA alterations in EGFR-mutant negative lung cancer patientsSomatic variants, copy number gains and clonality of alterations detected in cfDNA of EGFR-mutant negative patients.

  3. 3.

    Supplementary Data Set 3

    Demographic information and genomic alterations identified in cfDNA of EGFR-mutant lung cancer patients.Age, gender, smoking history, prior treatment and treatment outcomes for 137 samples from 97 patients with known clinical course.

  4. 4.

    Supplementary Data Set 4

    Demographic information, clinical response data, and genomic alterations identified in cfDNA of patients treated with an EGFR-TKI.Age, gender, smoking history, prior treatment and treatment outcomes for 73 patients treated with an EGFR TKI including 41 patients treated with osimertinib.

  5. 5.

    Supplementary Data Set 5

    Somatic mutations and clonality analysis of tumor samples described in Figure 5.Somatic variants identified in 7 tumor samples from patient with EGFR-mutant lung cancer throughout the course of her disease with clonality assessment for each variant determined by PyClone.

  6. 6.

    Supplementary Data Set 6

    Copy number alterations in tumor samples described in Figure 5 and Supplementary Figure 7.Chromosomal regions of copy number gain and loss identified in in 7 tumor samples from patient with EGFR-mutant lung cancer throughout the course of her disease.