Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA


Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection1,2. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity3,4. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy5,6,7. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1–2 years. For each case, exome sequencing was performed on 2–5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel8; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin9; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab10,11, and following subsequent treatment with lapatinib12,13, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib14. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

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Figure 1: Identification of treatment-associated mutational changes from exome sequencing of serial plasma samples.
Figure 2: Mutations showing evidence of genomic tumour evolution.
Figure 3: Genome-wide concordance between plasma DNA and tumour DNA.


  1. 1

    Diaz, L. A., Jr et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486, 537–540 (2012)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Aparicio, S. & Caldas, C. The implications of clonal genome evolution for cancer medicine. N. Engl. J. Med. 368, 842–851 (2013)

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    Shah, S. P. et al. The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature 486, 395–399 (2012)

    ADS  CAS  Article  PubMed  Google Scholar 

  5. 5

    Chan, K. C. et al. Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. Clin. Chem. 59, 211–224 (2013)

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Forshew, T. et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci. Transl. Med. 4, 136ra168 (2012)

    Article  Google Scholar 

  7. 7

    Leary, R. J. et al. Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Sci. Transl. Med. 4, 162ra154 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8

    Isakoff, S. J. et al. Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells. Cancer Res. 65, 10992–11000 (2005)

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Knudsen, E. S. & Knudsen, K. E. Tailoring to RB: tumour suppressor status and therapeutic response. Nature Rev. Cancer 8, 714–724 (2008)

    CAS  Article  Google Scholar 

  10. 10

    Cui, J. et al. Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells. Cancer Res. 72, 5625–5634 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Nagalingam, A. et al. Med1 plays a critical role in the development of tamoxifen resistance. Carcinogenesis 33, 918–930 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Liu, L. et al. Novel mechanism of lapatinib resistance in HER2-positive breast tumor cells: activation of AXL. Cancer Res. 69, 6871–6878 (2009)

    CAS  Article  PubMed  Google Scholar 

  13. 13

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

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Pao, W. et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73 (2005)

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Punnoose, E. A. et al. Evaluation of circulating tumor cells and circulating tumor DNA in non-small cell lung cancer: association with clinical endpoints in a phase II clinical trial of pertuzumab and erlotinib. Clin. Cancer Res. 18, 2391–2401 (2012)

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nature Med. 14, 985–990 (2008)

    ADS  CAS  Article  PubMed  Google Scholar 

  17. 17

    McBride, D. J. et al. Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors. Genes Chromosom. Cancer 49, 1062–1069 (2010)

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Yung, T. K. et al. Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients. Clin. Cancer Res. 15, 2076–2084 (2009)

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Lo, Y. M. et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci. Transl. Med. 2, 61ra91 (2010)

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genet. 43, 491–498 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Diehl, F. et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc. Natl Acad. Sci. USA 102, 16368–16373 (2005)

    ADS  CAS  Article  PubMed  Google Scholar 

  25. 25

    Siddique, H. R. & Saleem, M. Role of BMI1, a stem cell factor, in cancer recurrence and chemoresistance: preclinical and clinical evidences. Stem Cells 30, 372–378 (2012)

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Chang, H. et al. Identification of genes associated with chemosensitivity to SAHA/taxane combination treatment in taxane-resistant breast cancer cells. Breast Cancer Res. Treat. 125, 55–63 (2011)

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Sato, K. et al. Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair. EMBO J. 31, 3524–3536 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Haslehurst, A. M. et al. EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer. BMC Cancer 12, 91 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    VanderWeele, D. J., Zhou, R. & Rudin, C. M. Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin. Mol. Cancer Ther. 3, 1605–1613 (2004)

    CAS  PubMed  Google Scholar 

  30. 30

    Wu, C. C., Yu, C. T., Chang, G. C., Lai, J. M. & Hsu, S. L. Aurora-A promotes gefitinib resistance via a NF-κB signaling pathway in p53 knockdown lung cancer cells. Biochem. Biophys. Res. Commun. 405, 168–172 (2011)

    CAS  Article  PubMed  Google Scholar 

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We thank J. Langmore and K. Solomon (Rubicon Genomics) for early access to library preparation products. We thank L. Jones, S. Richardson, C. Hodgkin and H. Biggs for recruiting patients into the DETECT and CTCR-OVO4 studies, all medical and ancillary staff in the breast and gynaecological cancer clinic and patients for consenting to participate. We thank the Human Research Tissue Bank at Addenbrooke’s Hospital which is supported by the NIHR Cambridge Biomedical Research Centre. We thank the Cancer Science Institute, National University of Singapore, and the Hematology-Oncology Research Group, National University Health System, Singapore for their support. We acknowledge the support of Cancer Research UK, the University of Cambridge, National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge Experimental Cancer Medicine Centre, Hutchison Whampoa Limited, and the National Medical Research Council, Singapore. S.-J.D. is supported by an Australian NHMRC/RG Menzies Early Career Fellowship that is administered through the Peter MacCallum Cancer Centre, Victoria, Australia.

Author information




M.M., S.-J.D., T.F., D.W.Y.T., D.G., J.D.B., C.C. and N.R. designed the study. M.M., D.W.Y.T. and T.F. developed methods. S.-J.D., C.P., A.S.C.W., T.M.C., J.D.B. and C.C. designed and conducted the prospective clinical studies. M.M., S.-J.D., D.W.Y.T., D.G., T.F. and A.M.P. generated data. Z.K., S.H. and D.B. contributed sequencing data. M.M., F.M. and N.R. analysed sequencing data. S.-F.C. and J.H. contributed to experiments and data analysis. M.M., S.-J.D., D.W.Y.T., T.M.C., J.D.B., C.C. and N.R. interpreted data. M.M. and N.R. wrote the paper with assistance from S.-J.D., D.W.Y.T., C.C., J.D.B. and other authors. All authors approved the final manuscript. J.D.B., C.C. and N.R. are the project co-leaders and joint senior authors.

Corresponding authors

Correspondence to James D. Brenton or Carlos Caldas or Nitzan Rosenfeld.

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Competing interests

Z.K., S.H. and D.B. are full-time employees of Illumina, Inc., providers of the sequencing technology used in this study; the other authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Text, Supplementary Figures 1-8 and Supplementary Tables 1-3. (PDF 2709 kb)

Supplementary Tables

This file contains Supplementary Tables 4-9, which contain a list of mutations with increased representation in plasma over the course of treatment for Cases 1-6. (XLSX 103 kb)

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Murtaza, M., Dawson, SJ., Tsui, D. et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112 (2013).

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