Liquid biopsies come of age: towards implementation of circulating tumour DNA

Journal name:
Nature Reviews Cancer
Volume:
17,
Pages:
223–238
Year published:
DOI:
doi:10.1038/nrc.2017.7
Published online

Abstract

Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a 'liquid biopsy' for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.

At a glance

Figures

  1. Applications of circulating tumour DNA analysis during the course of disease management.
    Figure 1: Applications of circulating tumour DNA analysis during the course of disease management.

    a | A schematic time course for a hypothetical patient who undergoes surgery (or other initial treatment), has a disease relapse and then receives systemic therapy. The potential applications of liquid biopsies during this patient's care are indicated. The patient starts with one single disease focus, but multiple metastases and distinct clones (depicted in different colours) emerge following treatment. b | The information extracted from circulating tumour DNA (ctDNA) may be classified, broadly, as quantitative information (that is, relating to tumour burden) or genomic information. Quantification of ctDNA at a single time point may allow disease staging and prognostication, and genomic analysis can inform the selection of targeted therapies. Therefore, longitudinal analysis allows the quantitative tracking of tumour burden to monitor treatment response, for example, and by comparing genomic profiles over time, clonal evolution may be monitored.

  2. Origins and range of alterations in cell-free DNA.
    Figure 2: Origins and range of alterations in cell-free DNA.

    Cells release cell-free DNA (cfDNA) through a combination of apoptosis, necrosis and secretion. cfDNA can arise from cancerous cells but also from cells in the tumour microenvironment, immune cells or other body organs. In the bloodstream, cfDNA may exist as either free or associated with extracellular vesicles such as exosomes2. Multiple classes of genetic and epigenetic alterations can be found in cfDNA. Adapted with permission from Ref. 215, Macmillan Publishers Ltd.

  3. Current and future paradigms for the sensitive detection of circulating tumour DNA.
    Figure 3: Current and future paradigms for the sensitive detection of circulating tumour DNA.

    a | The analysis of cell-free (cfDNA) can range from the interrogation of individual loci to analysis of the whole genome (Table 1). Off-the-shelf digital PCR (dPCR) assays can achieve high sensitivity with a simple workflow but are limited by a low multiplexing capability. Targeted sequencing can allow the interrogation of multiple loci with high sensitivity through the use of methods that suppress background noise242.b | In molecular barcoding, unique molecular sequences are added to each molecule during library preparation so that sequencing reads originating from the same starting molecule can be identified. By comparing all reads from the same molecule, a single consensus sequence can be taken, thus suppressing errors arising from PCR or sequencing. c | To improve the sensitivity of the analysis — for example, for disease diagnosis or detection of minimal residual disease — other body fluids may be considered in combination with, or instead of, plasma. Sampling of body fluids or cytological specimens proximal to the tumour site may yield a higher concentration of DNA of tumour origin than is found in plasma. d | Circulating tumour DNA (ctDNA) has been shown to be shorter than cfDNA4, 80, 81, 84. Thus, experimental or in silico selection of shorter fragments may enrich for sequences of cancer origin81, and can improve sensitivity for samples with low proportions of ctDNA. In part a, the targeted sequencing image is adapted from Ref. 34 (Forshew, T. et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci. Transl Med. 4, 136ra68 (2012)). Reproduced with permission from the AAAS. The whole-genome sequencing image is courtesy of Dennis Lo (The Chinese University of Hong Kong) and is based on data published in Ref. 37.

  4. Leveraging multiple mutations to detect low-burden disease and overcome sampling noise.
    Figure 4: Leveraging multiple mutations to detect low-burden disease and overcome sampling noise.

    Even with a perfectly sensitive assay, the probability of detecting circulating tumour DNA (ctDNA) decreases as ctDNA concentration declines, as any single mutation of interest may not be present in a given volume of sample. At low ctDNA concentrations, some mutations will be detected while others are missed owing to sampling error. Sampling multiple pre-specified mutations in each reaction may improve the detection of low levels of ctDNA, as every target provides an independent opportunity to test for the presence of a mutant molecule in the set of DNA molecules at that locus34, 114. Sensitivity can be further improved by analysing multiple replicates that each contain few molecules, so that the mutant allele — where present in a reaction — will constitute a large proportion of the DNA template116. The dashed boxes below the graph show hypothetical examples of sets of molecules that may be captured by each replicate in the analysis of a sample.

  5. Adaptive or reactive treatment paradigms using liquid biopsies.
    Figure 5: Adaptive or reactive treatment paradigms using liquid biopsies.

    a | During systemic anticancer therapy, serial liquid biopsies may identify biochemical response or progression. If progression is identified, the clinician may be able to switch therapy or select a therapy to target arising clones that carry additional mutations that were identified by the analysis. b | This adaptive or reactive monitoring and treatment may continue as a loop, which would be facilitated by a fast turnaround time for circulating tumour DNA (ctDNA) analysis, for example through the use of point-of-care diagnostics. The time frames for this analysis can vary between hours and months; a time frame of hours could allow the analysis of early kinetics in response to therapy. EGFR, epidermal growth factor receptor; NSCLC, non-small-cell lung cancer; PIK3CAMUT, mutant PI3K catalytic subunit-α.

References

  1. Mandel, P. & Métais, P. Les acides nucléiques du plasma sanguin chez l'homme. C. R. Seances Soc. Biol. Fil. 142, 241243 (in French) (1948).
  2. Thierry, A. R., El Messaoudi, S., Gahan, P. B., Anker, P. & Stroun, M. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 35, 347376 (2016).
  3. Mouliere, F., El Messaoudi, S., Pang, D., Dritschilo, A. & Thierry, A. R. Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer. Mol. Oncol. 8, 927941 (2014).
  4. Mouliere, F. et al. High fragmentation characterizes tumour-derived circulating DNA. PLoS ONE 6, e23418 (2011).
    This study specifically characterizes ctDNA fragmentation patterns and the biological properties of ctDNA using an animal model system and clinical samples.
  5. Leon, S. A., Shapiro, B., Sklaroff, D. M. & Yaros, M. J. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 37, 646650 (1977).
  6. Rodrigues Filho, E. M. et al. Elevated cell-free plasma DNA level as an independent predictor of mortality in patients with severe traumatic brain injury. J. Neurotrauma 31, 16391646 (2014).
  7. Tsai, N.-W. et al. The value of serial plasma nuclear and mitochondrial DNA levels in patients with acute ischemic stroke. Clin. Chim. Acta 412, 476479 (2011).
  8. Breitbach, S., Sterzing, B., Magallanes, C., Tug, S. & Simon, P. Direct measurement of cell-free DNA from serially collected capillary plasma during incremental exercise. J. Appl. Physiol. 117, 119130 (2014).
  9. De Vlaminck, I. et al. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci. Transl Med. 6, 241ra77 (2014).
  10. De Vlaminck, I. et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc. Natl Acad. Sci. USA 112, 1333613341 (2015).
  11. Lo, Y. M. D. et al. Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485487 (1997).
  12. Hyett, J. A. et al. Reduction in diagnostic and therapeutic interventions by non-invasive determination of fetal sex in early pregnancy. Prenat. Diagn. 25, 11111116 (2005).
  13. Saito, H., Sekizawa, A., Morimoto, T., Suzuki, M. & Yanaihara, T. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma. Lancet 356, 1170 (2000).
  14. Lo, Y. M. D. et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc. Natl Acad. Sci. USA 104, 1311613121 (2007).
  15. Allyse, M. et al. Non-invasive prenatal testing: a review of international implementation and challenges. Int. J. Womens Health 7, 113126 (2015).
  16. Hill, M. et al. Evaluation of non-invasive prenatal testing (NIPT) for aneuploidy in an NHS setting: a reliable accurate prenatal non-invasive diagnosis (RAPID) protocol. BMC Pregnancy Childbirth 14, 229 (2014).
  17. Stroun, M. et al. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 46, 318322 (1989).
  18. Sidransky, D. et al. Identification of p53 gene mutations in bladder cancers and urine samples. Science 252, 706709 (1991).
  19. Sidransky, D. et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 256, 102105 (1992).
  20. Caldas, C. et al. Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res. 54, 35683573 (1994).
  21. Mao, L., Hruban, R. H., Boyle, J. O., Tockman, M. & Sidransky, D. Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res. 54, 16341637 (1994).
  22. Takeda, S., Ichii, S. & Nakamura, Y. Detection of K-ras mutation in sputum by mutant-allele-specific amplification (MASA). Hum. Mutat. 2, 112117 (1993).
  23. Sorenson, G. D. et al. Soluble normal and mutated DNA-sequences from single-copy genes in human blood. Cancer Epidemiol. Biomarkers Prev. 3, 6771 (1994).
  24. Swisher, E. M. et al. Tumor-specific p53 sequences in blood and peritoneal fluid of women with epithelial ovarian cancer. Am. J. Obstet. Gynecol. 193, 662667 (2005).
  25. Kimura, H. et al. Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer. Clin. Cancer Res. 12, 39153921 (2006).
  26. Sozzi, G., Musso, K., Ratcliffe, C., Goldstraw, P. & Pierotti, M. A. Detection of microsatellite alterations in plasma DNA of non-small cell lung cancer patients: a prospect for early diagnosis. Clin. Cancer Res. 5, 26892692 (1999).
  27. Lecomte, T. et al. Detection of free-circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis. Int. J. Cancer 100, 542548 (2002).
  28. Vogelstein, B. & Kinzler, K. W. Digital PCR. Proc. Natl Acad. Sci. USA 96, 92369241 (1999).
  29. Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl Acad. Sci. USA 100, 88178822 (2003).
  30. Diehl, F. et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc. Natl Acad. Sci. USA 102, 1636816373 (2005).
  31. Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985990 (2008).
    This study demonstrates that ctDNA dynamics in patients undergoing treatment for colorectal cancer reflect tumour responses and progression, and that ctDNA detection after surgery represented a marker of residual disease and a prognostic factor.
  32. Kuang, Y. et al. Noninvasive detection of EGFR T790M in gefitinib or erlotinib resistant non-small cell lung cancer. Clin. Cancer Res. 15, 26302636 (2009).
  33. Qian, X. et al. Circulating cell-free DNA has a high degree of specificity to detect exon 19 deletions and the single-point substitution mutation L858R in non-small cell lung cancer. Oncotarget 7, 2915429165 (2016).
  34. Forshew, T. et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci. Transl Med. 4, 136ra68 (2012).
    This study identifies cancer mutations directly in plasma using a next-generation sequencing-based assay, and applies this method to quantify and monitor multiple mutations.
  35. Dawson, S.-J. et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N. Engl. J. Med. 368, 11991209 (2013).
  36. 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).
    In this study, WGS of cfDNA from plasma identified copy number alterations and rearrangements.
  37. Chan, K. C. A. 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, 211224 (2013).
  38. Heitzer, E. et al. Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing. Genome Med. 5, 30 (2013).
    This study demonstrates that WGS of plasma cfDNA at a shallow sequencing depth can establish copy number profiles in patients with prostate cancer with a rapid turnaround time.
  39. Murtaza, M. et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108112 (2013).
    This paper describes the use of exome sequencing of cfDNA from serial plasma samples to study clonal evolution and to track ctDNA dynamics in high-burden disease.
  40. Overman, M. J. et al. Use of research biopsies in clinical trials: are risks and benefits adequately discussed? J. Clin. Oncol. 31, 1722 (2013).
  41. VanderLaan, P. A. et al. Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer. Lung Cancer 84, 3944 (2014).
  42. Ellis, P. M. et al. Dacomitinib compared with placebo in pretreated patients with advanced or metastatic non-small-cell lung cancer (NCIC CTG BR.26): a double-blind, randomised, phase 3 trial. Lancet Oncol. 15, 13791388 (2014).
  43. Popper, H. H. Commentary on tumor heterogeneity. Transl Lung Cancer Res. 5, 433435 (2016).
  44. De Mattos-Arruda, L. et al. Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle. Ann. Oncol. 25, 17291735 (2014).
  45. Lebofsky, R. et al. Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types. Mol. Oncol. 9, 783790 (2015).
  46. Jamal-Hanjani, M. et al. Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer. Ann. Oncol. 27, 862867 (2016).
  47. Murtaza, M. et al. Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer. Nat. Commun. 6, 8760 (2015).
  48. Duffy, M. J. Serum tumor markers in breast cancer: are they of clinical value? Clin. Chem. 52, 345351 (2006).
  49. Fazel, R. et al. Exposure to low-dose ionizing radiation from medical imaging procedures. N. Engl. J. Med. 361, 849857 (2009).
  50. Thakur, B. K. et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24, 766769 (2014).
  51. Kahlert, C. et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J. Biol. Chem. 289, 38693875 (2014).
  52. Jahr, S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 16591665 (2001).
  53. Stroun, M., Lyautey, J., Lederrey, C., Olson-Sand, A. & Anker, P. About the possible origin and mechanism of circulating DNA: apoptosis and active DNA release. Clin. Chim. Acta 313, 139142 (2001).
  54. Botezatu, I. et al. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin. Chem. 46, 10781084 (2000).
  55. Chan, K. C. A., Leung, S. F., Yeung, S. W., Chan, A. T. C. & Lo, Y. M. D. Quantitative analysis of the transrenal excretion of circulating EBV DNA in nasopharyngeal carcinoma patients. Clin. Cancer Res. 14, 4809 (2008).
  56. Birkenkamp-Demtröder, K. et al. Genomic alterations in liquid biopsies from patients with bladder cancer. Eur. Urol. 70, 7582 (2016).
  57. Su, Y.-H. et al. Human urine contains small, 150 to 250 nucleotide- sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. J. Mol. Diagn. 6, 101107 (2004).
  58. Reckamp, K. L. et al. A highly sensitive and quantitative test platform for detection of NSCLC EGFR mutations in urine and plasma. J. Thorac. Oncol. 11, 16901700 (2016).
  59. Wang, Y. et al. Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc. Natl Acad. Sci. USA 112, 97049709 (2015).
  60. Pan, W., Gu, W., Nagpal, S., Gephart, M. H. & Quake, S. R. Brain tumor mutations detected in cerebral spinal fluid. Clin. Chem. 61, 514522 (2015).
  61. De Mattos-Arruda, L. et al. Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat. Commun. 6, 8839 (2015).
  62. Rhodes, C. H., Honsinger, C. & Sorenson, G. D. PCR-detection of tumor-derived p53 DNA in cerebrospinal fluid. Am. J. Clin. Pathol. 103, 404408 (1995).
  63. Sriram, K. B. et al. Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas. BMC Cancer 12, 428 (2012).
  64. Mithani, S. K. et al. Mitochondrial resequencing arrays detect tumor-specific mutations in salivary rinses of patients with head and neck cancer. Clin. Cancer Res. 13, 73357340 (2007).
  65. Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M. & Shendure, J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164, 5768 (2016).
  66. Lehmann-Werman, R. et al. Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proc. Natl Acad. Sci. USA 113, E1826E1834 (2016).
  67. Sun, K. et al. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proc. Natl Acad. Sci. USA 112, E5503E5512 (2015).
    In this study the relative contributions of different tissues to the total cfDNA pool were analysed using methylation deconvolution.
  68. Lui, Y. Y. N. et al. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin. Chem. 48, 421427 (2002).
  69. Tug, S. et al. Exercise-induced increases in cell free DNA in human plasma originate predominantly from cells of the haematopoietic lineage. Exerc. Immunol. Rev. 21, 164173 (2015).
  70. To, E. W. H. et al. Rapid clearance of plasma Epstein–Barr virus DNA after surgical treatment of nasopharyngeal carcinoma. Clin. Cancer Res. 9, 32543259 (2003).
  71. Lo, Y. M. et al. Rapid clearance of fetal DNA from maternal plasma. Am. J. Hum. Genet. 64, 218224 (1999).
  72. Wang, Y., Chen, M., Xiao, N. & Liu, H. Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: a qualitative study. Gene 590, 142148 (2016).
  73. Tamkovich, S. N. et al. Circulating DNA and DNase activity in human blood. Ann. NY Acad. Sci. 1075, 191196 (2006).
  74. Tsui, N. B. Y. et al. High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing. PLoS ONE 7, e48319 (2012).
  75. Chused, T. M., Steinberg, A. D. & Talal, N. The clearance and localization of nucleic acids by New Zealand and normal mice. Clin. Exp. Immunol. 12, 465476 (1972).
  76. Giacona, M. B. et al. Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls. Pancreas 17, 8997 (1998).
  77. Lo, Y. M. D. et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci. Transl Med. 2, 61ra91 (2010).
  78. Thierry, A. R. et al. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res. 38, 61596175 (2010).
  79. Nadano, D., Yasuda, T. & Kishi, K. Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method. Clin. Chem. 39, 448452 (1993).
  80. Mouliere, F. et al. Circulating cell-free DNA from colorectal cancer patients may reveal high KRAS or BRAF mutation load. Transl Oncol. 6, 319328 (2013).
  81. Underhill, H. R. et al. Fragment length of circulating tumor DNA. PLoS Genet. 12, 426437 (2016).
  82. Gorges, T. M. et al. Cancer therapy monitoring in xenografts by quantitative analysis of circulating tumor DNA. Biomarkers 17, 498506 (2012).
  83. Zheng, Y. W. L. et al. Nonhematopoietically derived DNA is shorter than hematopoietically derived DNA in plasma: a transplantation model. Clin. Chem. 58, 549558 (2012).
  84. Jiang, P. & Lo, Y. M. D. The long and short of circulating cell-free DNA and the ins and outs of molecular diagnostics. Trends Genet. 32, 360371 (2016).
  85. Cheng, S. H. et al. Noninvasive prenatal testing by nanopore sequencing of maternal plasma DNA: feasibility assessment. Clin. Chem. 61, 13051306 (2015).
  86. Breitbach, S. et al. Direct quantification of cell-free, circulating DNA from unpurified plasma. PLoS ONE 9, e87838 (2014).
  87. Beránek, M. et al. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer. Acta Medica (Hradec Kralove) 59, 5458 (2016).
  88. Gansauge, M.-T. & Meyer, M. Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA. Nat. Protoc. 8, 737748 (2013).
  89. Burnham, P. et al. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma. Sci. Rep. 6, 27859 (2016).
  90. Kostyuk, S. V. et al. Fragments of cell-free DNA increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway, and suppress apoptosis. Biochem. (Mosc.) Suppl. Ser. B Biomed. Chem. 6, 6874 (2012).
  91. Nishimoto, S. et al. Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Sci. Adv. 2, e1501332 (2016).
  92. Dvor˘áková, M. et al. DNA released by leukemic cells contributes to the disruption of the bone marrow microenvironment. Oncogene 32, 52015209 (2013).
  93. Gartler, S. M. Cellular uptake of deoxyribonucleic acid by human tissue culture cells. Nature 184, 15051506 (1959).
  94. García-Olmo, D. C. et al. Cell-free nucleic acids circulating in the plasma of colorectal cancer patients induce the oncogenic transformation of susceptible cultured cells. Cancer Res. 70, 560567 (2010).
  95. Ju, Y. S. et al. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells. Genome Res. 25, 814824 (2015).
  96. Wu, D. Y., Ugozzoli, L., Pal, B. K. & Wallace, R. B. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc. Natl Acad. Sci. USA 86, 27572760 (1989).
  97. Newton, C. R. et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 25032516 (1989).
  98. QIAGEN. Therascreen EGFR Plasma RGQ PCR Kit Handbook — Version 2. https://www.qiagen.com/gb/resources/resourcedetail?id=eb32e329-3422-4eda-b3d6-e44ed787002a&lang=en (2014).
  99. US Food & Drug Administration. Premarket approval P150044 — Cobas EGFR MUTATION TEST V2. FDA http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpma/pma.cfm?id=P150044 (2016).
  100. Schiavon, G. et al. Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer. Sci. Transl Med. 7, 313ra182 (2015).
  101. Taly, V. et al. Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin. Chem. 59, 17221731 (2013).
  102. Sacher, A. G. et al. Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol. 2, 10141022 (2016).
  103. Yung, T. K. F. 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, 20762084 (2009).
  104. Mosko, M. J. et al. Ultrasensitive detection of multiplexed somatic mutations using MALDI-TOF mass spectrometry. J. Mol. Diagn. 18, 2331 (2016).
  105. Marziali, A., Pel, J., Bizzotto, D. & Whitehead, L. A. Novel electrophoresis mechanism based on synchronous alternating drag perturbation. Electrophoresis 26, 8290 (2005).
  106. Thompson, J. D., Shibahara, G., Rajan, S., Pel, J. & Marziali, A. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment. PLoS ONE 7, e31597 (2012).
  107. Song, C. et al. Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment. Nucleic Acids Res. 44, 111 (2016).
  108. Li, J. et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat. Med. 14, 579584 (2008).
  109. Guha, M., Castellanos-Rizaldos, E., Liu, P., Mamon, H. & Makrigiorgos, G. M. Differential strand separation at critical temperature: a minimally disruptive enrichment method for low-abundance unknown DNA mutations. Nucleic Acids Res. 41, e50 (2013).
  110. Newman, A. M. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat. Med. 20, 548554 (2014).
  111. Lanman, R. B. 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).
  112. Frenel, J.-S. et al. Serial next generation sequencing of circulating cell free DNA evaluating tumour clone response to molecularly targeted drug administration. Clin. Cancer Res. 21, 45864596 (2015).
  113. Narayan, A. et al. Ultrasensitive measurement of hotspot mutations in tumor DNA in blood using error-suppressed multiplexed deep sequencing. Cancer Res. 72, 34923498 (2012).
  114. Newman, A. M. et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat. Biotechnol. 34, 547555 (2016).
    This paper describes the use of an error suppression method for capture-enrichment sequencing that uses a combination of in silico elimination of stereotyped background artefacts and molecular barcoding.
  115. Kinde, I., Wu, J., Papadopoulos, N., Kinzler, K. W. & Vogelstein, B. Detection and quantification of rare mutations with massively parallel sequencing. Proc. Natl Acad. Sci. USA 108, 95309535 (2011).
  116. Rosenfeld, N., Forshew, T., Marass, F. & Murtaza, M. A method for detecting a genetic variant. World Intellectual Property Organization patent WO2016009224A1 (2016).
  117. Gale, D. et al. Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay. Inivata http://inivata.com/wp-content/uploads/2016/06/AACRposter2016.pdf (2016).
  118. Schwaederle, M. C. et al. Detection rate of actionable mutations in diverse cancers using a biopsy-free (blood) circulating tumor DNA assay. Oncotarget 33, 11004 (2015).
  119. Belic, J. et al. Rapid identification of plasma DNA samples with increased ctDNA levels by a modified FAST-SeqS approach. Clin. Chem. 61, 838849 (2015).
  120. Kirkizlar, E. et al. Detection of clonal and subclonal copy-number variants in cell-free DNA from patients with breast cancer using a massively multiplexed PCR methodology. Transl Oncol. 8, 407416 (2015).
  121. Parkinson, C. A. et al. Exploratory analysis of TP53 mutations in circulating tumour DNA as biomarkers of treatment response for patients with relapsed high-grade serous ovarian carcinoma: a retrospective study. PLoS Med. 13, e1002198 (2016).
  122. Thierry, A. R. et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat. Med. 20, 430435 (2014).
    This blinded prospective multicentre study compared KRAS and BRAF mutation status in tumours and plasma using an allele-specific quantitative PCR approach.
  123. Kamat, A. A. et al. Circulating cell-free DNA: a novel biomarker for response to therapy in ovarian carcinoma. Cancer Biol. Ther. 5, 13691374 (2006).
  124. Bettegowda, C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci. Transl Med. 6, 224ra24 (2014).
    In this study, a comprehensive quantitative analysis finds varying levels of ctDNA across patients with distinct cancer types and provides indicative ranges of ctDNA levels across the stages of disease.
  125. Nygaard, A. D., Garm Spindler, K.-L., Pallisgaard, N., Andersen, R. F. & Jakobsen, A. The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer. Lung Cancer 79, 312317 (2013).
  126. Wang, S. et al. Potential clinical significance of a plasma-based KRAS mutation analysis in patients with advanced non-small cell lung cancer. Clin. Cancer Res. 16, 13241330 (2010).
  127. Gautschi, O. et al. Origin and prognostic value of circulating KRAS mutations in lung cancer patients. Cancer Lett. 254, 265273 (2007).
  128. Santiago-Walker, A. et al. Correlation of BRAF mutation status in circulating-free DNA and tumor and association with clinical outcome across four BRAFi and MEKi clinical trials. Clin. Cancer Res. 22, 567574 (2016).
  129. Gray, E. S. et al. Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma. Oncotarget 6, 4200842018 (2015).
  130. Lipson, E. J. et al. Circulating tumor DNA analysis as a real-time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade. J. Immunother. Cancer 2, 42 (2014).
  131. Oshiro, C. et al. PIK3CA mutations in serum DNA are predictive of recurrence in primary breast cancer patients. Breast Cancer Res. Treat. 150, 299307 (2015).
  132. Scherer, F. et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci. Transl Med. 8, 364ra155 (2016).
  133. Office for National Statistics. Cancer survival by stage at diagnosis for England (experimental statistics): adults diagnosed 2012, 2013 and 2014 and followed up to 2015. ONS https://www.ons.gov.uk/peoplepopulationandcommunity/healthandsocialcare/conditionsanddiseases/bulletins/cancersurvivalbystageatdiagnosisforenglandexperimentalstatistics/adultsdiagnosed20122013and2014andfollowedupto2015 (2016).
  134. Gormally, E. et al. TP53 and KRAS2 mutations in plasma DNA of healthy subjects and subsequent cancer occurrence: a prospective study. Cancer Res. 66, 68716876 (2006).
  135. Amant, F. et al. Presymptomatic identification of cancers in pregnant women during noninvasive prenatal testing. JAMA Oncol. 1, 814 (2015).
  136. Bianchi, D. W. et al. Noninvasive prenatal testing and incidental detection of occult maternal malignancies. JAMA 314, 162 (2015).
  137. Beaver, J. A. et al. Detection of cancer DNA in plasma of patients with early-stage breast cancer. Clin. Cancer Res. 20, 26432650 (2014).
  138. Cohen, P. A. et al. Abnormal plasma DNA profiles in early ovarian cancer using a non-invasive prenatal testing platform: implications for cancer screening. BMC Med. 14, 126 (2016).
  139. Gorges, T. M. et al. Enumeration and molecular characterization of tumor cells in lung cancer patients using a novel in vivo device for capturing circulating tumor cells. Clin. Cancer Res. 22, 21972206 (2016).
  140. Kinde, I. et al. Evaluation of DNA from the Papanicolaou test to detect ovarian and endometrial cancers. Sci. Transl Med. 5, 167ra4 (2013).
  141. Nair, N. et al. Genomic analysis of uterine lavage fluid detects early endometrial cancers and reveals a prevalent landscape of driver mutations in women without histopathologic evidence of cancer: a prospective cross-sectional study. PLoS Med. 13, e1002206 (2016).
  142. Ross-Innes, C. S. et al. Risk stratification of Barrett's oesophagus using a non-endoscopic sampling method coupled with a biomarker panel: a cohort study. Lancet Gastroenterol. Hepatol. 2, 2331 (2016).
  143. Chan, K. C. A. et al. Early detection of nasopharyngeal carcinoma by plasma Epstein–Barr virus DNA analysis in a surveillance program. Cancer 119, 18381844 (2013).
  144. Guerrero-Preston, R. et al. Molecular triage of premalignant lesions in liquid-based cervical cytology and circulating cell-free DNA from urine, using a panel of methylated human papilloma virus and host genes. Cancer Prev. Res. 9, 915924 (2016).
  145. Martincorena, I. et al. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 348, 880886 (2015).
  146. Genovese, G. et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N. Engl. J. Med. 371, 24772487 (2014).
  147. Fernandez-Cuesta, L. et al. Identification of circulating tumor DNA for the early detection of small-cell lung cancer. eBioMedicine 10, 612 (2016).
  148. Rosenfeld, N. et al. MicroRNAs accurately identify cancer tissue origin. Nat. Biotechnol. 26, 462469 (2008).
  149. Olsen, J. A. et al. A minimally-invasive blood-derived biomarker of oligodendrocyte cell-loss in multiple sclerosis. eBioMedicine 10, 227235 (2016).
  150. Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883892 (2012).
  151. Yates, L. R. et al. Subclonal diversification of primary breast cancer revealed by multiregion sequencing. Nat. Med. 21, 751759 (2015).
  152. Turajlic, S. & Swanton, C. Metastasis as an evolutionary process. Science 352, 169175 (2016).
  153. Piotrowska, Z. 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, 3754 (2015).
  154. De Mattos-Arruda, L. et al. Establishing the origin of metastatic deposits in the setting of multiple primary malignancies: the role of massively parallel sequencing. Mol. Oncol. 8, 150158 (2014).
  155. Chan, K. C. A. et al. Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing. Proc. Natl Acad. Sci. USA 110, 1876118768 (2013).
  156. Kuo, Y.-B., Chen, J. S., Fand, C. W., Li, Y. S. & Chan, E. C. Comparison of KRAS mutation analysis of primary tumors and matched circulating cell-free DNA in plasmas of patients with colorectal cancer. Clin. Chim. Acta 433, 284289 (2014).
  157. de Bruin, E. C. et al. Spatial and temporal diversity in genomic instability processes defines lung cancer evolution. Science 346, 251256 (2014).
  158. Zhang, J. et al. Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing. Science 346, 256259 (2014).
  159. Oxnard, G. R. et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J. Clin. Oncol. 34, 33753382 (2016).
    In this retrospective analysis, patients with NSCLC who were positive for EGFRT790M in plasma show outcomes with osimertinib that were equivalent to those of patients classified as positive by a tissue-based assay, supporting the use of plasma analysis to avoid tumour biopsies in patients who are positive for EGFRT790M in plasma.
  160. Siravegna, G. et al. Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. Nat. Med. 21, 795801 (2015).
  161. Tie, J. et al. Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. Ann. Oncol. 26, 17151722 (2015).
  162. Chabon, J. J. et al. Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients. Nat. Commun. 7, 11815 (2016).
    In this study, ctDNA analysis by screening of a large panel of genes identifies multiple heterogeneous resistance mechanisms in patients with NSCLC treated with the EGFR inhibitor rociletinib.
  163. Karachaliou, N. et al. Association of EGFR L858R mutation in circulating free DNA with survival in the EURTAC trial. JAMA Oncol. 1, 149157 (2015).
  164. Reck, M. et al. Circulating free tumor-derived DNA (ctDNA) determination of EGFR mutation status in European and Japanese patients with advanced NSCLC: the ASSESS study. J. Thorac. Oncol. 11, 16821689 (2016).
  165. Han, B. et al. Determining the prevalence of EGFR mutations in Asian and Russian patients (pts) with advanced non-small-cell lung cancer (aNSCLC) of adenocarcinoma (ADC) and non-ADC histology: IGNITE study. Ann. Oncol. 26, i29i44 (2015).
  166. Douillard, J.-Y. et al. Gefitinib treatment in EGFR mutated Caucasian NSCLC. J. Thorac. Oncol. 9, 13451353 (2014).
  167. Normanno, N., Denis, M. G., Thress, K. S., Ratcliffe, M. & Reck, M. Guide to detecting epidermal growth factor receptor (EGFR) mutations in ctDNA of patients with advanced non-small-cell lung cancer. Oncotarget http://dx.doi.org/10.18632/oncotarget.13915 (2016).
  168. Douillard, J.-Y. et al. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study. Br. J. Cancer 110, 5562 (2014).
  169. Mok, T. S. et al. Osimertinib or platinum–pemetrexed in EGFR T790M-positive lung cancer. N. Engl. J. Med. http://dx.doi.org/10.1056/NEJMoa1612674 (2016).
  170. Sorensen, B. S. et al. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib. Cancer 120, 38963901 (2014).
  171. Oxnard, G. R. et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin. Cancer Res. 20, 1698705 (2014).
  172. Thress, K. S. et al. EGFR mutation detection in ctDNA from NSCLC patient plasma: a cross-platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer 90, 509515 (2015).
  173. Remon, J. et al. Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA. Annc. Oncol. doi:doi.dx.org:/10.1093/annonc/mdx017 (2017).
  174. European Medicines Agency. Iressa: public assessment report — product information. EMA http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/001016/WC500036358.pdf (2016).
  175. European Medicines Agency. Tagrisso: public assessment report — product information. EMA http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004124/WC500202022.pdf (2016).
  176. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02693535 (2017).
  177. Schwaederle, M. et al. Use of liquid biopsies in clinical oncology: pilot experience in 168 patients. Clin. Cancer Res. 22, 54975505 (2016).
  178. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02140463 (2015).
  179. Remon, J. et al. Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer (NSCLC) patients lacking tissue samples. Inivata http://inivata.com/wp-content/uploads/2016/09/MAP2016posterFINAL.pdf (2016).
  180. Institut Gustave Roussy. The prospective MOSCATO 01 trial demonstrates that molecular “portraits” improve outcome of patients with metastatic cancer. PRNewswire http://www.prnewswire.com/news-releases/the-prospective-moscato-01-trial-demonstrates-that-molecular-portraits-improve-outcome-of-patients-with-metastatic-cancer-594507371.html (2016).
  181. Pishvaian, M. J. et al. A pilot study evaluating concordance between blood-based and patient-matched tumor molecular testing within pancreatic cancer patients participating in the Know Your Tumor (KYT) initiative. Oncotarget http://dx.doi.org/10.18632/oncotarget.13225 (2016).
  182. Heitzer, E. et al. Establishment of tumor-specific copy number alterations from plasma DNA of patients with cancer. Int. J. Cancer 133, 346356 (2013).
  183. Mohan, S. et al. Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing. PLoS Genet. 10, e1004271 (2014).
  184. Kinde, I., Papadopoulos, N., Kinzler, K. W. & Vogelstein, B. FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing. PLoS ONE 7, e41162 (2012).
  185. Abdueva, D. et al. Detection, frequency and actionability of recurrent copy number gains detected by non-invasive liquid biopsy of 3,942 lung and breast cancer samples. J. Clin. Oncol. 34, abstr. 11541 (2016).
  186. Jiang, P. et al. Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc. Natl Acad. Sci. USA 112, E1317E1325 (2015).
    In this study, a genome-wide analysis of plasma DNA from patients with hepatocellular carcinoma finds that short molecules of cfDNA are enriched for tumour-associated copy number alterations.
  187. Romanel, A. et al. Plasma AR and abiraterone-resistant prostate cancer. Sci. Transl Med. 7, 312re10 (2015).
  188. Paweletz, C. P. et al. Bias-corrected targeted next-generation sequencing for rapid, multiplexed detection of actionable alterations in cell-free DNA from advanced lung cancer patients. Clin. Cancer Res. 22, 915922 (2016).
  189. Schreuer, M. et al. Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors. J. Transl Med. 14, 95 (2016).
  190. Marchetti, A. et al. Early prediction of response to tyrosine kinase inhibitors by quantification of EGFR mutations in plasma of NSCLC patients. J. Thorac. Oncol. 10, 14371443 (2015).
  191. Xi, L. et al. Circulating tumor DNA as an early indicator of response to T-cell transfer immunotherapy in metastatic melanoma. Clin. Cancer Res. 22, 54805486 (2016).
  192. Xin, Y. et al. Method for detecting BCR and TCR immune repertoire in blood plasma cfDNA. Chinese patent CN105087789A (2015).
  193. Tie, J. et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci. Transl Med. 8, 346ra92 (2016).
    This prospective cohort study of 230 patients with resected stage II colon cancer demonstrates that post-operative ctDNA detection predicts poor recurrence-free survival.
  194. Garcia-Murillas, I. et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci. Transl Med. 7, 302ra133 (2015).
    This study demonstrates the use of ctDNA for the detection of MRD in early-stage breast cancer following treatment with curative intent.
  195. Reinert, T. et al. Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery. Gut 65, 625634 (2015).
  196. Olsson, E. et al. Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. EMBO Mol. Med. 7, 10341047 (2015).
  197. Leary, R. J. et al. Development of personalized tumor biomarkers using massively parallel sequencing. Sci. Transl Med. 2, 20ra14 (2010).
  198. Mcbride, D. J. et al. Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors. Genes Chromosomes Cancer 49, 10621069 (2011).
  199. Russo, M. et al. Acquired resistance to the TRK inhibitor entrectinib in colorectal cancer. Cancer Discov. 6, 3644 (2016).
  200. Misale, S. et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 486, 532536 (2012).
    In this study, serial ctDNA analysis identifies KRAS-mutant alleles in the plasma of cetuximab-treated patients 10 months before disease relapse was identified by imaging.
  201. Diaz, L. A. J. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486, 537540 (2012).
  202. Ilié, M. & Hofman, P. Pros: can tissue biopsy be replaced by liquid biopsy? Transl Lung Cancer Res. 5, 420423 (2016).
  203. Thress, K. S. et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat. Med. 21, 560562 (2015).
    In this study, serial ctDNA monitoring of patients with lung cancer who were treated with AZD9291, an EGFR inhibitor, revealed a diversity of resistance mechanisms to therapy.
  204. Russo, M. et al. Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer. Cancer Discov. 6, 147153 (2015).
  205. Morelli, M. P. et al. Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment. Ann. Oncol. 26, 731736 (2015).
  206. Gremel, G. et al. Distinct sub-clonal tumour responses to therapy revealed by circulating cell-free DNA. Ann. Oncol. 27, 19591965 (2016).
  207. Ulz, P. et al. Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer. Nat. Commun. 7, 12008 (2016).
  208. European Medicines Agency. Draft guideline on good pharmacogenomic practice. EMA (2016).
  209. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02284633 (2015).
  210. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02743910 (2016).
  211. International Standard Randomised Controlled Trials Number Registry. isrctn.com http://www.isrctn.com/ISRCTN16945804 (2016).
  212. Naik, S. H., Schumacher, T. N. & Perie, L. Cellular barcoding: a technical appraisal. Exp. Hematol. 42, 598608 (2014).
  213. El Messaoudi, S. et al. Circulating DNA as a strong multimarker prognostic tool for metastatic colorectal cancer patient management care. Clin. Cancer Res. 22, 30673077 (2016).
  214. Spindler, K. L. G., Pallisgaard, N., Andersen, R. F. & Jakobsen, A. Changes in mutational status during third-line treatment for metastatic colorectal cancer — results of consecutive measurement of cell free DNA, KRAS and BRAF in the plasma. Int. J. Cancer 135, 22152222 (2014).
  215. Schwarzenbach, H., Hoon, D. S. B. & Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426437 (2011).
  216. Sullivan, R. J. et al. Plasma-based monitoring of BRAF mutations during therapy for malignant melanoma using combined exosomal RNA and cell-free DNA analysis. J. Clin. Oncol. 33, 9017 (2015).
  217. Ulz, P. et al. Inferring expressed genes by whole-genome sequencing of plasma DNA. Nat. Genet. 48, 12731278 (2016).
  218. Shao, H. et al. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat. Commun. 6, 6999 (2015).
  219. Alix-Panabières, C. & Pantel, K. Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy. Cancer Discov. 6, 479491 (2016).
  220. Best, M. G. et al. RNA-seq of tumor-educated platelets enables blood-based pan-cancer, multiclass, and molecular pathway cancer diagnostics. Cancer Cell 28, 666676 (2015).
  221. Gormally, E., Caboux, E., Vineis, P. & Hainaut, P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat. Res. 635, 105117 (2007).
  222. Webb, S. The cancer bloodhounds. Nat. Biotechnol. 34, 10901094 (2016).
  223. Long-Mira, E., Washetine, K. & Hofman, P. Sense and nonsense in the process of accreditation of a pathology laboratory. Virchows Arch. 468, 4349 (2016).
  224. Melchior, L. et al. Multi-center evaluation of the novel fully-automated PCR-based Idylla BRAF mutation test on formalin-fixed paraffin-embedded tissue of malignant melanoma. Exp. Mol. Pathol. 99, 485491 (2015).
  225. Janku, F. et al. BRAF mutation testing in cell-free DNA from the plasma of patients with advanced cancers using a rapid, automated molecular diagnostics system. Mol. Cancer Ther. 15, 13971404 (2016).
  226. Norris, A. L., Workman, R. E., Fan, Y., Eshleman, J. R. & Timp, W. Nanopore sequencing detects structural variants in cancer. Cancer Biol. Ther. 17, 246253 (2016).
  227. Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530, 228232 (2016).
  228. Wei, S. & Williams, Z. Rapid short-read sequencing and aneuploidy detection using minION nanopore technology. Genetics 202, 3744 (2016).
  229. Ip, C. L. C. et al. MinION Analysis and Reference Consortium: phase 1 data release and analysis. F1000Res. 4, 1075 (2015).
  230. Loose, M., Malla, S. & Stout, M. Real-time selective sequencing using nanopore technology. Nat. Methods 13, 751754 (2016).
  231. Umetani, N., Hiramatsu, S. & Hoon, D. S. B. Higher amount of free circulating DNA in serum than in plasma is not mainly caused by contaminated extraneous DNA during separation. Ann. NY Acad. Sci. 1075, 299307 (2006).
  232. Willems, M., Moshage, H., Nevens, F., Fevery, J. & Yap, S. H. Plasma collected from heparinized blood is not suitable for HCV-RNA detection by conventional RT-PCR assay. J. Virol. Methods 42, 127130 (1993).
  233. Jung, M., Klotzek, S., Lewandowski, M., Fleischhacker, M. & Jung, K. Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin. Chem. 49, 10281029 (2003).
  234. Parpart-Li, S. et al. The effect of preservative and temperature on the analysis of circulating tumor DNA. Clin. Cancer Res. http://dx.doi.org/10.1158/1078-0432.CCR-16-1691 (2016).
  235. El Messaoudi, S., Rolet, F., Mouliere, F. & Thierry, A. R. Circulating cell free DNA: preanalytical considerations. Clin. Chim. Acta 424, 222230 (2013).
  236. Sherwood, J. L. et al. Optimised pre-analytical methods improve KRAS mutation detection in circulating tumour DNA (ctDNA) from patients with non-small cell lung cancer (NSCLC). PLoS ONE 11, 114 (2016).
  237. Toro, P. V. et al. Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA. Clin. Biochem. 48, 993998 (2015).
  238. Kang, Q. et al. Comparative analysis of circulating tumor DNA stability in K3EDTA, Streck and CellSave blood collection tubes. Clin. Biochem. 49, 13541360 (2016).
  239. Medina Diaz, I. et al. Performance of Streck cfDNA blood collection tubes for liquid biopsy testing. PLoS ONE 11, e0166354 (2016).
  240. Norton, S. E., Lechner, J. M., Williams, T. & Fernando, M. R. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin. Biochem. 46, 15611565 (2013).
  241. Dietz, S. et al. Low input whole-exome sequencing to determine the representation of the tumor exome in circulating DNA of non-small cell lung cancer patients. PLoS ONE 11, e0161012 (2016).
  242. Schmitt, M. W. et al. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl Acad. Sci. USA 109, 1450814513 (2012).
  243. Zonta, E., Garlan, F., Perez-Toralla, K. & Taly, V. Multiplex detection of rare mutations by picoliter droplet based digital PCR: sensitivity and specificity considerations. PLoS ONE 11, e0159094 (2016).
  244. Kidess, E. et al. Mutation profiling of tumor DNA from plasma and tumor tissue of colorectal cancer patients with a novel, high-sensitivity multiplexed mutation detection platform. Oncotarget 6, 25492561 (2015).

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Author information

  1. These authors contributed equally to this work.

    • Richard Baird &
    • Nitzan Rosenfeld

Affiliations

  1. Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

    • Jonathan C. M. Wan,
    • Charles Massie,
    • Florent Mouliere,
    • James D. Brenton,
    • Carlos Caldas &
    • Nitzan Rosenfeld
  2. Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK.

    • Jonathan C. M. Wan,
    • Charles Massie,
    • Florent Mouliere,
    • James D. Brenton,
    • Carlos Caldas,
    • Simon Pacey,
    • Richard Baird &
    • Nitzan Rosenfeld
  3. Clinical Trials Unit, Clinic Institute of Haematological and Oncological Diseases, Hospital Clinic de Barcelona, IDIBAPs, Carrer de Villarroel, 170 Barcelona 08036, Spain.

    • Javier Garcia-Corbacho
  4. Department of Oncology, University of Cambridge Hutchison–MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK.

    • Carlos Caldas,
    • Simon Pacey &
    • Richard Baird

Competing interests statement

N.R. is the Chief Scientific Officer of Inivata (Cambridge, UK, and Research Triangle Park, North Carolina, USA). N.R. and J.D.B. are co-founders and shareholders of Inivata. N.R. and F.M. are co-inventors of patent applications that describe methods for the analysis of DNA fragments and applications of circulating tumour DNA.

Corresponding author

Correspondence to:

Author details

  • Jonathan C. M. Wan

    Jonathan C. M. Wan is an M.D.–Ph.D. student at Trinity College, University of Cambridge, UK. He completed his preclinical medical studies at King's College London, UK. At present, he is carrying out his Ph.D. in the group of Nitzan Rosenfeld at the Cancer Research UK Cambridge Institute, University of Cambridge (2015–18), where he is working on developing individualized circulating tumour DNA assays to monitor patients with melanoma with high sensitivity.

  • Charles Massie

    Charles Massie trained as a biochemist and then obtained a Ph.D. in Oncology at the University of Cambridge, UK. With a particular interest in the biology and early detection of prostate cancer, his research combines computational approaches, cancer genomics and high-sensitivity approaches for the analysis of tumour DNA in peripheral fluids.

  • Javier Garcia-Corbacho

    Javier García-Corbacho completed his medical oncology training at Hospital Universitario Reina Sofía (Córdoba, Spain) and his research training at the University of Cambridge, UK. He is currently the Head of the Clinical Trials Unit of the Clinic Institute of Haematological and Oncological Diseases (ICMHO, Barcelona, Spain), where he leads the Early Phase Clinical Trials Unit. His translational research interests include gene profiling and circulating tumour DNA.

  • Florent Mouliere

    Florent Mouliere is a postdoctoral research associate at the Cancer Research UK Cambridge Institute, University of Cambridge, UK. He has an interest in tailoring new sequencing technologies to the study of the biology of circulating nucleic acids and to improve the sensitivity of liquid biopsy in difficult pathological contexts (for example, brain tumours, early response kinetics and early-stage cancers). He received his Ph.D. from the University of Montpellier, France.

  • James D. Brenton

    James D. Brenton is a medical oncologist and senior group leader at the Cancer Research UK Cambridge Institute, University of Cambridge, UK, where he leads the Functional Genomics of Ovarian Cancer Laboratory. His research focuses on identifying predictive genomic biomarkers for therapy in ovarian cancer and elucidating mechanisms of drug resistance.

  • Carlos Caldas

    Carlos Caldas is the Professor of Cancer Medicine at the University of Cambridge, UK; Head of the Breast Cancer Functional Genomics Laboratory at the Cancer Research UK Cambridge Institute; and Director of the Breast Cancer Programme at Cancer Research UK Cambridge Centre. His laboratory has published extensively on breast cancer genomics and he is a pioneer of circulating tumour DNA applications.

  • Simon Pacey

    Simon Pacey is an academic consultant in medical oncology who specializes in early-phase and phase I clinical trials. His site-specific interest is prostate malignancy. His research interests include leading a portfolio of early-phase oncology trials and translational work to study the effects of drugs in men with localized, hormone-sensitive prostate cancer.

  • Richard Baird

    Richard Baird is an academic medical oncologist at the Cancer Research UK Cambridge Centre. He runs a portfolio of early-phase clinical trials that are focused on novel breast cancer therapeutics and biomarker-directed treatment strategies. He is the chief investigator on three investigator-initiated trials and is UK Academic Lead for the Association of Cancer Physicians.

  • Nitzan Rosenfeld

    Nitzan Rosenfeld trained in physics, systems biology and biotechnology. Since 2009, he has been leading a translational research group at the Cancer Research UK Cambridge Institute, University of Cambridge, UK, that is devoted to circulating tumour DNA (ctDNA)-based molecular diagnostics, establishing techniques for ctDNA analysis and the applications of ctDNA in oncology. He is co-founder and Chief Scientific Officer of Inivata (Cambridge, UK, and Research Triangle Park, North Carolina, USA), which applies ctDNA sequencing for precision medicine.

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