Circulating Cell-Free DNA and Cancer Therapy Monitoring: Methods and Potential

  • Peter B. Gahan
Part of the Methods in Molecular Biology book series (MIMB, volume 1909)


The monitoring of therapy during the treatment of cancer patients is currently assessed by the levels of circulating tumor cells or by PET/CT scans. Neither approach has the sensitivity or specificity to be very sure of the efficacy of the treatment. Moreover, PET/CT scans can be both comparatively expensive and produce low levels of radiation for the patient. The advent of the possibility of using circulating DNA released from the tumor permits (1) a possible early marker of the presence of the cancer, (2) an indication of the success of the primary treatment, (3) an indication of the early presence of possible metastasis, (4) a marker of the success of secondary subsequent treatment, (5) determining which patients can benefit from a particular treatment, and (6) offering a prognosis. These aspects will be discussed concerning the application of circulating tumor DNA analysis to the monitoring of cancer patients undergoing therapy.

Key words

cfDNA ctDNA Cancer therapy monitoring Methodology Current limitations Prognosis 


  1. 1.
    Economopoulos P, Georgoulias V, Kotsakis A (2017) Classifying circulating tumor cells to monitor cancer progression. Expert Rev Mol Diagn 17:153–165CrossRefGoogle Scholar
  2. 2.
    Yan W-T, Cui X, Chen Q et al (2017) Circulating tumor cell status monitors the treatment responses in breast cancer patients: a meta-analysis. Sci Rep 7:43464PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Horn P, Jakobsen EH, Madsen JS et al (2016) New approach for interpreting changes in circulating tumour cells (CTC) for evaluation of treatment effect in metastatic breast cancer. Trans Oncol 7:694–701CrossRefGoogle Scholar
  4. 4.
    Tibbe AG, Miller MC, Terstappen LW (2007) Statistical considerations for enumeration of circulating tumor cells. Cytometry A 71:154–162PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Allan AL, Keeney M (2010) Circulating tumor cell analysis: technical and statistical considerations for application to the clinic. J Oncol 2010:426218, 10 p.PubMedCrossRefGoogle Scholar
  6. 6.
    Kowalik A, Kowalewska M, Gózdz S (2017) Current approaches for avoiding the limitations of circulating tumor cells detection methods—implications for diagnosis and treatment of patients with solid tumors. Trans Res 185:58–84CrossRefGoogle Scholar
  7. 7.
    Ashworth TR (1869) A case of cancer in which cells similar to those in tumours were seen in blood after death. Aust Med J 14:146–147Google Scholar
  8. 8.
    McNamara D (2016) Hidden costs of cancer from CT scans add up. Medscape. Apr 25Google Scholar
  9. 9.
    Gahan PB (2018) Introduction—liquid biopsies in cancer studies. Trans Cancer Res 7(Suppl 2):S101–S104. Scholar
  10. 10.
    Gahan PB (2010) Circulating nucleic acids in plasma and serum: diagnosis and prognosis in cancer. EPMA J 1:503–512PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Beck J, Urnovitz HB, Riggert J et al (2009) Profile of the circulating DNA in apparently healthy individuals. Clin Chem 55:730–738PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Holdenrieder S, Eichhorn P, Beuers U (2006) Nucleosomal DNA fragments in autoimmune diseases. In: Swaminathan R, Butt A, Gahan PB (eds) Circulating nucleic acids in plasma and serum IV, Ann N Y Acad Sci, vol 1075, Blackwell Publishing, Boston, MA, pp 318–327Google Scholar
  13. 13.
    Thierry AR, Mouliere F, Gongora C et al (2010) Origin and quantification of circulating DNA in mice and human colorectal cancer xenografts. Nucleic Acid Res 38:6159–6175PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Thierry AR, El Messaoudi S, Gahan PB et al (2016) Origins, structures and functions of circulating DNA in oncology. Cancer Metastasis Rev 35:347–376PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Stroun M, Anker P, Maurice P et al (1997) Circulating nucleic acids in higher organisms. Int Rev Cytol 51:1–48Google Scholar
  16. 16.
    Gahan PB, Stroun M (2010) The biology of circulating nucleic acids in plasma and serum. In: Rykova EY, Kikuchi Y (eds) Extracellular nucleic acids, Nucleic acids and molecular biology. Springer, BerlinGoogle Scholar
  17. 17.
    Diehl F, Li M, Dressman D et al (2005) Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Natl Acad Sci U S A 102(45):16368–16373PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Suzuki N, Kamataki A, Yamaki J et al (2008) Characterisation of circulating DNA in healthy human plasma. Clin Chim Acta 387:55–58PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Zhong XY, Ladewig A, Schmid S et al (2007) Elevated level of cell free plasma DNA is associated with breast cancer. Arch Gynecol Obstet 276:327–331PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Divella R, Tommasi S, Lacalamita R et al (2009) Circulating hTERT DNA in early breast cancer. Anticancer Res 29:2845–2849PubMedGoogle Scholar
  21. 21.
    Schwarzenbach H, Stoehlmacher J, Pantel K et al (2008) Detection and monitoring of cell-free DNA in blood of patients with colorectal cancer. Ann N Y Acad Sci 1137:190196CrossRefGoogle Scholar
  22. 22.
    Mouliere F, El Messaoudi S, Gongora C et al (2013) Circulating cell-free DNA from colorectal cancer patients may reveal high KRAS or BRAF mutation load. Transl Oncol 6:319–328PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Thierry AR, Mouliere F, Gongora C et al (2010) Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res 38:6159–6175PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    El Messaoudi S, Mouliere F, Du Manoir S et al (2016) Circulating DNA as a strong multi-marker prognostic tool for metastatic colorectal cancer patient management care. Clin Cancer Res 22:3067–3077PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Nygaard AD, Holdgaard PC, Spindler KL et al (2014) The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC. Br J Cancer 110:363–368PubMedCrossRefGoogle Scholar
  26. 26.
    El Messaoudi S, Thierry A (2015) Pre-analytical requirements for analysing nucleic acids from blood. In: Gahan PB (ed) Circulating nucleic acids in early diagnosis, prognosis and treatment monitoring, Advances in predictive, preventive medicine, vol 5. Springer, Dordrecht, pp 45–70Google Scholar
  27. 27.
    Holdenrieder S (2015) CNAPS in therapy monitoring. In: Gahan PB (ed) Circulating nucleic acids in early diagnosis, prognosis and treatment monitoring, Advances in predictive, preventive medicine, vol 5. Springer, Dordrecht, pp 325–370Google Scholar
  28. 28.
    Tamkovich SN, Bryzgunova OE, Rykova EY (2005) Circulating nucleic acids in blood of healthy male and female donors. Clin Chem 51:1317–1319PubMedCrossRefGoogle Scholar
  29. 29.
    Ledoux L, Charles P (1972) Fate of exogenous DNA in mammals. In: Ledoux L (ed) Uptake of informative molecules by living cells. North-Holland Publishing Co., Amsterdam, pp 397–413Google Scholar
  30. 30.
    Gosse C, Le Pecq JB, Defrance P et al (1965) Initial degradation of deoxyribonucleic acid after injection in mammals. Cancer Res 25:877–883PubMedPubMedCentralGoogle Scholar
  31. 31.
    Tsumita T, Iwagana M (1963) Fate of injected deoxyribonucleic acid in mice. Nature 198:1088–1089PubMedCrossRefGoogle Scholar
  32. 32.
    Gauthier VJ, Tyler LN, Mannik M (1996) Blood clearance kinetics and liver uptake of mononucleosomes in mice. J Immunol 156:1151–1156PubMedGoogle Scholar
  33. 33.
    Lo YMD, Zhang J, Leung TN et al (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64:218–224PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Adams DH, Gahan PB (1982) Stimulated and non-stimulated rat spleen cells release different DNA-complexes. Differentiation 22:47–52PubMedCrossRefGoogle Scholar
  35. 35.
    Adams DH, Gahan PB (1983) The DNA extruded by rat spleen cells in culture. Int J Biochem 15:547–552PubMedCrossRefGoogle Scholar
  36. 36.
    Adams DH, Diaz N, Gahan PB (1997) In vitro stimulation by tumour cell media of [3H]thymidine incorporation by mouse spleen lymphocytes. Cell Biochem Funct 15:119–126PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Gahan PB, Stroun M (2010) Biology of CNAPS. In: Yo Kikuchi Y, Rykova EY (eds) Extracellular nucleic acids, Nucleic acids and molecular biology. Springer, Berlin, pp 167–189CrossRefGoogle Scholar
  38. 38.
    Zhang R, Nakahira K, Guo X et al (2016) Very short mitochondrial DNA fragments and heteroplasmy in human plasma. Sci Rep 6:36097PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Strydom C, Robinson C, Pretorius E et al (2006) The effect of selected metals on the central metabolic pathways in biology. Water SA 32:543–554Google Scholar
  40. 40.
    Mouliere F, Robert B, Peyrotte EA et al (2011) High fragmentation characterizes tumour-derived circulating DNA. PLoS One 6:e23418PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Holdenrieder S, Stieber P, Bodenmueller H et al (2001) Nucleosomes in serum of patients with benign and malignant diseases. Int J Cancer 95:114–120PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Holdenrieder S, Stieber P, von Pawel J et al (2004) Circulating nucleosomes predict the response to chemotherapy in patients with advanced non-small cell lung cancer. Clin Cancer Res 10:5981–5987PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Holdenrieder S, Stieber P, von Pawel J et al (2006) Early and specific prediction of the therapeutic efficiency in lung cancer by nucleosomal DNA and cytokeratin fragments. Ann N Y Acad Sci 1075:244–257PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Kremer A, Holdenrieder S, Stieber P et al (2006) Nucleosomes in colorectal cancer patients during radiochemotherapy. Tumour Biol 27:235–242PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Wittwer C, Boeck S, Heinemann V et al (2013) Circulating nucleosomes and immunogenic cell death markers HMGB1, sRAGE and DNAse in patients with advanced pancreatic cancer undergoing chemotherapy. Int J Cancer 133:2619–2630PubMedPubMedCentralGoogle Scholar
  46. 46.
    Stoetzer OJ, Ferching DM, Salat C et al (2013) Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating nucleosomes. Cancer Lett 336:140–148PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Nagata S, Nagase H, Kawane K et al (2003) Apoptosis at a glance: death or life? Cell Death Differ 10:108–116PubMedCrossRefPubMedCentralGoogle Scholar
  48. 48.
    Nagata S (2005) DNA degradation in development and programmed cell death. Ann Rev Immunol 23:853–875CrossRefGoogle Scholar
  49. 49.
    Grunt M, Hillebrand T, Schwarzenbach H (2018) Clinical relevance of size selection of circulating DNA. Transl Cancer Res 7:S171CrossRefGoogle Scholar
  50. 50.
    Schwarzenbach H, Hoon DS, Pantel K (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11:426–437CrossRefGoogle Scholar
  51. 51.
    Jiang P, Chan CW, Chan KC et al (2015) Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci U S A 112:E1317–E1325PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Zheng YW, Chan KC, Sun H et al (2012) Nonhematopoietically derived DNA is shorter than hematopoietically derived DNA in plasma: a transplantation model. Clin Chem 58:549–558PubMedCrossRefGoogle Scholar
  53. 53.
    Schwarzenbach H, Pantel K (2015) Circulating DNA as biomarker in breast cancer. Breast Cancer Res 17:136PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Chandrananda D, Thorne NP, Bahlo M (2015) High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA. BMC Med Genet 8:29Google Scholar
  55. 55.
    Lo YM, Chan KC, Sun H et al (2010) Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med 2:61ra91PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Wang M, Block TM, Steel L et al (2004) Preferential isolation of fragmented DNA enhances the detection of circulating k-ras DNA. Clin Chem 50:211–213PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Moser T, Ulz P, Zhou Q et al (2017) Single-stranded DNA library preparation does not preferentially enrich circulating tumor DNA. Clin Chem 63:1656–1659PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Heidary M, Auer M, Ulz P et al (2014) The dynamic range of circulating tumor DNA in metastatic breast cancer. Breast Cancer Res 16:421PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Heitzer E, Auer M, Hoffmann EM et al (2013) Establishment of tumor-specific copy number alterations from plasma DNA of patients with cancer. Int J Cancer 133:346–356PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Oellerich M, Schütz E, Beck J et al (2017) Using circulating cell-free DNA to monitor personalized cancer therapy. Crit Rev Clin Lab Sci 54:205PubMedCrossRefGoogle Scholar
  61. 61.
    Schmidt B, Fleischhacker M (2018) Is liquid biopsy ready for the litmus test and what has been achieved so far to deal with pre-analytical issues? Transl Cancer Res 7:S130CrossRefGoogle Scholar
  62. 62.
    Ladas I, Fitarelli-Kiehl M, Song C (2017) Multiplexed elimination of wild-type DNA and high-resolution melting prior to targeted resequencing of liquid biopsies. Clin Chem 63:1605–1613PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Gahan PB (2015) A brief history and the present and future status of CNAPS. In: Gahan PB (ed) Circulating nucleic acids in early diagnosis, prognosis and treatment monitoring, Advances in predictive, preventive medicine, vol 5. Springer, Dordrecht, pp 3–14Google Scholar
  64. 64.
    Pan W, Quake SR (2015) Genomic approaches to the analysis of cell free nucleic acids. In: Gahan PB (ed) Circulating nucleic acids in early diagnosis, prognosis and treatment monitoring, Advances in predictive, preventive medicine, vol 5. Springer, Dordrecht, pp 113–139Google Scholar
  65. 65.
    Zhou Q, Moser T, Perakis S et al (2018) Untargeted profiling of cell-free circulating DNA. Transl Cancer Res 7:S140–S152. Scholar
  66. 66.
    Roschewski M, Staudt LM, Wilson WH (2016) Dynamic monitoring of circulating tumor DNA in non-Hodgkin lymphoma. Blood 127:3127–3132PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Berger AW, Schwerdel D, Welz H et al (2017) Treatment monitoring in metastatic colorectal cancer patients by quantification and KRAS genotyping of circulating cell-free DNA. PLoS One 12(3):e0174308. Scholar
  68. 68.
    Swisher EM, Wollan M, Mahtani SM et al (2005) Tumor-specific p53 sequences in blood and peritoneal fluid of women with epithelial ovarian cancer. Am J Obstet Gynecol 193:662–667PubMedPubMedCentralCrossRefGoogle Scholar
  69. 69.
    Thålin C, Lundström S, Seignez C et al (2018) Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer. PLoS One 13:e0191231PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Cohen JD, Javed AA, Thoburn C et al (2017) Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers. Proc Natl Acad Sci U S A 114:10202–10207PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Cohen JD, Li L, Wang Y et al (2018) Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 359:926–930PubMedPubMedCentralCrossRefGoogle Scholar
  72. 72.
    Snyder MW, Kircher M, Hill AJ et al (2016) Cell-free DNA compromises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164:57–68PubMedPubMedCentralCrossRefGoogle Scholar
  73. 73.
    Seufferlein T, Schwerdel D, Welz H et al (2017) Treatment monitoring of metastatic colorectal cancer by quantification and genotyping of mutated KRAS in circulating cell-free DNA. J Clin Oncol 35:e15037. Scholar
  74. 74.
    Chen T, He R, Hu X et al (2017) Circulating tumour DNA: a potential biomarker from solid tumors’ monitor to anticancer therapies. Cancer Transl Med 3:64–67CrossRefGoogle Scholar
  75. 75.
    Shu Y, Wu X, Tong X et al (2017) Circulating tumor DNA mutation profiling by targeted next generation sequencing provides guidance for personalized treatments in multiple cancer types. Sci Rep 7:583PubMedPubMedCentralCrossRefGoogle Scholar
  76. 76.
    Forshew T, Murtaza M, Parkinson C et al (2012) Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med 4:136ra68. Scholar
  77. 77.
    Abbosh C, Birkbak NJ, Wilson GA et al (2017) Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 545:446–451PubMedPubMedCentralCrossRefGoogle Scholar
  78. 78.
    Perets P, Greenberg O, Shenter T et al (2018) Mutant KRAS circulating tumor DNA is an accurate tool for pancreatic cancer monitoring. Oncologist 23:566–572. Scholar
  79. 79.
    Schiavon G, Hrebien S, Garcia-Murillas I et al (2015) Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer. Sci Transl Med 7:313ra182. Scholar
  80. 80.
    Mohan S, Heitzer E, Ulz P et al (2014) Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing. PLoS Genet 10:e1004271. Scholar
  81. 81.
    Thierry AR, El Messaoudi S, Mollevi C et al (2017) Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment. Ann Oncol 28:2149–2159PubMedCrossRefPubMedCentralGoogle Scholar
  82. 82.
    Diaz LA, Williams R, Wu J et al (2012) The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486:537–540PubMedPubMedCentralCrossRefGoogle Scholar
  83. 83.
    Diaz LA, Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol 32:579–586PubMedPubMedCentralCrossRefGoogle Scholar
  84. 84.
    Gerlinger M, Rowan AJ, Horswell S et al (2012) Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883–892PubMedPubMedCentralCrossRefGoogle Scholar
  85. 85.
    Misale S, Yaeger R, Hobor S et al (2012) Emergence of KRAS mutations and acquired resistance to anti EGFR therapy in colorectal cancer. Nature 486:532–536PubMedPubMedCentralCrossRefGoogle Scholar
  86. 86.
    Murtaza M, Dawson S-J, Tsui DW et al (2013) Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497:108–112PubMedCrossRefPubMedCentralGoogle Scholar
  87. 87.
    Bettegowda C, Sausen M, Leary RL et al (2014) Detection of circulating tumor DNA in early- and late-stage malignancies. Sci Trans Med 6:224ra24. Scholar
  88. 88.
    Singh AP, Li S, Cheng H (2017) Circulating DNA in EGFR-mutated lung cancer. Ann Transl Med 5:379. Scholar
  89. 89.
    Dawson S-J, Tsui DWY, Murtaza M et al (2013) Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368:1199–1209PubMedPubMedCentralCrossRefGoogle Scholar
  90. 90.
    Sirera R, Bremnes RM, Cabrera A et al (2011) Circulating DNA is a useful prognostic factor in patients with advanced non-small cell lung cancer. J Thorac Oncol 6:286–290PubMedCrossRefGoogle Scholar
  91. 91.
    Tissot C, Toffart A-C, Villar S et al (2015) Circulating free DNA concentration is an independent prognostic biomarker in lung cancer. Eur Respir J 46:1773–1780PubMedCrossRefGoogle Scholar
  92. 92.
    Sunami E, Shinozaki M, Higano CS et al (2009) Multimarker circulating DNA assay for assessing blood of prostate cancer patients. Clin Chem 55:559–567PubMedCrossRefGoogle Scholar
  93. 93.
    Singh N, Gupta S, Pandey RM et al (2015) High levels of cell-free circulating nucleic acids in pancreatic cancer are associated with vascular encasement, metastasis and poor survival. Cancer Investig 33:78–85CrossRefGoogle Scholar
  94. 94.
    Gootenberg JS, Abudayyeh OO, Kellner MJ et al (2018) Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a and Csm6. Science 360:439–444. Scholar
  95. 95.
    Gröbner SN, Worst BC, Weischenfeldt J et al (2018) The landscape of genomic alterations across childhood cancers. Nature 555:321–327PubMedCrossRefGoogle Scholar
  96. 96.
    Ma X, Yu L, Yanling L et al (2018) Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours. Nature 555:371–376PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Peter B. Gahan
    • 1
  1. 1.Fondazione Enrico PuccinelliPerugiaItaly

Personalised recommendations