Analysis of Circulating Tumor DNA

  • Sridurga Mithraprabhu
  • Andrew Spencer
Part of the Methods in Molecular Biology book series (MIMB, volume 1792)


Circulating tumor DNA (ctDNA) analysis is currently gaining momentum as an innovative methodology for characterizing the tumor genome and monitoring therapeutic efficacy in the multifocal, genetically and spatially heterogeneous plasma cell malignancy, multiple myeloma (MM). Circulating cell-free DNA (cfDNA), which consists of a combination of DNA derived from both tumor and normal cells, is present in extracellular bodily fluids. The presence of ctDNA within this admixture has been demonstrated recently in MM. In this chapter, we describe the routinely utilized methodology for the extraction and longitudinal analysis of specific mutations present in ctDNA derived from peripheral blood plasma of MM patients.

Key words

Circulating tumor DNA Circulating cell-free nucleic acids Cell-free DNA Multiple myeloma Peripheral blood plasma Droplet digital PCR Liquid biopsy 


  1. 1.
    Mandel P, Metais P (1948) Les acides nucléiques du plasma sanguin chez l'homme. C R Seances Soc Biol Fil 142(3–4):241–243PubMedGoogle Scholar
  2. 2.
    Chen XQ et al (1996) Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 2(9):1033–1035PubMedCrossRefGoogle Scholar
  3. 3.
    Vasioukhin V et al (1994) Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br J Haematol 86(4):774–779PubMedCrossRefGoogle Scholar
  4. 4.
    Chan KC et al (2013) 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(1):211–224PubMedCrossRefGoogle Scholar
  5. 5.
    Forshew T et al (2012) Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med 4(136):136–168CrossRefGoogle Scholar
  6. 6.
    Heitzer E et al (2013) Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing. Genome Med 5(4):30PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Leary RJ et al (2012) Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Sci Transl Med 4(162):162–154CrossRefGoogle Scholar
  8. 8.
    Murtaza M et al (2013) Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497(7447):108–112PubMedCrossRefGoogle Scholar
  9. 9.
    Plagnol V et al. (2015) Assessment of clinical applications of circulating tumor DNA using an enhanced TAm-Seq platform. J Clin Oncol. 33(15)Google Scholar
  10. 10.
    Kidess E et al (2015) 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(4):2549–2561PubMedCrossRefGoogle Scholar
  11. 11.
    Demetri GD et al (2013) Mutational analysis of plasma DNA from patients (pts) in the phase III GRID study of regorafenib (REC) versus placebo (PL) in tyrosine kinase inhibitor (TKI)-refractory GIST: correlating genotype with clinical outcomes. J Clin Oncol 31(15):10503CrossRefGoogle Scholar
  12. 12.
    Demetri GD, et al. (2013) Detection of oncogenic kinase mutations in circulating plasma DNA and correlation with clinical benefit in the phase III GRID study of regorafenib vs placebo in TKI-refractory metastatic GIST. Cancer Res. 73(8)Google Scholar
  13. 13.
    Dawson SJ et al (2013) Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368(13):1199–1209PubMedCrossRefGoogle Scholar
  14. 14.
    Tsao SC et al (2015) Monitoring response to therapy in melanoma by quantifying circulating tumor DNA with droplet digital PCR for BRAF and NRAS mutations. Sci Rep 5:11198PubMedCrossRefGoogle Scholar
  15. 15.
    Olsson E et al (2015) Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. EMBO Mol Med 7(8):1034–1047PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Oxnard GR et al (2014) 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(6):1698–1705PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Mithraprabhu S et al (2017) Circulating tumor DNA analysis demonstrates spatial mutational heterogeneity that coincides with disease relapse in myeloma. Leukemia 31(8):1695–1705PubMedCrossRefGoogle Scholar
  18. 18.
    Sanmamed MF et al (2015) Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors. Clin Chem 61(1):297–304PubMedCrossRefGoogle Scholar
  19. 19.
    Kis O et al (2017) Circulating tumor DNA sequence analysis as an alternative to multiple myeloma bone marrow aspirates. Nat Commun 8:15086PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Rustad EH et al (2017) Monitoring multiple myeloma by quantification of recurrent mutations in serum. Haematologica 102(7):1266–1272PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Oberle A et al (2017) Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA. Haematologica 102(6):1105–1111PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Jen J, Wu L, Sidransky D (2000) An overview on the isolation and analysis of circulating tumor DNA in plasma and serum. Ann N Y Acad Sci 906:8–12PubMedCrossRefGoogle Scholar
  23. 23.
    Jung M et al (2003) Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem 49(6 Pt 1):1028–1029PubMedCrossRefGoogle Scholar
  24. 24.
    Lee TH et al (2001) Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfusion 41(2):276–282PubMedCrossRefGoogle Scholar
  25. 25.
    Umetani N, Hiramatsu S, Hoon DS (2006) Higher amount of free circulating DNA in serum than in plasma is not mainly caused by contaminated extraneous DNA during separation. Ann N Y Acad Sci 1075:299–307PubMedCrossRefGoogle Scholar
  26. 26.
    Ulz P, Auer M, Heitzer E (2016) Detection of circulating tumor DNA in the blood of cancer patients: an important tool in cancer chemoprevention. Methods Mol Biol 1379:45–68PubMedCrossRefGoogle Scholar
  27. 27.
    Norton SE et al (2013) 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(15):1561–1565PubMedCrossRefGoogle Scholar
  28. 28.
    Norton SE et al (2013) A new blood collection device minimizes cellular DNA release during sample storage and shipping when compared to a standard device. J Clin Lab Anal 27(4):305–311PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Fong SL et al (2009) Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients. Clin Chem 55(3):587–589PubMedCrossRefGoogle Scholar
  30. 30.
    Devonshire AS et al (2014) Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem 406(26):6499–6512PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Mauger F et al (2015) Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma. Anal Bioanal Chem 407(22):6873–6878PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sridurga Mithraprabhu
    • 1
    • 2
  • Andrew Spencer
    • 1
    • 2
    • 3
  1. 1.Myeloma Research Group, Australian Centre for Blood Diseases, Alfred Hospital—Monash UniversityMelbourneAustralia
  2. 2.Malignant Haematology and Stem Cell TransplantationAlfred HospitalMelbourneAustralia
  3. 3.Department of Clinical HaematologyMonash UniversityMelbourneAustralia

Personalised recommendations