Enrichment and Analysis of ctDNA

  • Pauline GilsonEmail author
Part of the Recent Results in Cancer Research book series (RECENTCANCER, volume 215)


ctDNA provided by liquid biopsy offers a promising alternative to tumor biopsy as it gives a non-invasive and «real-time» access to the cancer genome and reflects tumor intra and extra heterogeneity. ctDNA has shown growing clinical interest for cancer diagnosis, prognosis, theragnostics, therapeutic monitoring, and clonal evolution tracking. A major technical limit for ctDNA analysis from body fluids is the extremely low proportion of ctDNA compared to non-malignant cell-free DNA, underscoring the need for highly sensitive and specific detection techniques. The control of pre-analytical procedures appears essential for optimal ctDNA analysis and need to be standardized for clinical research applications. This chapter provides insights into major current technologies for ctDNA detection. Overall, PCR-based techniques are able to detect limited molecular alterations and have a high sensitivity suitable for monitoring purposes while NGS-based approaches are broad range molecular screening assays more specifically indicated for treatment selection. We briefly reviewed new technical innovations that are now available for ctDNA detection.


Circulating tumor DNA Liquid biopsy Cell-free DNA Polymerase chain reaction Next-generation sequencing 


  1. Aguado C, Giménez-Capitán A, Karachaliou N, Pérez-Rosado A, Viteri S, Morales-Espinosa D et al (2016) Fusion gene and splice variant analyses in liquid biopsies of lung cancer patients. Transl Lung Cancer Res 5(5):525–531PubMedPubMedCentralCrossRefGoogle Scholar
  2. Baker M (2012) Digital PCR hits its stride. Nat Methods 9:541–544, 30 May 2012CrossRefGoogle Scholar
  3. Basu AS (2017) Digital assays part I: partitioning statistics and digital PCR. SLAS Technol 22(4):369–386PubMedGoogle Scholar
  4. Beutler E, Gelbart T, Kuhl W (1990) Interference of heparin with the polymerase chain reaction. BioTechniques 9(2):166PubMedGoogle Scholar
  5. Bratman SV, Newman AM, Alizadeh AA, Diehn M (2015) Potential clinical utility of ultrasensitive circulating tumor DNA detection with CAPP-Seq. Expert Rev Mol Diagn 15(6):715–719PubMedPubMedCentralCrossRefGoogle Scholar
  6. Breitbach S, Tug S, Helmig S, Zahn D, Kubiak T, Michal M et al (2014) Direct quantification of cell-free, circulating DNA from unpurified plasma. PLoS One 9(3):e87838PubMedPubMedCentralCrossRefGoogle Scholar
  7. Bronkhorst AJ, Aucamp J, Pretorius PJ (2015) Cell-free DNA: preanalytical variables. Clin Chim Acta 23(450):243–253CrossRefGoogle Scholar
  8. Butler TM, Johnson-Camacho K, Peto M, Wang NJ, Macey TA, Korkola JE et al (2015) Exome sequencing of cell-free DNA from metastatic cancer patients identifies clinically actionable mutations distinct from primary disease. PloS One 10:e0136407PubMedPubMedCentralCrossRefGoogle Scholar
  9. Butler KS, Young MYL, Li Z, Elespuru RK, Wood SC (2016) Performance characteristics of the AmpliSeq cancer hotspot panel v2 in combination with the ion torrent next generation sequencing personal genome machine. Regul Toxicol Pharmacol 74:178–186PubMedCrossRefGoogle Scholar
  10. Chan KCA, Yeung S-W, Lui W-B, Rainer TH, Lo YMD (2005) Effects of preanalytical factors on the molecular size of cell-free DNA in blood. Clin Chem 51(4):781–784PubMedCrossRefGoogle Scholar
  11. Chan KCA, Jiang P, Zheng YWL, Liao GJW, Sun H, Wong J 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
  12. Cheng F, Su L, Qian C (2016) Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer. Oncotarget. 7(30):48832–48841PubMedPubMedCentralGoogle Scholar
  13. Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM (2001) Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 47(9):1607–1613PubMedGoogle Scholar
  14. Cui S, Ye L, Wang H, Chu T, Zhao Y, Gu A et al (2018) Use of superARMS EGFR mutation detection kit to detect EGFR in plasma cell-free DNA of patients with lung adenocarcinoma. Clin Lung Cancer 19(3):e313–e322PubMedCrossRefGoogle Scholar
  15. Davis AA, Chae YK, Agte S, Pan A, Iams WT, Cruz MRDS et al (2017) Association of circulating tumor DNA (ctDNA) tumor mutational burden (TMB) with DNA repair mutations and response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer (NSCLC). J Clin Oncol 35(15):11537CrossRefGoogle Scholar
  16. Dawson S-J, Tsui DWY, Murtaza M, Biggs H, Rueda OM, Chin S-F et al (2013) Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368(13):1199–1209CrossRefGoogle Scholar
  17. De Maio G, Rengucci C, Zoli W, Calistri D (2014) Circulating and stool nucleic acid analysis for colorectal cancer diagnosis. World J Gastroenterol 20(4):957–967PubMedPubMedCentralCrossRefGoogle Scholar
  18. De Mattos-Arruda L, Mayor R, Ng CKY, Weigelt B, Martínez-Ricarte F, Torrejon D et al (2015) Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat Commun 6:8839PubMedPubMedCentralCrossRefGoogle Scholar
  19. Denis JA, Guillerm E, Coulet F, Larsen AK, Lacorte J-M (2017) The role of BEAMing and digital PCR for multiplexed analysis in molecular oncology in the era of next-generation sequencing. Mol Diagn Ther 21(6):587–600PubMedCrossRefGoogle Scholar
  20. Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA 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
  21. Diaz LA, Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol Off J Am Soc Clin Oncol 32(6):579–586CrossRefGoogle Scholar
  22. Diehl F, Smergeliene E (2013) BEAMing for cancer. Genet Eng Biotechnol News 33(15):48–49CrossRefGoogle Scholar
  23. Diehl F, Li M, Dressman D, He Y, Shen D, Szabo S 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
  24. Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D (2006) BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods 3(7):551–559PubMedCrossRefPubMedCentralGoogle Scholar
  25. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M et al (2008a) Circulating mutant DNA to assess tumor dynamics. Nat Med 14(9):985–990PubMedCrossRefGoogle Scholar
  26. Diehl F, Schmidt K, Durkee KH, Moore KJ, Goodman SN, Shuber AP et al (2008b) Analysis of Mutations in DNA Isolated From Plasma and Stool of Colorectal Cancer Patients. Gastroenterology. 135(2):489–498.e7PubMedPubMedCentralCrossRefGoogle Scholar
  27. Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci 100(15):8817–8822PubMedCrossRefGoogle Scholar
  28. El Messaoudi S, Rolet F, Mouliere F, Thierry AR (2013) Circulating cell free DNA: preanalytical considerations. Clin Chim Acta Int J Clin Chem 424:222–230CrossRefGoogle Scholar
  29. Ellinger J, Bastian PJ, Haan KI, Heukamp LC, Buettner R, Fimmers R et al (2008) Noncancerous PTGS2 DNA fragments of apoptotic origin in sera of prostate cancer patients qualify as diagnostic and prognostic indicators. Int J Cancer. 122(1):138–143PubMedCrossRefGoogle Scholar
  30. Feng Q, Gai F, Sang Y, Zhang J, Wang P, Wang Y et al (2018) A comparison of QuantStudioTM 3D digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients. Cancer Manag Res 10:115–121PubMedPubMedCentralCrossRefGoogle Scholar
  31. Fleischhacker M, Schmidt B, Weickmann S, Fersching DMI, Leszinski GS, Siegele B et al (2011) Methods for isolation of cell-free plasma DNA strongly affect DNA yield. Clin Chim Acta Int J Clin Chem 412(23–24):2085–2088CrossRefGoogle Scholar
  32. Fong SL, Zhang JT, Lim CK, Eu KW, Liu Y (2009) Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients. Clin Chem 55(3):587–589PubMedCrossRefGoogle Scholar
  33. Forshew T, Murtaza M, Parkinson C, Gale D, Tsui DWY, Kaper F et al (2012) Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med 4(136):136ra68PubMedCrossRefGoogle Scholar
  34. Fredebohm J, Mehnert DH, Löber A-K, Holtrup F, van Rahden V, Angenendt P et al (2016) Detection and quantification of KIT mutations in ctDNA by Plasma Safe-SeqS. Adv Exp Med Biol 924:187–189PubMedCrossRefGoogle Scholar
  35. Gale D, Lawson ARJ, Howarth K, Madi M, Durham B, Smalley S et al (2018) Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA. PloS One 13:e0194630 PubMedPubMedCentralCrossRefGoogle Scholar
  36. García-Foncillas J, Alba E, Aranda E, Díaz-Rubio E, López-López R, Tabernero J et al (2017) Incorporating BEAMing technology as a liquid biopsy into clinical practice for the management of colorectal cancer patients: an expert taskforce review. Ann Oncol Off J Eur Soc Med Oncol 28(12):2943–2949CrossRefGoogle Scholar
  37. Gray ES, Rizos H, Reid AL, Boyd SC, Pereira MR, Lo J et al (2015) Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma. Oncotarget 6(39):42008–420018PubMedPubMedCentralCrossRefGoogle Scholar
  38. Haber DA, Velculescu VE (2014) Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA. Cancer Discov 4(6):650–661PubMedPubMedCentralCrossRefGoogle Scholar
  39. Haselmann V, Ahmad-Nejad P, Geilenkeuser WJ, Duda A, Gabor M, Eichner R et al (2018) Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA). Clin Chem Lab Med 56(2):220–228PubMedCrossRefGoogle Scholar
  40. Hauser S, Zahalka T, Fechner G, Müller SC, Ellinger J (2013) Serum DNA hypermethylation in patients with kidney cancer: results of a prospective study. Anticancer Res 33(10):4651–4656PubMedGoogle Scholar
  41. Heitzer E, Ulz P, Belic J, Gutschi S, Quehenberger F, Fischereder K 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
  42. Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 93(18):9821–9826PubMedPubMedCentralCrossRefGoogle Scholar
  43. Heyries KA, Tropini C, Vaninsberghe M, Doolin C, Petriv OI, Singhal A et al (2011) Megapixel digital PCR. Nat Methods 8(8):649–651PubMedCrossRefGoogle Scholar
  44. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 83(22):8604–8610PubMedPubMedCentralCrossRefGoogle Scholar
  45. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ et al (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10):1003–1005PubMedPubMedCentralCrossRefGoogle Scholar
  46. Hirotsu Y, Ooka Y, Sakamoto I, Nakagomi H, Omata M (2017) Simultaneous detection of genetic and copy number alterations in BRCA1/2 genes. Oncotarget 8(70):114463–114473PubMedPubMedCentralCrossRefGoogle Scholar
  47. How-Kit A, Lebbé C, Bousard A, Daunay A, Mazaleyrat N, Daviaud C et al (2014) Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen, FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR. Anal Bioanal Chem 406(22):5513–5520PubMedCrossRefGoogle Scholar
  48. Ilié M, Hofman P (2016) Pros: can tissue biopsy be replaced by liquid biopsy? Transl Lung Cancer Res 5(4):420–423PubMedPubMedCentralCrossRefGoogle Scholar
  49. Jacobs B, Claes B, Bachet J-B, Bouche O, Sablon E, Maertens GG et al (2017) Evaluation of a fully automated extended RAS-BRAF test on prospectively collected plasma samples from patients with metastatic colorectal cancer. J Clin Oncol. 35(15_suppl):e15127CrossRefGoogle Scholar
  50. Janku F, Claes B, Huang HJ, Falchook GS, Devogelaere B, Kockx M et al (2015) BRAF mutation testing with a rapid, fully integrated molecular diagnostics system. Oncotarget 6(29):26886–26894PubMedPubMedCentralCrossRefGoogle Scholar
  51. Janku F, Huang HJ, Claes B, Falchook GS, Fu S, Hong D et al (2016) 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(6):1397–1404PubMedCrossRefGoogle Scholar
  52. Jones S, Zhang X, Parsons DW, Lin JC-H, Leary RJ, Angenendt P et al (2008) Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321(5897):1801–1806PubMedPubMedCentralCrossRefGoogle Scholar
  53. Jones S, Hruban RH, Kamiyama M, Borges M, Zhang X, Parsons DW et al (2009) Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science 324(5924):217PubMedPubMedCentralCrossRefGoogle Scholar
  54. Jovelet C, Madic J, Remon J, Honoré A, Girard R, Rouleau E et al (2017) Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer. PLoS One 12(8):e0183319PubMedPubMedCentralCrossRefGoogle Scholar
  55. Jung M, Klotzek S, Lewandowski M, Fleischhacker M, Jung K (2003) Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem 49(6):1028–1029PubMedCrossRefGoogle Scholar
  56. Kaisaki PJ, Cutts A, Popitsch N, Camps C, Pentony MM, Wilson G et al (2016) Targeted next-generation sequencing of plasma DNA from cancer patients: factors influencing consistency with tumour DNA and prospective investigation of its utility for diagnosis. PloS One 11:e0162809PubMedPubMedCentralCrossRefGoogle Scholar
  57. Keppens C, Palma JF, Das PM, Scudder S, Wen W, Normanno N et al (2018) Detection of EGFR variants in plasma: a multilaboratory comparison of a real-time PCR EGFR mutation test in Europe. J Mol Diagn JMD 20(4):483–494PubMedCrossRefGoogle Scholar
  58. Kimura H, Fujiwara Y, Sone T, Kunitoh H, Tamura T, Kasahara K et al (2006) EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib. Br J Cancer 95(10):1390–1395PubMedPubMedCentralCrossRefGoogle Scholar
  59. Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B (2011) Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A 108(23):9530–9535PubMedPubMedCentralCrossRefGoogle Scholar
  60. Kinde I, Bettegowda C, Wang Y, Wu J, Agrawal N, Shih I-M et al (2013) Evaluation of DNA from the papanicolaou test to detect ovarian and endometrial cancers. Sci Transl Med 5(167):167ra4PubMedPubMedCentralCrossRefGoogle Scholar
  61. Klevebring D, Neiman M, Sundling S, Eriksson L, Darai Ramqvist E, Celebioglu F et al (2018) Evaluation of exome sequencing to estimate tumor burden in plasma. PloS One 9:e104417PubMedPubMedCentralCrossRefGoogle Scholar
  62. Kloten V, Rüchel N, Brüchle NO, Gasthaus J, Freudenmacher N, Steib F et al (2017) Liquid biopsy in colon cancer: comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR. Oncotarget 8(49):86253–86263PubMedPubMedCentralCrossRefGoogle Scholar
  63. Lam NYL, Rainer TH, Chiu RWK, Lo YMD (2004) EDTA is a better anticoagulant than heparin or citrate for delayed blood processing for plasma DNA analysis. Clin Chem 50(1):256–257PubMedCrossRefGoogle Scholar
  64. Lanman RB, Mortimer SA, Zill OA, Sebisanovic D, Lopez R, Blau S et al (2015) Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumor DNA. PLoS One 10(10):e0140712PubMedPubMedCentralCrossRefGoogle Scholar
  65. Leary RJ, Sausen M, Kinde I, Papadopoulos N, Carpten JD, Craig D et al (2012) Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Sci Transl Med 4(162):162ra154PubMedPubMedCentralCrossRefGoogle Scholar
  66. Lee TH, Montalvo L, Chrebtow V, Busch MP (2001) Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfus (Paris) 41(2):276–282CrossRefGoogle Scholar
  67. Lee C-Y, Chiu Y-C, Wang L-B, Kuo Y-L, Chuang EY, Lai L-C et al (2013) Common applications of next-generation sequencing technologies in genomic research. Transl Cancer Res 2(1):33–45Google Scholar
  68. Li M, Diehl F, Dressman D, Vogelstein B, Kinzler KW (2006) BEAMing up for detection and quantification of rare sequence variants. Nat Methods 3(2):95–97PubMedCrossRefGoogle Scholar
  69. Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14(5):579–584PubMedCrossRefGoogle Scholar
  70. Li J, Wang L, Jänne PA, Makrigiorgos GM (2009) Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR. Clin Chem 55(4):748–756PubMedPubMedCentralCrossRefGoogle Scholar
  71. Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D et al (2001) DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem 47(4):635–644PubMedGoogle Scholar
  72. Lissa D, Robles AI (2016) Methylation analyses in liquid biopsy. Transl Lung Cancer Res 5(5):492–504PubMedPubMedCentralCrossRefGoogle Scholar
  73. Little S (2001) Amplification-refractory mutation system (ARMS) analysis of point mutations. Curr Protoc Hum Genet 7(1):8–9 (Chapter 9: Unit 9.8)Google Scholar
  74. Liu Q, Sommer SS (2000) Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification. BioTechn 29(5):1072–1076 1078, 1080 passimCrossRefGoogle Scholar
  75. Liu Q, Nguyen VQ, Li X, Sommer SS (2006) Multiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions. BioTechn 40(5):661–668CrossRefGoogle Scholar
  76. Lo YM (1998) The amplification refractory mutation system. Methods Mol Med 16:61–69PubMedGoogle Scholar
  77. Loeb LA, Loeb KR, Anderson JP (2003) Multiple mutations and cancer. Proc Natl Acad Sci U S A. 100(3):776–781PubMedPubMedCentralCrossRefGoogle Scholar
  78. Lu J-L, Liang Z-Y (2016) Circulating free DNA in the era of precision oncology: Pre- and post-analytical concerns. Chronic Dis Transl Med 2(4):223–230PubMedPubMedCentralCrossRefGoogle Scholar
  79. Luca CD, Vigliar E, d’Anna M, Pisapia P, Bellevicine C, Malapelle U et al (2017) KRAS detection on archival cytological smears by the novel fully automated polymerase chain reaction-based Idylla mutation test. Cyto J 14(1):5Google Scholar
  80. Madic J, Piperno-Neumann S, Servois V, Rampanou A, Milder M, Trouiller B et al (2012) Pyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma. Clin Cancer Res Off J Am Assoc Cancer Res 18(14):3934–3941CrossRefGoogle Scholar
  81. Madic J, Zocevic A, Senlis V, Fradet E, Andre B, Muller S et al (2016) Three-color crystal digital PCR. Biomol Detect Quantif 3(10):34–46CrossRefGoogle Scholar
  82. Majewski J, Schwartzentruber J, Lalonde E, Montpetit A, Jabado N (2011) What can exome sequencing do for you? J Med Genet 48(9):580–589PubMedCrossRefGoogle Scholar
  83. Malapelle U, Pisapia P, Sgariglia R, Vigliar E, Biglietto M, Carlomagno C et al (2016) Less frequently mutated genes in colorectal cancer: evidences from next-generation sequencing of 653 routine cases. J Clin Pathol 69(9):767–771PubMedPubMedCentralCrossRefGoogle Scholar
  84. Malapelle U, Sirera R, Jantus-Lewintre E, Reclusa P, Calabuig-Fariñas S, Blasco A et al (2017) Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer. Expert Rev Mol Diagn 17(3):209–215PubMedCrossRefGoogle Scholar
  85. Manier S, Park J, Capelletti M, Bustoros M, Freeman SS, Ha G et al (2018) Whole-exome sequencing of cell-free DNA and circulating tumor cells in multiple myeloma. Nat Commun 9:1–11Google Scholar
  86. Mao L, Hruban RH, Boyle JO, Tockman M, Sidransky D (1994) Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 54(7):1634–1637PubMedGoogle Scholar
  87. Masago K, Fujita S, Hata A, Okuda C, Yoshizumi Y, Kaji R et al (2018) Validation of the digital PCR system in tyrosine kinase inhibitor-resistant EGFR mutant non-small-cell lung cancer. Pathol Int 68(3):167–173PubMedCrossRefGoogle Scholar
  88. Mauger F, Daunay A, Deleuze J-F, Tost J, How-Kit A (2016) Multiplexing of E-ice-COLD-PCR assays for mutation detection and identification. Clin Chem 62:1155–1158PubMedCrossRefGoogle Scholar
  89. Mauger F, How-Kit A, Tost J (2017) COLD-PCR technologies in the area of personalized medicine: methodology and applications. Mol Diagn Ther 21(3):269–283PubMedCrossRefGoogle Scholar
  90. Meienberg J, Zerjavic K, Keller I, Okoniewski M, Patrignani A, Ludin K et al (2015) New insights into the performance of human whole-exome capture platforms. Nucleic Acids Res 43(11):e76PubMedPubMedCentralCrossRefGoogle Scholar
  91. Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55(4):632–640PubMedPubMedCentralCrossRefGoogle Scholar
  92. Milbury CA, Li J, Liu P, Makrigiorgos GM (2011a) COLD-PCR: improving the sensitivity of molecular diagnostics assays. Expert Rev Mol Diagn 11(2):159–169PubMedPubMedCentralCrossRefGoogle Scholar
  93. Milbury CA, Li J, Makrigiorgos GM (2011b) Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucl Acids Res 39(1):e2PubMedCrossRefGoogle Scholar
  94. Mouliere F, El Messaoudi S, Gongora C, Guedj A-S, Robert B, Del Rio M et al (2013) Circulating cell-free DNA from colorectal cancer patients may reveal high KRAS or BRAF mutation load. Transl Oncol 6(3):319–328PubMedPubMedCentralCrossRefGoogle Scholar
  95. Mouliere F, El Messaoudi S, Pang D, Dritschilo A, Thierry AR (2014) Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer. Mol Oncol 8(5):927–941PubMedPubMedCentralCrossRefGoogle Scholar
  96. Murtaza M, Dawson S-J, Tsui DWY, Gale D, Forshew T, Piskorz AM et al (2013) Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497(7447):108–112CrossRefGoogle Scholar
  97. Newman AM, Bratman SV, To J, Wynne JF, Eclov NCW, Modlin LA et al (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med 20(5):548–554PubMedPubMedCentralCrossRefGoogle Scholar
  98. Newman AM, Lovejoy AF, Klass DM, Kurtz DM, Chabon JJ, Scherer F et al (2016) Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol 34(5):547–555PubMedPubMedCentralCrossRefGoogle Scholar
  99. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N et al (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucl Acids Res 17(7):2503–2516PubMedCrossRefPubMedCentralGoogle Scholar
  100. Ng EKO, Leung CPH, Shin VY, Wong CLP, Ma ESK, Jin HC et al (2011) Quantitative analysis and diagnostic significance of methylated SLC19A3 DNA in the plasma of breast and gastric cancer patients. PLoS One 6(7):e22233PubMedPubMedCentralCrossRefGoogle Scholar
  101. Nikolaev S, Lemmens L, Koessler T, Blouin J-L, Nouspikel T (2018) Circulating tumoral DNA: Preanalytical validation and quality control in a diagnostic laboratory. Anal Biochem 542:34–39PubMedCrossRefPubMedCentralGoogle Scholar
  102. Parsons DW, Jones S, Zhang X, Lin JC-H, Leary RJ, Angenendt P et al (2008) An integrated genomic analysis of human glioblastoma multiforme. Science 321(5897):1807–1812PubMedPubMedCentralCrossRefGoogle Scholar
  103. Pécuchet N, Rozenholc Y, Zonta E, Pietrasz D, Didelot A, Combe P et al (2016) Analysis of base-position error rate of next-generation sequencing to detect tumor mutations in circulating DNA. Clin Chem 62(11):1492–1503PubMedCrossRefGoogle Scholar
  104. Pekin D, Skhiri Y, Baret J-C, Le Corre D, Mazutis L, Salem CB et al (2011) Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip. 11(13):2156–2166PubMedCrossRefPubMedCentralGoogle Scholar
  105. Perakis S, Auer M, Belic J, Heitzer E (2017) Advances in circulating tumor DNA analysis. Adv Clin Chem 80:73–153Google Scholar
  106. Perez-Toralla K, Pekin D, Bartolo J-F, Garlan F, Nizard P, Laurent-Puig P et al (2015) PCR digitale en micro-compartiments—I. Détection sensible de séquences d’acides nucléiques rares. médecine. Sciences 31(1):84–92Google Scholar
  107. Perkins G, Lu H, Garlan F, Taly V (2017) Droplet-based digital PCR: application in cancer research. Adv Clin Chem 79:43–91Google Scholar
  108. Phallen J, Sausen M, Adleff V, Leal A, Hruban C, White J et al (2017) Direct detection of early-stage cancers using circulating tumor DNA. Sci Transl Med 9(403):eaan2415PubMedPubMedCentralCrossRefGoogle Scholar
  109. Rabbani B, Tekin M, Mahdieh N (2014) The promise of whole-exome sequencing in medical genetics. J Hum Genet 59(1):5–15PubMedCrossRefGoogle Scholar
  110. Reckamp KL, Melnikova VO, Karlovich C, Sequist LV, Camidge DR, Wakelee H, Perol M, Oxnard GR, Kosco K, Croucher P et al (2016) A highly sensitive and quantitative test platform for detection of NSCLC EGFR mutations in urine and plasma. J Thorac Oncol Off Publ Int Assoc Study Lung Cancer 11:1690–1700PubMedCrossRefGoogle Scholar
  111. Rothé F, Laes J-F, Lambrechts D, Smeets D, Vincent D, Maetens M et al (2014) Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer. Ann Oncol Off J Eur Soc Med Oncol 25(10):1959–1965CrossRefGoogle Scholar
  112. Sanmamed MF, Fernández-Landázuri S, Rodríguez C, Zárate R, Lozano MD, Zubiri L 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
  113. Sasaki M, Anast J, Bassett W, Kawakami T, Sakuragi N, Dahiya R (2003) Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation. Biochem Biophys Res Commun 309(2):305–309PubMedCrossRefGoogle Scholar
  114. Schmiegel W, Scott RJ, Dooley S, Lewis W, Meldrum CJ, Pockney P et al (2017) Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing. Mol Oncol 11(2):208–219PubMedPubMedCentralCrossRefGoogle Scholar
  115. Schwarze K, Buchanan J, Taylor JC, Wordsworth S (2018) Are whole-exome and whole-genome sequencing approaches cost-effective? A systematic review of the literature. Genet Med Off J Am Coll Med Genet 20:1122–1130PubMedCrossRefGoogle Scholar
  116. Siravegna G, Marsoni S, Siena S, Bardelli A (2017) Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol 14(9):531–548PubMedPubMedCentralCrossRefGoogle Scholar
  117. Sjöblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD et al (2006) The consensus coding sequences of human breast and colorectal cancers. Science 314(5797):268–274PubMedCrossRefGoogle Scholar
  118. Spindler K-LG, Pallisgaard N, Vogelius I, Jakobsen A (2012) Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clin Cancer Res 18(4):1177–1185PubMedCrossRefGoogle Scholar
  119. Stroun M, Lyautey J, Lederrey C, Olson-Sand A, Anker P (2001) About the possible origin and mechanism of circulating DNA apoptosis and active DNA release. Clin Chim Acta Int J Clin Chem 313(1–2):139–142CrossRefGoogle Scholar
  120. Swinkels DW, Wiegerinck E, Steegers EAP, de Kok JB (2003) Effects of blood-processing protocols on cell-free DNA quantification in Plasma. Clin Chem 49(3):525–526PubMedCrossRefGoogle Scholar
  121. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA (1992) Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13(3):444–449Google Scholar
  122. Takai E, Totoki Y, Nakamura H, Morizane C, Nara S, Hama N et al (2015) Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer. Sci Rep 5:18425PubMedPubMedCentralCrossRefGoogle Scholar
  123. Taly V, Pekin D, Benhaim L, Kotsopoulos SK, Corre DL, Li X et al (2013) Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem 59(12):1722–1731PubMedCrossRefGoogle Scholar
  124. Thierry AR (2016) A targeted Q-PCR-based method for point mutation testing by analyzing circulating DNA for cancer management care. Methods Mol Biol Clifton NJ 1392:1–16Google Scholar
  125. Thierry AR, Mouliere F, Messaoudi SE, Mollevi C, Lopez-Crapez E, Rolet F et al (2014) Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med 20(4):430–435PubMedPubMedCentralCrossRefGoogle Scholar
  126. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M (2016) Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev 35(3):347–376PubMedPubMedCentralCrossRefGoogle Scholar
  127. Tie J, Kinde I, Wang Y, Wong HL, Roebert J, Christie M et al (2015) Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. Ann Oncol Off J Eur Soc Med Oncol 26(8):1715–1722CrossRefGoogle Scholar
  128. Tost J (2016) The clinical potential of enhanced-ice-COLD-PCR. Expert Rev Mol Diagn 16(3):265–268PubMedCrossRefGoogle Scholar
  129. Tsao SC-H, Weiss J, Hudson C, Christophi C, Cebon J, Behren A et al (2015) Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations. Sci Rep 5:11198PubMedCrossRefGoogle Scholar
  130. Vallée A, Marcq M, Bizieux A, Kouri CE, Lacroix H, Bennouna J et al (2013) Plasma is a better source of tumor-derived circulating cell-free DNA than serum for the detection of EGFR alterations in lung tumor patients. Lung Cancer Amst Neth 82(2):373–374CrossRefGoogle Scholar
  131. Vidal J, Muinelo L, Dalmases A, Jones F, Edelstein D, Iglesias M et al (2017) Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients. Ann Oncol Off J Eur Soc Med Oncol 28(6):1325–1332CrossRefGoogle Scholar
  132. Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96(16):9236–9241PubMedPubMedCentralCrossRefGoogle Scholar
  133. Wang Y, Springer S, Mulvey CL, Silliman N, Schaefer J, Sausen M et al (2015) Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med. 7(293):293ra104PubMedPubMedCentralCrossRefGoogle Scholar
  134. Wang Y, Li L, Han R, Jiao L, Zheng J, He Y (2018) Clinical analysis by next-generation sequencing for NSCLC patients with MET amplification resistant to osimertinib. Lung Cancer Amst Neth 118:105–110CrossRefGoogle Scholar
  135. Warr A, Robert C, Hume D, Archibald A, Deeb N, Watson M (2015) Exome sequencing: current and future perspectives. G3 Genes Genom Genet 5(8):1543–1550Google Scholar
  136. Warton K, Lin V, Navin T, Armstrong NJ, Kaplan W, Ying K et al (2014) Methylation-capture and next-generation sequencing of free circulating DNA from human plasma. BMC Genom 15:476CrossRefGoogle Scholar
  137. Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S et al (2012) Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res. 40(11):e82PubMedPubMedCentralCrossRefGoogle Scholar
  138. Wielscher M, Vierlinger K, Kegler U, Ziesche R, Gsur A, Weinhäusel A (2015) Diagnostic performance of plasma DNA methylation profiles in lung cancer, pulmonary fibrosis and COPD. EBioMedicine 2(8):929–936PubMedPubMedCentralCrossRefGoogle Scholar
  139. Yi X, Ma J, Guan Y, Chen R, Yang L, Xia X (2017) The feasibility of using mutation detection in ctDNA to assess tumor dynamics. Int J Cancer 140(12):2642–2647PubMedPubMedCentralCrossRefGoogle Scholar
  140. Yu Q, Yu Q, Huang F, Huang F, Zhang M, Zhang M et al (2017) Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients. Mol Med Rep. 16(2):1157–1166PubMedPubMedCentralCrossRefGoogle Scholar
  141. Yung TKF, Chan KCA, Mok TSK, Tong J, To K-F, Lo YMD (2009) 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 Off J Am Assoc Cancer Res 15(6):2076–2084CrossRefGoogle Scholar
  142. Zhong Q, Bhattacharya S, Kotsopoulos S, Olson J, Taly V, Griffiths AD et al (2011) Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Lab Chip. 11(13):2167–2174PubMedCrossRefGoogle Scholar

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© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Université de Lorraine, CNRS UMR 7039 CRAN, Institut de Cancérologie de Lorraine, Service de BiopathologieNancyFrance

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