Lymphoma pp 383-435 | Cite as

Ultrasensitive Detection of Circulating Tumor DNA in Lymphoma via Targeted Hybridization Capture and Deep Sequencing of Barcoded Libraries

  • Miguel Alcaide
  • Christopher Rushton
  • Ryan D. MorinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1956)


Liquid biopsies are rapidly emerging as powerful tools for the early detection of cancer, noninvasive genomic profiling of localized or metastatic tumors, prompt detection of treatment resistance-associated mutations, and monitoring of therapeutic response and minimal residual disease in patients during clinical follow-up. Growing evidence strongly supports the utility of circulating tumor DNA (ctDNA) as a biomarker for the stratification and clinical management of lymphoma patients. However, ctDNA is diluted by variable amounts of cell-free DNA (cfDNA) shed by nonneoplastic cells causing a background signal of wild-type DNA that limits the sensitivity of methods that rely on DNA sequencing. Here, we describe an error suppression method for single-molecule counting that relies on targeted sequencing of cfDNA libraries constructed with semi-degenerate barcode adapters. Custom pools of biotinylated DNA baits for target enrichment can be designed to specifically track somatic mutations in one patient, survey mutation hotspots with diagnostic and prognostic value or be comprised of comprehensive gene panels with broad patient coverage in lymphoma. Such methods are amenable to track ctDNA levels during longitudinal liquid biopsy testing with high specificity and sensitivity and characterize, in real time, the genetic profiles of tumors without the need of standard invasive biopsies. The analysis of ultra-deep sequencing data according to the bioinformatics pipelines also described in this chapter affords to harness lower limits of detection for ctDNA below 0.1%.

Key words

Liquid biopsy Noninvasive genetic profiling Mutation detection Cell-free DNA Minimal residual disease Therapeutic response Tumor burden Targeted enrichment Duplex sequencing DNA damage 


  1. 1.
    Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 10:472–484CrossRefGoogle Scholar
  2. 2.
    Heitzer E, Ulz P, Geigl JB (2015) Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem 61:112–123. Scholar
  3. 3.
    Wan JCM, Massie C, Garcia-Corbacho J et al (2017) Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 17:223–238. Scholar
  4. 4.
    Siravegna G, Marsoni S, Siena S, Bardelli A (2017) Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol 14:531–548. Scholar
  5. 5.
    Dominguez-Vigil IG, Moreno-Martinez AK, Wang JY et al (2018) The dawn of the liquid biopsy in the fight against cancer. Oncotarget 9:2912–2922. Scholar
  6. 6.
    Stewart CM, Kothari PD, Mouliere F et al (2018) The value of cell-free DNA for molecular pathology. J Pathol 244:616–627. Scholar
  7. 7.
    Volik S, Alcaide M, Morin RD, Collins CC (2016) Cell-free DNA (cfDNA): clinical significance and utility in cancer shaped by emerging technologies. Mol Cancer Res 14:898–908. Scholar
  8. 8.
    Volckmar A-L, Sultmann H, Riediger A et al (2018) A field guide for cancer diagnostics using cell-free DNA: from principles to practice and clinical applications. Genes Chromosomes Cancer 57:123–139. Scholar
  9. 9.
    Jahr S, Hentze H, Englisch S et al (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61:1659–1665PubMedGoogle Scholar
  10. 10.
    Schwarzenbach H (2013) Circulating nucleic acids as biomarkers in breast cancer. Breast Cancer Res 15:211. Scholar
  11. 11.
    Kahlert C, Melo SA, Protopopov A et al (2014) 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 289:3869–3875. Scholar
  12. 12.
    Allenson K, Castillo J, San Lucas FA et al (2017) High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients. Ann Oncol 28:741–747. Scholar
  13. 13.
    Wang W, Kong P, Ma G et al (2017) Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget 8:43180–43191. Scholar
  14. 14.
    Bronkhorst AJ, Wentzel JF, Aucamp J et al (2016) Characterization of the cell-free DNA released by cultured cancer cells. Biochim Biophys Acta 1863:157–165. Scholar
  15. 15.
    Snyder MW, Kircher M, Hill AJ et al (2016) Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164:57–68. Scholar
  16. 16.
    Zhivotosky B, Orrenius S (2001) Assessment of apoptosis and necrosis by DNA fragmentation and morphological criteria. Curr Protoc Cell Biol Chapter 18:Unit 18.3. doi:
  17. 17.
    Campos CDM, Jackson JM, Witek MA, Soper SA (2018) Molecular profiling of liquid biopsy samples for precision medicine. Cancer J 24:93–103. Scholar
  18. 18.
    Rodda AE, Parker BJ, Spencer A, Corrie SR (2018) Extending circulating tumor DNA analysis to ultralow abundance mutations: techniques and challenges. ACS Sensors 3:540–560. Scholar
  19. 19.
    Mouliere F, Rosenfeld N (2015) Circulating tumor-derived DNA is shorter than somatic DNA in plasma. Proc Natl Acad Sci U S A 112:3178–3179. Scholar
  20. 20.
    Underhill HR, Kitzman JO, Hellwig S et al (2016) Fragment length of circulating tumor DNA. PLoS Genet 12:e1006162. Scholar
  21. 21.
    Peng M, Chen C, Hulbert A et al (2017) Non-blood circulating tumor DNA detection in cancer. Oncotarget 8:69162–69173. Scholar
  22. 22.
    Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775:181–232PubMedGoogle Scholar
  23. 23.
    Lo YM, Zhang J, Leung TN et al (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64:218–224CrossRefGoogle Scholar
  24. 24.
    Bettegowda C, Sausen M, Leary RJ et al (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6:224ra24. Scholar
  25. 25.
    Phallen J, Sausen M, Adleff V et al (2017) Direct detection of early-stage cancers using circulating tumor DNA. Sci Transl Med 9.
  26. 26.
    Burgener JM, Rostami A, De Carvalho DD, Bratman SV (2017) Cell-free DNA as a post-treatment surveillance strategy: current status. Semin Oncol 44:330–346. Scholar
  27. 27.
    De Mattos-Arruda L, Mayor R, Ng CKY et al (2015) Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat Commun 6:8839CrossRefGoogle Scholar
  28. 28.
    Wang Y, Springer S, Zhang M et al (2015) Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci 112:9704 LP–9709709CrossRefGoogle Scholar
  29. 29.
    Abbosh C, Birkbak NJ, Wilson GA et al (2017) Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 545:446–451. Scholar
  30. 30.
    Newman AM, Bratman SV, To J et al (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med 20:548–554. Scholar
  31. 31.
    Riediger AL, Dietz S, Schirmer U et al (2016) Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients. Sci Rep 6:33505. Scholar
  32. 32.
    Sloan DB, Broz AK, Sharbrough J, Wu Z (2018) Detecting rare mutations and DNA damage with sequencing-based methods. Trends Biotechnol 36:729–740. Scholar
  33. 33.
    Salk JJ, Schmitt MW, Loeb LA (2018) Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations. Nat Rev Genet 19:269–285. Scholar
  34. 34.
    Olmedillas-López S, García-Arranz M, García-Olmo D (2017) Current and emerging applications of droplet digital PCR in oncology. Mol Diagn Ther 21:493–510. Scholar
  35. 35.
    Camus V, Bohers E, Dubois S et al (2018) Circulating tumor DNA: an important tool in precision medicine for lymphoma. Expert Rev Precis Med Drug Dev 3:11–21. Scholar
  36. 36.
    Scherer F, Kurtz DM, Diehn M, Alizadeh AA (2017) High-throughput sequencing for noninvasive disease detection in hematologic malignancies. Blood 130:440–452. Scholar
  37. 37.
    Hiemcke-Jiwa LS, Minnema MC, Radersma-van Loon JH et al (2017) The use of droplet digital PCR in liquid biopsies: a highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid. Hematol Oncol 36:429–435. Scholar
  38. 38.
    Chase ML, Armand P (2018) Minimal residual disease in non-Hodgkin lymphoma—current applications and future directions. Br J Haematol 180:177–188. Scholar
  39. 39.
    Camus V, Jardin F, Tilly H (2017) The value of liquid biopsy in diagnosis and monitoring of diffuse large b-cell lymphoma: recent developments and future potential. Expert Rev Mol Diagn 17:557–566. Scholar
  40. 40.
    Assouline SE, Nielsen TH, Yu S et al (2016) Phase 2 study of panobinostat with or without rituximab in relapsed diffuse large B-cell lymphoma. Blood 128:185–194CrossRefGoogle Scholar
  41. 41.
    Scherer F, Kurtz DM, Newman AM et al (2016) Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Transl Med 8:364ra155. Scholar
  42. 42.
    Kurtz DM, Green MR, Bratman SV et al (2015) Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin high-throughput sequencing. Blood 125:3679–3687CrossRefGoogle Scholar
  43. 43.
    Roschewski M, Staudt LM, Wilson WH (2016) Dynamic monitoring of circulating tumor DNA in non-Hodgkin lymphoma. Blood 127:3127–3132. Scholar
  44. 44.
    Schmitt MW, Kennedy SR, Salk JJ et al (2012) Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A 109:14508–14513. Scholar
  45. 45.
    Kennedy SR, Schmitt MW, Fox EJ et al (2014) Detecting ultralow-frequency mutations by Duplex Sequencing. Nat Protoc 9:2586–2606. Scholar
  46. 46.
    Alcaide M, Yu S, Davidson J et al (2017) Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits. Sci Rep 7:10574. Scholar
  47. 47.
    Lavoie J-M, Alcaide M, Fisher RA et al (2017) Targeted error-suppressed detection of circulating paternal DNA to establish a diagnosis of gestational trophoblastic neoplasm. JCO Precis Oncol:1–6.
  48. 48.
    Alcaide M, Yu S, Bushell K et al (2016) Multiplex droplet digital PCR quantification of recurrent somatic mutations in diffuse large B-cell and follicular lymphoma. Clin Chem 62:1238–1247. Scholar
  49. 49.
    Gordon LI (2016) Precision monitoring by next-generation sequencing in lymphoma: circulating tumor DNA as a new biomarker. Oncology (Williston Park) 30:745–746Google Scholar
  50. 50.
    Spina V, Bruscaggin A, Cuccaro A et al (2018) Circulating tumor DNA reveals genetics, clonal evolution and residual disease in classical Hodgkin lymphoma. Blood 131:2413–2425. Scholar
  51. 51.
    Arthur SE, Jiang A, Grande BM et al (2018) Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma. Nat Commun 9:4001.
  52. 52.
    Hung SS, Meissner B, Chavez EA et al (2018) Assessment of capture and amplicon-based approaches for the development of a targeted next-generation sequencing pipeline to personalize lymphoma management. J Mol Diagn 20:203–214. Scholar
  53. 53.
    Norton SE, Luna KK, Lechner JM 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 27:305–311. Scholar
  54. 54.
    Medina Diaz I, Nocon A, Mehnert DH et al (2016) Performance of streck cfDNA blood collection tubes for liquid biopsy testing. PLoS One 11:e0166354. Scholar
  55. 55.
    Board RE, Williams VS, Knight L et al (2008) Isolation and extraction of circulating tumor DNA from patients with small cell lung cancer. Ann N Y Acad Sci 1137:98–107. Scholar
  56. 56.
    Newman AM, Lovejoy AF, Klass DM et al (2016) Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol 34:547–555CrossRefGoogle Scholar
  57. 57.
    MacConaill LE, Burns RT, Nag A et al (2018) Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC Genomics 19:30. Scholar
  58. 58.
    Li YS, Jiang BY, Yang JJ et al (2018) Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small cell lung cancer: a new medium of liquid biopsy. Ann Oncol 29:945–952. Scholar
  59. 59.
    Maggi EC, Gravina S, Cheng H et al (2018) Development of a method to implement whole-genome bisulfite sequencing of cfDNA from cancer patients and a mouse tumor model. Front Genet 9:6. Scholar
  60. 60.
    Mussolin L, Burnelli R, Pillon M et al (2013) Plasma cell-free DNA in paediatric lymphomas. J Cancer 4:323–329. Scholar
  61. 61.
    Primerano S, Burnelli R, Carraro E et al (2016) Kinetics of circulating plasma cell-free DNA in paediatric classical Hodgkin lymphoma. J Cancer 7:364–366. Scholar
  62. 62.
    Sozzi G, Roz L, Conte D et al (2005) Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays. J Natl Cancer Inst 97:1848–1850. Scholar
  63. 63.
    Schmitt MW, Fox EJ, Prindle MJ et al (2015) Sequencing small genomic targets with high efficiency and extreme accuracy. Nat Methods 12:423–425. Scholar
  64. 64.
    Stoler N, Arbeithuber B, Guiblet W et al (2016) Streamlined analysis of duplex sequencing data with Du Novo. Genome Biol 17:180. Scholar

Copyright information

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

Authors and Affiliations

  • Miguel Alcaide
    • 1
  • Christopher Rushton
    • 1
  • Ryan D. Morin
    • 1
    Email author
  1. 1.Department of Molecular Biology and BiochemistrySimon Fraser UniversityBurnabyCanada

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