Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Lymphoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1956))

Abstract

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%.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 10:472–484

    Article  CAS  Google Scholar 

  2. Heitzer E, Ulz P, Geigl JB (2015) Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem 61:112–123. https://doi.org/10.1373/clinchem.2014.222679

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1038/nrc.2017.7

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1038/nrclinonc.2017.14

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.18632/oncotarget.23131

    Article  PubMed  Google Scholar 

  6. Stewart CM, Kothari PD, Mouliere F et al (2018) The value of cell-free DNA for molecular pathology. J Pathol 244:616–627. https://doi.org/10.1002/path.5048

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1158/1541-7786.MCR-16-0044

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1002/gcc.22517

    Article  CAS  PubMed  Google Scholar 

  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 61:1659–1665

    CAS  PubMed  Google Scholar 

  10. Schwarzenbach H (2013) Circulating nucleic acids as biomarkers in breast cancer. Breast Cancer Res 15:211. https://doi.org/10.1186/bcr3446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1074/jbc.C113.532267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1093/annonc/mdx004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.18632/oncotarget.17858

    Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1016/j.bbamcr.2015.10.022

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1016/j.cell.2015.11.050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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: https://doi.org/10.1002/0471143030.cb1803s12

  17. Campos CDM, Jackson JM, Witek MA, Soper SA (2018) Molecular profiling of liquid biopsy samples for precision medicine. Cancer J 24:93–103. https://doi.org/10.1097/PPO.0000000000000311

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1021/acssensors.7b00953

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1073/pnas.1501321112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Underhill HR, Kitzman JO, Hellwig S et al (2016) Fragment length of circulating tumor DNA. PLoS Genet 12:e1006162. https://doi.org/10.1371/journal.pgen.1006162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Peng M, Chen C, Hulbert A et al (2017) Non-blood circulating tumor DNA detection in cancer. Oncotarget 8:69162–69173. https://doi.org/10.18632/oncotarget.19942

    Article  PubMed  PubMed Central  Google Scholar 

  22. Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775:181–232

    CAS  PubMed  Google Scholar 

  23. Lo YM, Zhang J, Leung TN et al (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64:218–224

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1126/scitranslmed.3007094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Phallen J, Sausen M, Adleff V et al (2017) Direct detection of early-stage cancers using circulating tumor DNA. Sci Transl Med 9. https://doi.org/10.1126/scitranslmed.aan2415

  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. https://doi.org/10.1053/j.seminoncol.2018.01.009

    Article  PubMed  Google Scholar 

  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 6:8839

    Article  Google Scholar 

  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–9709709

    Article  Google Scholar 

  29. Abbosh C, Birkbak NJ, Wilson GA et al (2017) Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 545:446–451. https://doi.org/10.1038/nature22364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1038/nm.3519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1038/srep33505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1016/j.tibtech.2018.02.009

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1038/nrg.2017.117

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1007/s40291-017-0278-8

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1080/23808993.2018.1412798

    Article  Google Scholar 

  36. Scherer F, Kurtz DM, Diehn M, Alizadeh AA (2017) High-throughput sequencing for noninvasive disease detection in hematologic malignancies. Blood 130:440–452. https://doi.org/10.1182/blood-2017-03-735639

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1002/hon.2489

    Article  CAS  PubMed  Google Scholar 

  38. Chase ML, Armand P (2018) Minimal residual disease in non-Hodgkin lymphoma—current applications and future directions. Br J Haematol 180:177–188. https://doi.org/10.1111/bjh.14996

    Article  PubMed  Google Scholar 

  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. https://doi.org/10.1080/14737159.2017.1319765

    Article  CAS  PubMed  Google Scholar 

  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 128:185–194

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1126/scitranslmed.aai8545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kurtz DM, Green MR, Bratman SV et al (2015) Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin high-throughput sequencing. Blood 125:3679–3687

    Article  CAS  Google Scholar 

  43. Roschewski M, Staudt LM, Wilson WH (2016) Dynamic monitoring of circulating tumor DNA in non-Hodgkin lymphoma. Blood 127:3127–3132. https://doi.org/10.1182/blood-2016-03-635219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1073/pnas.1208715109

    Article  PubMed  PubMed Central  Google Scholar 

  45. Kennedy SR, Schmitt MW, Fox EJ et al (2014) Detecting ultralow-frequency mutations by Duplex Sequencing. Nat Protoc 9:2586–2606. https://doi.org/10.1038/nprot.2014.170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1038/s41598-017-10269-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1200/PO.17.00154

  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. https://doi.org/10.1373/clinchem.2016.255315

    Article  CAS  PubMed  Google Scholar 

  49. Gordon LI (2016) Precision monitoring by next-generation sequencing in lymphoma: circulating tumor DNA as a new biomarker. Oncology (Williston Park) 30:745–746

    Google Scholar 

  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. https://doi.org/10.1182/blood-2017-11-812073

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1038/s41467-018-06354-3

  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. https://doi.org/10.1016/j.jmoldx.2017.11.010

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1002/jcla.21603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1371/journal.pone.0166354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1196/annals.1448.020

    Article  CAS  PubMed  Google Scholar 

  56. Newman AM, Lovejoy AF, Klass DM et al (2016) Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol 34:547–555

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1186/s12864-017-4428-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1093/annonc/mdy009

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.3389/fgene.2018.00006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Mussolin L, Burnelli R, Pillon M et al (2013) Plasma cell-free DNA in paediatric lymphomas. J Cancer 4:323–329. https://doi.org/10.7150/jca.6226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.7150/jca.13593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1093/jnci/dji432

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1038/nmeth.3351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Stoler N, Arbeithuber B, Guiblet W et al (2016) Streamlined analysis of duplex sequencing data with Du Novo. Genome Biol 17:180. https://doi.org/10.1186/s13059-016-1039-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada

    Miguel Alcaide, Christopher Rushton & Ryan D. Morin

Authors
  1. Miguel Alcaide
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher Rushton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryan D. Morin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ryan D. Morin .

Editor information

Editors and Affiliations

  1. Institute of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Nordrhein-Westfalen, Essen, Germany

    Ralf Küppers

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Alcaide, M., Rushton, C., Morin, R.D. (2019). Ultrasensitive Detection of Circulating Tumor DNA in Lymphoma via Targeted Hybridization Capture and Deep Sequencing of Barcoded Libraries. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 1956. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9151-8_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9151-8_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9150-1

  • Online ISBN: 978-1-4939-9151-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation