Advertisement

Digital PCR pp 303-322 | Cite as

Identification and Use of Personalized Genomic Markers for Monitoring Circulating Tumor DNA

  • Yilun Chen
  • Anthony M. George
  • Eleonor Olsson
  • Lao H. SaalEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1768)

Abstract

Digital PCR techniques are ideally suited for accurately quantifying trace amounts of target DNA sequences, such as tumor-derived mutant DNA that is present in the blood circulation of patients with cancer. Here, we describe an approach marrying low-coverage whole-genome sequencing of tumor tissues, to enumerate chromosomal rearrangement breakpoints, together with droplet digital PCR (ddPCR)-based personalized rearrangement assays to cost-effectively monitor circulating tumor DNA levels at multiple time-points during the clinical course. The method is generally applicable to essentially any cancer patient, as all cancers harbor unstable genomes, and may have uses for measuring minimal residual disease, response to therapy, and early detection of metastasis.

Key words

Cell-free circulating tumor DNA Personalized medicine Liquid biopsy Noninvasive diagnosis Whole-genome sequencing Droplet digital PCR 

Notes

Acknowledgments

We thank members of the Translational Oncogenomics Unit, Division of Oncology and Pathology for assistance, and in particular to Christof Winter and Robert Rigo for bioinformatics work. This work was supported by the Swedish Cancer Society, Swedish Research Council, Swedish Foundation for Strategic Research, Knut and Alice Wallenberg Foundation, VINNOVA, and Governmental Funding of Clinical Research within National Health Service, Swedish Breast Cancer Group, Crafoord Foundation, Lund University Medical Faculty, Gunnar Nilsson Cancer Foundation, Skåne University Hospital Foundation, BioCARE Research Program, King Gustav Vth Jubilee Foundation, Krapperup Foundation, and the Mrs. Berta Kamprad Foundation.

References

  1. 1.
    Haber DA, Velculescu VE (2014) Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA. Cancer Discov 4:650–661CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ignatiadis M, Dawson SJ (2014) Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality? Ann Oncol 25:2304–2313CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Schwarzenbach H, Hoon DS, Pantel K (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11:426–437CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong SM, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SK, Netto GJ, Oliner KS, Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih l M, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang TL, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6:224ra224CrossRefGoogle Scholar
  5. 5.
    Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, Dunning MJ, Gale D, Forshew T, Mahler-Araujo B, Rajan S, Humphray S, Becq J, Halsall D, Wallis M, Bentley D, Caldas C, Rosenfeld N (2013) Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368:1199–1209CrossRefPubMedGoogle Scholar
  6. 6.
    Jung K, Fleischhacker M, Rabien A (2010) Cell-free DNA in the blood as a solid tumor biomarker—a critical appraisal of the literature. Clin Chim Acta 411:1611–1624CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, Thornton K, Agrawal N, Sokoll L, Szabo SA, Kinzler KW, Vogelstein B, Diaz LA Jr (2008) Circulating mutant DNA to assess tumor dynamics. Nat Med 14:985–990CrossRefGoogle Scholar
  8. 8.
    Olsson E, Winter C, George A, Chen Y, Howlin J, Tang M-HE, Dahlgren M, Schulz R, Grabau D, van Westen D, Ferno M, Ingvar C, Rose C, Bendahl PO, Ryden L, Borg A, Gruvberger-Saal SK, Jernstrom H, Saal LH (2015) Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. EMBO Mol Med 7:1034–1047CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ, Cheang M, Osin P, Nerurkar A, Kozarewa I, Garrido JA, Dowsett M, Reis-Filho JS, Smith IE, Turner NC (2015) Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med 7:302ra133CrossRefGoogle Scholar
  10. 10.
    Leary RJ, Kinde I, Diehl F, Schmidt K, Clouser C, Duncan C, Antipova A, Lee C, McKernan K, De La Vega FM, Kinzler KW, Vogelstein B, Diaz LA Jr, Velculescu VE (2010) Development of personalized tumor biomarkers using massively parallel sequencing. Sci Transl Med 2:20ra14CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Diehl F, Li M, Dressman D, He Y, Shen D, Szabo S, Diaz LA Jr, Goodman SN, David KA, Juhl H, Kinzler KW, Vogelstein B (2005) Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Natl Acad Sci U S A 102:16368–16373CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chin K, de Solorzano CO, Knowles D, Jones A, Chou W, Rodriguez EG, Kuo WL, Ljung BM, Chew K, Myambo K, Miranda M, Krig S, Garbe J, Stampfer M, Yaswen P, Gray JW, Lockett SJ (2004) In situ analyses of genome instability in breast cancer. Nat Genet 36:984–988CrossRefPubMedGoogle Scholar
  13. 13.
    Alkner S, Tang MH, Brueffer C, Dahlgren M, Chen Y, Olsson E, Winter C, Baker S, Ehinger A, Ryden L, Saal LH, Ferno M, Gruvberger-Saal SK (2015) Contralateral breast cancer can represent a metastatic spread of the first primary tumor: determination of clonal relationship between contralateral breast cancers using next-generation whole genome sequencing. Breast Cancer Res 17:102CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Tang MH, Dahlgren M, Brueffer C, Tjitrowirjo T, Winter C, Chen Y, Olsson E, Wang K, Torngren T, Sjostrom M, Grabau D, Bendahl PO, Ryden L, Nimeus E, Saal LH, Borg A, Gruvberger-Saal SK (2015) Remarkable similarities of chromosomal rearrangements between primary human breast cancers and matched distant metastases as revealed by whole-genome sequencing. Oncotarget 6:37169–37184PubMedPubMedCentralGoogle Scholar
  15. 15.
    Saal LH, Vallon-Christersson J, Hakkinen J, Hegardt C, Grabau D, Winter C, Brueffer C, Tang MH, Reutersward C, Schulz R, Karlsson A, Ehinger A, Malina J, Manjer J, Malmberg M, Larsson C, Ryden L, Loman N, Borg A (2015) The Sweden Cancerome Analysis Network - Breast (SCAN-B) Initiative: a large-scale multicenter infrastructure towards implementation of breast cancer genomic analyses in the clinical routine. Genome Med 7:20CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chen K, Wallis JW, McLellan MD, Larson DE, Kalicki JM, Pohl CS, McGrath SD, Wendl MC, Zhang Q, Locke DP, Shi X, Fulton RS, Ley TJ, Wilson RK, Ding L, Mardis ER (2009) BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nat Methods 6:677–681CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Boeva V, Zinovyev A, Bleakley K, Vert JP, Janoueix-Lerosey I, Delattre O, Barillot E (2011) Control-free calling of copy number alterations in deep-sequencing data using GC-content normalization. Bioinformatics 27:268–269CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Yilun Chen
    • 1
  • Anthony M. George
    • 1
  • Eleonor Olsson
    • 1
  • Lao H. Saal
    • 1
    Email author
  1. 1.Translational Oncogenomics Unit, Division of Oncology and Pathology, Department of Clinical SciencesLund UniversityLundSweden

Personalised recommendations