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Circulating DNA Analysis

Protocols and Clinical Applications Using Taqman Assays

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Molecular Diagnosis of Cancer

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 97))

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Abstract

In recent years, the application of circulating nucleic acids as a clinical diagnostic tool has aroused the interest of many clinicians and biomedical scientists. Circulating nucleic acids refer to the DNA and RNA species that are present in the plasma or serum portion of a blood sample. Although the existence of circulating nucleic acids was described more than half a century ago, the potential clinical implication of circulating nucleic acid analysis has only been realized by the global scientific community in the past 5 yr. In this chapter, current developments of circulating DNA analysis will be discussed. The clinical applications of circulating Epstein-Barr virus (EBV) DNA in cancer detection and monitoring will be discussed in detail to illustrate the important technical issues of performing circulating DNA analysis.

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References

  1. Mandel, P. and Metais, P. (1948) Les acides nucleiques du plasma sanguin chez I’homme. C.R. Acad. Sci. Paris 142, 241–243.

    CAS  Google Scholar 

  2. Watson, J. and Crick, F. (1953) A structure for deoxyribose nucleic acid. Nature 171, 737.

    Article  PubMed  CAS  Google Scholar 

  3. Leon, S. A., Shapiro, B., Sklaroff, D. M., et al. (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 37, 646–650.

    PubMed  CAS  Google Scholar 

  4. Stroun, M., Anker, P., Maurice, P., et al. (1989) Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 46, 318–322.

    Article  PubMed  CAS  Google Scholar 

  5. Sorenson, G. D., Pribish, D. M., Valone, F. H., et al. (1994) Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol. Biomarkers Prev. 3, 67–71.

    PubMed  CAS  Google Scholar 

  6. Vasioukhin, V., Anker, P., Maurice, P., 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, 774–779.

    Article  PubMed  CAS  Google Scholar 

  7. Chen, X. Q., Stroun, M., Magnenat, J. L., et al. (1996) Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nature Med. 2, 1033–1035.

    Article  PubMed  CAS  Google Scholar 

  8. Nawroz, H., Koch, W., Anker, P., et al. (1996) Microsatellite alterations in serum DNA of head and neck cancer patients. Nature Med. 2, 1035–1037.

    Article  PubMed  CAS  Google Scholar 

  9. Jackson, P. E., Qian, G. S., Friesen, M. D., et al. (2001) Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry. Cancer Res. 61, 33–35.

    PubMed  CAS  Google Scholar 

  10. Lo, Y. M., Chan, L. Y., Lo, K. W., et al. (1999) Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res. 59, 1188–1191.

    PubMed  CAS  Google Scholar 

  11. Lo, Y. M., Wong, I. H., Zhang, J., et al. (1999) Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res. 59, 3899–3903.

    PubMed  CAS  Google Scholar 

  12. Gonzalez, R., Silva, J. M., Sanchez, A., et al. (2000) Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: follow-up study and prognostic significance. Ann. Oncol. 11, 1097–1104.

    Article  PubMed  CAS  Google Scholar 

  13. Lo, Y. M., Corbetta, N., Chamberlain, P. F., et al. (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485–487.

    Article  PubMed  CAS  Google Scholar 

  14. Lo, Y. M. D., Hjelm, N. M., Fidler, C., et al. (1998) Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N. Engl. J. Med. 339, 1734–1738.

    Article  PubMed  CAS  Google Scholar 

  15. Lo, Y. M. D., Lau, T. K., Zhang, J., et al. (1999) Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin. Chem. 45, 1747–1751.

    PubMed  CAS  Google Scholar 

  16. Lo, Y. M., Tein, M. S., Pang, C. C., et al. (1998) Presence of donor-specific DNA in plasma of kidney and liver-transplant recipients. Lancet 351, 1329–1330.

    Article  PubMed  CAS  Google Scholar 

  17. Lo, Y. M., Rainer, T. H., Chan, L. Y., et al. (2000) Plasma DNA as a prognostic marker in trauma patients. Clin. Chem. 46, 319–323.

    PubMed  CAS  Google Scholar 

  18. Sorenson, G. D. (2000) A review of studies on the detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer. Ann. NY Acad. Sci. 906, 13–16.

    Article  PubMed  CAS  Google Scholar 

  19. Gocke, C. D., Benko, F. A., Kopreski, M. S., et al. (2000) p53 and APC mutations are detectable in the plasma and serum of patients with colorectal cancer (CRC) or adenomas. Ann. NY Acad. Sci. 906, 44–50.

    Article  PubMed  CAS  Google Scholar 

  20. Mayall, F., Jacobson, G., Wilkins, R., et al. (1998) Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma. J. Clin. Pathol. 51, 611–613.

    Article  PubMed  CAS  Google Scholar 

  21. Yamada, T., Nakamori, S., Ohzato, H., et al. (1998) Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features. Clin. Cancer Res. 4, 1527–1532.

    PubMed  CAS  Google Scholar 

  22. Silva, J. M., Dominguez, G., Silva, J., et al. (2001) Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics. Clin. Cancer Res. 7, 2821–2825.

    PubMed  CAS  Google Scholar 

  23. Kopreski, M. S., Benko, F. A., Kwee, C., et al. (1997) Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer. Br. J. Cancer. 76, 1293–1299.

    Article  PubMed  CAS  Google Scholar 

  24. Castells, A., Puig, P., Mora, J., et al. (1999) K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic significance. J. Clin. Oncol. 17, 578–584.

    PubMed  CAS  Google Scholar 

  25. Nunes, D. N., Kowalski, L. P., and Simpson, A. J. (2001) Circulating tumor-derived DNA may permit the early diagnosis of head and neck squamous cell carcinomas. Int. J. Cancer. 92, 214–219.

    Article  PubMed  CAS  Google Scholar 

  26. Chen, X., Bonnefoi, H., Diebold-Berger, S., et al. (1999) Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer. Clin. Cancer Res. 5, 2297–2303.

    PubMed  CAS  Google Scholar 

  27. Coulet, F., Blons, H., Cabelguenne, A., et al. (2000) Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis. Cancer Res. 60, 707–711.

    PubMed  CAS  Google Scholar 

  28. Fujiwara, Y., Chi, D. D., Wang, H., et al. (1999) Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients. Cancer Res. 59, 1567–1571.

    PubMed  CAS  Google Scholar 

  29. Taback, B., Fujiwara, Y., Wang, H. J., et al. (2001) Prognostic significance of circulating microsatellite markers in the plasma of melanoma patients. Cancer Res. 61, 5723–5726.

    PubMed  CAS  Google Scholar 

  30. Silva, J. M., Dominguez, G., Garcia, J. M., et al. (1999) Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Cancer Res. 59, 3251–3256.

    PubMed  CAS  Google Scholar 

  31. Penta, J. S., Johnson, F. M., Wachsman, J. T., et al. (2001) Mitochondrial DNA in human malignancy. Mutat. Res. 488, 119–133.

    Article  PubMed  CAS  Google Scholar 

  32. Zhong, S., Ng, M. C., Lo, Y. M., et al. (2000) Presence of mitochondrial tRNA(Leu(UUR)) A to G 3243 mutation in DNA extracted from serum and plasma of patients with type 2 diabetes mellitus. J. Clin. Pathol. 53, 466–469.

    Article  PubMed  CAS  Google Scholar 

  33. Jeronimo, C., Nomoto, S., Caballero, O. L., et al. (2001) Mitochondrial mutations in early stage prostate cancer and bodily fluids. Oncogene 20, 5195–5198.

    Article  PubMed  CAS  Google Scholar 

  34. 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.

    PubMed  CAS  Google Scholar 

  35. Giacona, M. B., Ruben, G. C., Iczkowski, K. A., et al. (1998) Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls. Pancreas 17, 89–97.

    Article  PubMed  CAS  Google Scholar 

  36. Nomoto, S., Yamashita, K., Koshikawa, K., et al. (2002) Mitochondrial D-loop mutations as clonal markers in multicentric hepatocellular carcinoma and plasma. Clin. Cancer Res. 8, 481–487.

    PubMed  CAS  Google Scholar 

  37. Herman, J. G., Graff, J. R., Myohanen, S., et al. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93, 9821–9826.

    Article  PubMed  CAS  Google Scholar 

  38. Esteller, M., Sanchez-Cespedes, M., Rosell, R., et al. (1999) Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res. 59, 67–70.

    PubMed  CAS  Google Scholar 

  39. Hibi, K., Taguchi, M., Nakayama, H., et al. (2001) Molecular detection of p16 promoter methylation in the serum of patients with esophageal squamous cell carcinoma. Clin. Cancer Res. 7, 3135–3138.

    PubMed  CAS  Google Scholar 

  40. Kawakami, K., Brabender, J., Lord, R. V., et al. (2000) Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J. Natl. Cancer Inst. 92, 1805–1811.

    Article  PubMed  CAS  Google Scholar 

  41. Wong, I. H., Lo, Y. M., Zhang, J., et al. (1999) Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res. 59, 71–73.

    PubMed  CAS  Google Scholar 

  42. Silva, J. M., Dominguez, G., Villanueva, M. J., et al. (1999) Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients. Br. J. Cancer 80, 1262–1264.

    Article  PubMed  CAS  Google Scholar 

  43. Wong, I. H., Ng, M. H., Huang, D. P., et al. (2000) Aberrant p15 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications. Blood 95, 1942–1949.

    PubMed  CAS  Google Scholar 

  44. Grunau, C., Clark, S. J., and Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, E65–E65.

    Article  PubMed  CAS  Google Scholar 

  45. Rickinson, A. B. and Kieff, E. (1996) Epstein-Barr virus, in Fields Virology (Fields, B. N., Knipe, D. M., and Howley, P. M., eds.), Lippincott-Raven, Philadelphia, pp. 2397–2446.

    Google Scholar 

  46. Tam, J. S. and Murray, H. G. (1990) Nasopharyngeal carcinoma and Epstein-Barr virus—associated serologic markers. Ear Nose Throat J. 69, 261–267.

    PubMed  CAS  Google Scholar 

  47. Mutirangura, A., Pornthanakasem, W., Theamboonlers, A., et al. (1998) Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin. Cancer Res. 4, 665–669.

    PubMed  CAS  Google Scholar 

  48. Lo, Y. M., Chan, L. Y., Chan, A. T., et al. (1999) Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res. 59, 5452–5455.

    PubMed  CAS  Google Scholar 

  49. Lo, Y. M., Leung, S. F., Chan, L. Y., et al. (2000) Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma. Cancer Res. 60, 2351–2355.

    PubMed  CAS  Google Scholar 

  50. Lo, Y. M., Chan, A. T., Chan, L. Y., et al. (2000) Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res. 60, 6878–6881.

    PubMed  CAS  Google Scholar 

  51. Klein, G., Dombos, L., and Gothoskar, B. (1972) Sensitivity of Epstein-Barr virus (EBV) producer and non-producer human lymphoblastoid cell lines to superin-fection with EB-virus. Int. J. Cancer 10, 44–57.

    Article  PubMed  CAS  Google Scholar 

  52. Heid, C. A., Stevens, J., Livak, K. J., et al. (1996) Real time quantitative PCR. Genome Res. 6, 986–994.

    Article  PubMed  CAS  Google Scholar 

  53. Higuchi, R., Fockler, C., Dollinger, G., et al. (1993) Kinetic PCR analysis: realtime monitoring of DNA amplification reactions. Biotechnology (NY) 11, 1026–1030.

    Article  CAS  Google Scholar 

  54. Lo, Y. M. D., Tein, M. S., Lau, T. K., et al. (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am. J. Hum. Genet. 62, 768–775.

    Article  PubMed  CAS  Google Scholar 

  55. Stryer, L. (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846.

    Article  PubMed  CAS  Google Scholar 

  56. Holland, P. M., Abramson, R. D., Watson, R., et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.

    Article  PubMed  CAS  Google Scholar 

  57. Lui, Y. Y., Chik, K. W., Chiu, R. W., et al. (2002) Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin. Chem. 48, 421–427.

    PubMed  CAS  Google Scholar 

  58. Lee, T. H., Montalvo, L., Chrebtow, V., 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, 276–282.

    Article  PubMed  CAS  Google Scholar 

  59. Hsiao, J. R., Jin, Y. T., and Tsai, S. T. (2002) Detection of cell free Epstein-Barr virus DNA in sera from patients with nasopharyngeal carcinoma. Cancer 94, 723–729.

    Article  PubMed  CAS  Google Scholar 

  60. Shotelersuk, K., Khorprasert, C., Sakdikul, S., et al. (2000) Epstein-Barr virus DNA in serum/plasma as a tumor marker for nasopharyngeal cancer. Clin. Cancer Res. 6, 1046–1051.

    PubMed  CAS  Google Scholar 

  61. Chiu, R. W. K., Poon, L. L. M., Lau, T. K., et al. (2001) Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin. Chem. 47, 1607–1613.

    PubMed  CAS  Google Scholar 

  62. Pang, J., Modlin, J., and Yolken, R. (1992) Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. Mol. Cell Probes 6, 251–256.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, New Territories, Hong Kong

    Kwan-Chee Allen Chan & Yuk-Ming Dennis Lo

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  1. Kwan-Chee Allen Chan
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  2. Yuk-Ming Dennis Lo
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Editors and Affiliations

  1. Division of Reproductive and Developmental Sciences, The University of Edinburgh, The Royal Infirmary, Edinburgh, Scotland, UK

    Joseph E. Roulston

  2. Division of Cancer and Molecular Pathology, University Department of Surgery, Glasgow Royal Infirmary, Glasgow, Scotland, UK

    John M. S. Bartlett

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© 2004 Humana Press Inc., Totowa, NJ

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Chan, KC.A., Lo, YM.D. (2004). Circulating DNA Analysis. In: Roulston, J.E., Bartlett, J.M.S. (eds) Molecular Diagnosis of Cancer. Methods in Molecular Medicine, vol 97. Humana Press. https://doi.org/10.1385/1-59259-760-2:217

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  • DOI: https://doi.org/10.1385/1-59259-760-2:217

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-160-8

  • Online ISBN: 978-1-59259-760-4

  • eBook Packages: Springer Protocols

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