Pathology & Oncology Research

, Volume 18, Issue 2, pp 271–276 | Cite as

Quantitation of Plasma Circulating DNA Using Quantitative PCR for the Detection of Hepatocellular Carcinoma

  • Zhaohui Huang
  • Dong Hua
  • Yu Hu
  • Zhihong Cheng
  • Xike Zhou
  • Qigen Xie
  • Qiongyao Wang
  • Feng Wang
  • Xiang Du
  • Yanjun Zeng


Circulating DNA is a potential biomarker for tumor diagnosis and prognosis. This study was aimed to quantify the circulating DNA in plasma from patients with hepatocellular carcinoma (HCC) using quantitative PCR and evaluate its potential clinical value. Blood samples were collected from 72 patients with HCC, 37 with liver cirrhosis or chronic hepatitis and 41 healthy volunteers. Plasma DNA was extracted and quantified by a real-time quantitative PCR method. The diagnostic and prognostic value of plasma DNA analysis for HCC was evaluated. DNA levels in the HCC plasma (median: 173 ng/mL) were significantly higher than those in the healthy controls (9 ng/mL) or control benign patients (46 ng/mL) (P < 0.001). The area under the receiver-operation characteristic (ROC) curve (AUC) assessing plasma DNA was 0.949 for healthy controls and 0.874 for control patients. Plasma DNA detection could discriminate HCC from normal controls with 90.2% sensitivity and 90.3% specificity at the cut-off value of 18.2 ng/mL. Combined ROC analyses using plasma DNA and serum AFP revealed an elevated AUC of 0.974 with 95.1% sensitivity and 94.4% specificity in discriminating HCC from normal controls. The plasma DNA levels were positively associated with tumor size (P = 0.012), and were significantly elevated in HCC patients with intrahepatic spreading or vascular invasion (P = 0.035). The overall survival time of patients with high plasma DNA levels showed a shortened tread when compared with that of patients with low plasma DNA concentrations (P = 0.071). Plasma DNA may be a valuable noninvasive tool for the detecting and predicting the metastasis potential of HCC; and the prognostic value of plasma DNA needed further investigation.


Hepatocellular carcinoma Plasma Circulating DNA Quantitative PCR 



This study supported by a grant from the Natural Science Foundation of Jiangsu Province (Grant No. BK2008114).


  1. 1.
    El-Serag HB, Rudolph KL (2007) Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132(7):2557–2576PubMedCrossRefGoogle Scholar
  2. 2.
    Farazi PA, DePinho RA (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6(9):674–687PubMedCrossRefGoogle Scholar
  3. 3.
    Farinati F, Marino D, De Giorgio M et al (2006) Diagnostic and prognostic role of alpha-fetoprotein in hepatocellular carcinoma: both or neither? Am J Gastroenterol 101(3):524–532PubMedCrossRefGoogle Scholar
  4. 4.
    Butt AN, Swaminathan R (2008) Overview of circulating nucleic acids in plasma/serum. Ann N Y Acad Sci 1137:236–242PubMedCrossRefGoogle Scholar
  5. 5.
    Huang ZH, Li LH, Hua D (2006) Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients. Cancer Lett 243(1):64–70PubMedCrossRefGoogle Scholar
  6. 6.
    Gal S, Fidler C, Lo YM et al (2004) Quantitation of circulating DNA in the serum of breast cancer patients by real-time PCR. Br J Cancer 90(6):1211–1215PubMedCrossRefGoogle Scholar
  7. 7.
    Sozzi G, Conte D, Mariani L et al (2001) Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 61(12):4675–4678PubMedGoogle Scholar
  8. 8.
    Paci M, Maramotti S, Bellesia E et al (2009) Circulating plasma DNA as diagnostic biomarker in non-small cell lung cancer. Lung Cancer 64(1):92–97PubMedCrossRefGoogle Scholar
  9. 9.
    Kumar S, Guleria R, Singh V et al (2010) Efficacy of circulating plasma DNA as a diagnostic tool for advanced non-small cell lung cancer and its predictive utility for survival and response to chemotherapy. Lung Cancer 70(2):211–217PubMedCrossRefGoogle Scholar
  10. 10.
    Kamat AA, Baldwin M, Urbauer D et al (2010) Plasma cell-free DNA in ovarian cancer: an independent prognostic biomarker. Cancer 116(8):1918–1925PubMedCrossRefGoogle Scholar
  11. 11.
    Kamat AA, Sood AK, Dang D et al (2006) Quantification of total plasma cell-free DNA in ovarian cancer using real-time PCR. Ann N Y Acad Sci 1075:230–234PubMedCrossRefGoogle Scholar
  12. 12.
    Allen D, Butt A, Cahill D et al (2004) Role of cell-free plasma DNA as a diagnostic marker for prostate cancer. Ann N Y Acad Sci 1022:76–80PubMedCrossRefGoogle Scholar
  13. 13.
    Tomita H, Ichikawa D, Ikoma D et al (2007) Quantification of circulating plasma DNA fragments as tumor markers in patients with esophageal cancer. Anticancer Res 27(4C):2737–2741PubMedGoogle Scholar
  14. 14.
    Ren N, Qin LX, Tu H et al (2006) The prognostic value of circulating plasma DNA level and its allelic imbalance on chromosome 8p in patients with hepatocellular carcinoma. J Cancer Res Clin Oncol 132(6):399–407PubMedCrossRefGoogle Scholar
  15. 15.
    Sai S, Ichikawa D, Tomita H et al (2007) Quantification of plasma cell-free DNA in patients with gastric cancer. Anticancer Res 27(4C):2747–2751PubMedGoogle Scholar
  16. 16.
    Hauser S, Zahalka T, Ellinger J et al (2010) Cell-free circulating DNA: diagnostic value in patients with renal cell cancer. Anticancer Res 30(7):2785–2789PubMedGoogle Scholar
  17. 17.
    Huang ZH, Hua D, Du CH et al (2007) Quantitation of plasma circulating DNA and its clinical value in the diagnosis and prognosis of breast cancer. Maternal Child Health Care China 22(15):2095–2097Google Scholar
  18. 18.
    Leon SA, Shapiro B, Sklaroff DM et al (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37(3):646–650PubMedGoogle Scholar
  19. 19.
    Kohler C, Radpour R, Barekati Z et al (2009) Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors. Mol Cancer 8:105PubMedCrossRefGoogle Scholar
  20. 20.
    Yoon KA, Park S, Lee SH et al (2009) Comparison of circulating plasma DNA levels between lung cancer patients and healthy controls. J Mol Diagn 11(3):182–185PubMedCrossRefGoogle Scholar
  21. 21.
    Gordian E, Ramachandran K, Reis IM et al (2010) Serum free circulating DNA is a useful biomarker to distinguish benign versus malignant prostate disease. Cancer Epidemiol Biomarkers Prev 19(8):1984–1991PubMedCrossRefGoogle Scholar
  22. 22.
    Ostrow KL, Hoque MO, Loyo M et al (2010) Molecular analysis of plasma DNA for the early detection of lung cancer by quantitative methylation-specific PCR. Clin Cancer Res 16(13):3463–3472PubMedCrossRefGoogle Scholar
  23. 23.
    Lee TH, 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(2):276–282PubMedCrossRefGoogle Scholar
  24. 24.
    Thijssen MA, Swinkels DW, Ruers TJ et al (2002) Difference between free circulating plasma and serum DNA in patients with colorectal liver metastases. Anticancer Res 22(1A):421–425PubMedGoogle Scholar
  25. 25.
    Thierry AR, Mouliere F, Gongora C et al (2010) Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res 38(18):6159–6175PubMedCrossRefGoogle Scholar
  26. 26.
    Ren N, Ye QH, Qin LX et al (2006) Circulating DNA level is negatively associated with the long-term survival of hepatocellular carcinoma patients. World J Gastroenterol 12(24):3911–3914PubMedGoogle Scholar
  27. 27.
    Galeazzi M, Morozzi G, Piccini M et al (2003) Dosage and characterization of circulating DNA: present usage and possible applications in systemic autoimmune disorders. Autoimmun Rev 2(1):50–55PubMedCrossRefGoogle Scholar
  28. 28.
    Antonatos D, Patsilinakos S, Spanodimos S et al (2006) Cell-free DNA levels as a prognostic marker in acute myocardial infarction. Ann N Y Acad Sci 1075:278–281PubMedCrossRefGoogle Scholar
  29. 29.
    Lippmann ML, Morgan L, Fein A et al (1982) Plasma and serum concentrations of DNA in pulmonary thromboembolism. Am Rev Respir Dis 125(4):416–419PubMedGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2011

Authors and Affiliations

  • Zhaohui Huang
    • 1
  • Dong Hua
    • 1
  • Yu Hu
    • 1
  • Zhihong Cheng
    • 1
  • Xike Zhou
    • 1
  • Qigen Xie
    • 1
  • Qiongyao Wang
    • 1
  • Feng Wang
    • 1
  • Xiang Du
    • 2
  • Yanjun Zeng
    • 3
  1. 1.Wuxi Oncology Institute, The Fourth Affiliated Hospital of Suzhou UniversityWuxiChina
  2. 2.Department of PathologyFudan University Shanghai Cancer CenterShanghaiChina
  3. 3.Biomechanics and Medical Information InstituteBeijing University of TechnologyBeijingChina

Personalised recommendations