Next generation digital PCR measurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue.

BACKGROUND Hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus (HBV) infection. False-negative results are common in routine serological tests and quantitative real-time PCR because of HBV surface antigen (HBsAg) variation and low HBV copy number. Droplet digital PCR (ddPCR), a next generation digital PCR, is a novel, sensitive, and specific platform that can be used to improve HBV detection. METHODS A total of 131 HCC cases with different tumor stages and clinical features were initially classified with a serological test as HBsAg positive (n = 107) or negative (n = 24) for HBV infection. Next, DNA templates were prepared from the corresponding formalin-fixed paraffin-embedded (FFPE) tissues to determine HBV copy number by ddPCR. RESULTS HBV copy numbers, successfully determined for all clinical FFPE tissues (n = 131), ranged from 1.1 to 175.5 copies/μL according to ddPCR. The copy numbers of HBV were positively correlated with tumor-nodes-metastasis (P = 0.008) and Barcelona-Clinic Liver Cancer (P = 0.045) classification. Moreover, serum cholinesterase correlated with hepatitis B viral load (P = 0.006). CONCLUSIONS HBV infection is a key factor that influences tumorigenesis in HCC by regulating tumor occurrence and development. ddPCR improves the analytical sensitivity and specificity of measurements in nucleic acids at a single-molecule level and is suitable for HBV detection.

[1]  Mojca Milavec,et al.  Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR , 2013, PloS one.

[2]  Stephen R Quake,et al.  Digital PCR provides absolute quantitation of viral load for an occult RNA virus. , 2012, Journal of virological methods.

[3]  Angela M. Liu,et al.  Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma , 2012, Nature Genetics.

[4]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[5]  Alexandra S. Whale,et al.  Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation , 2012, Nucleic acids research.

[6]  Fan Shen,et al.  Quantification of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from hepatocellular carcinoma tissues by capillary hydrophilic-interaction liquid chromatography/quadrupole TOF mass spectrometry. , 2013, Clinical chemistry.

[7]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[8]  Z. Gu,et al.  Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus , 2012, Journal of Clinical Microbiology.

[9]  Pei-Jer Chen,et al.  Re-evaluation of the Carcinogenic Significance of Hepatitis B Virus Integration in Hepatocarcinogenesis , 2012, PloS one.

[10]  S. An.  Risk of Hepatocellular Carcinoma Across a Biological Gradient of Serum Hepatitis B Virus DNA Level. JAMA 2006;295:65~73 , 2006 .

[11]  Serge Saxonov,et al.  Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. , 2013, Methods.

[12]  C. Mathers,et al.  Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 , 2010, International journal of cancer.

[13]  G. Torzilli,et al.  Potential role of cholinesterases to predict short-term outcome after hepatic resection for hepatocellular carcinoma , 2013, Updates in Surgery.

[14]  EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. , 2012, Journal of hepatology.

[15]  O. Hino,et al.  Relationship between serum and histochemical markers for hepatitis B virus and rate of viral integration in hepatocellular carcinomas in Japan , 1985, International journal of cancer.

[16]  H. Katayama,et al.  Circulating microRNAs (cmiRNAs) as novel potential biomarkers for hepatocellular carcinoma. , 2013, Neoplasma.

[17]  Linos Vandekerckhove,et al.  Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA , 2014, PloS one.

[18]  M. Sherman.  Hepatocellular carcinoma: epidemiology, surveillance, and diagnosis. , 2010, Seminars in liver disease.

[19]  K. Fung,et al.  Transcriptional analysis of doxorubicin‐induced cytotoxicity and resistance in human hepatocellular carcinoma cell lines , 2009, Liver international : official journal of the International Association for the Study of the Liver.

[20]  Benjamin J Hindson,et al.  Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue. , 2013, Clinical chemistry.

[21]  J. Bruix,et al.  Hepatocellular carcinoma , 2012, The Lancet.

[22]  P. Walson,et al.  Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury. , 2013, Clinical chemistry.

[23]  Meng Li,et al.  The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma. , 2014, Journal of hepatology.

[24]  Guan-Tarn Huang,et al.  Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. , 2006, JAMA.

[25]  T. Liang,et al.  Specific and Nonhepatotoxic Degradation of Nuclear Hepatitis B Virus cccDNA , 2014, Science.

[26]  Z. Zou,et al.  Analysis of Hepatitis B Virus Intrahepatic Covalently Closed Circular DNA and Serum Viral Markers in Treatment-Naive Patients with Acute and Chronic HBV Infection , 2014, PloS one.

[27]  Chien-Jen Chen,et al.  Carriers of inactive hepatitis B virus are still at risk for hepatocellular carcinoma and liver-related death. , 2010, Gastroenterology.

[28]  O. O. Ogunkeye,et al.  Serum cholinesterase activity helps to distinguish between liver disease and non-liver disease aberration in liver function tests. , 2006, Pathophysiology : the official journal of the International Society for Pathophysiology.

[29]  Hanlee P. Ji,et al.  Correction to High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR , 2015, Analytical chemistry.

[30]  P. Loehrer,et al.  Risk of Hepatocellular Carcinoma Across a Biological Gradient of Serum Hepatitis B Virus DNA Level , 2007 .

[31]  R. Goldin,et al.  Management of chronic hepatitis B virus infection , 2004 .

[32]  M. Buendia,et al.  Mechanisms of HBV-related hepatocarcinogenesis. , 2010, Journal of hepatology.

[33]  T. Ide,et al.  A real-time quantitative polymerase chain reaction method for hepatitis B virus in patients with chronic hepatitis B treated with lamivudine , 2003, American Journal of Gastroenterology.