Application of new and improved biomarkers for the diagnosis of hepatocellular carcinoma HCC

Lead Research Organisation: MRC Unit, The Gambia

Abstract

Liver cancer is an important complication of chronic Hepatitis B infection and is the most common cause of death in adult men in the Gambia. Usually the diagnosis is made too late to offer effective treatment. This project aims to test whether a combination of different tests can predict the development of early liver cancer more accurately than current methods. The study will be carried out using stored blood samples of people with Hepatitis B infection who did or did not develop liver cancer, from a previously characterised study population called the Gambia Liver cancer study.

Technical Summary

Surveillance programs aimed at detecting early stage HCC are based on the use of ultrasound and the detection of elevated levels of alpha foetoprotein (AFP) in the serum.AFP as a diagnostic serologic test has low sensitivity (40-80%) and specificity (50-70%). It been shown that the detection of free AFP in parallel with complexed AFP (IC), serum squamous cell carcinoma antigen SCCA variants and SCCA immune complex (IC) can significantly improve (up to 90.8%) the sensitivity of HCC diagnosis.In addition, the serine/threonine kinase Aurora A has been found to be elevated in HCC. It is possible that the elevated levels of Aurora A protein in HCC cells could give rise to detectable levels of Aurora A appearing in patient serum. The detectability of Aurora A in serum using sensitive immunoblotting will be established. If Aurora A appears in HCC patient serum at levels above the detection limit, this may provide a useful diagnostic feature that can be further developed with more sensitive immunodetection techniques.The aim of this study is to:Validate SCCA, SCCA-IC, AFP-IC assays in diagnosing HCC.Initiate studies to determine the serum levels of Aurora A in normal vs. HCC patients.Investigate whether these markers represent useful tools for the detection of HCC.
 
Title HBV DNA assay 
Description Real-time PCR to quantify hepatitis B virus DNA in chronic carriers. DNA was extracted from 200 µL of serum using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was eluted into 100 µL nuclease-free water and 5?l added to a 25?l PCR reaction mixture. The reaction was carried out using a commercial SYBR-Green reaction mix (Qiagen, Hilden, Germany). The kit contains HotStarTaq polymerase which is included to avoid false positives in the quantitative PCR. The primer sequences were 5'-GTG TCT GCG GCG TTT TAT CA (sense) and 5' GAC AAA CGG GCA ACA TAC CTT (antisense) designed to amplify a 98 base pair product from positions 379 to 476 of the HBV genome. Thermal cycling was performed in an ABi 5700 sequence detection system (PE Applied Biosystems, Warrington, UK). Reaction conditions were: 95OC for 15 minutes followed by 40 cycles of 94OC for 15 seconds, 55OC for 30 seconds and 72OC for 30 seconds. A four point standard curve (1.5x108copies per millilitre (cpm), 1.5x106cpm, 1.5x104cpm, 1.5x102cpm) was generated from a high titre plasma donation quantified by end point dilution PCR. The calibration of this standard was confirmed by comparison with an International HBV DNA standard, (97/746) (NIBSC, Potters Bar, UK). Test samples falling above the top of the standard curve were re-assayed at a dilution of 1:100. Each test run included positive and negative controls. The performance of the assay was evaluated by comparison with a commercial assay (HBV Monitor, Roche Molecular Systems, Inc., Branchburg, NJ 08876 USA) performed according to the manufacturer's instructions. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Cheaper than commercial HBV DNA quantification assaya 
 
Title Ola assay for HBV pre core and basal core promoter mutation 
Description 1) A novel, rapid and sensitive oligonuclotide ligation assay for the detection of cancer predicting mutations in the pre-core and basal core. The probes used in the OLA assay were designed against the core gene sequence of genotype E (ayw4) (Genebank accession number X75657). Ligation primers specific for wild-type sequences were labeled at the 5' end with digoxigenin, mutant-specific primers were labeled with fluorescein. The common oligonucleotide which hybridises downstream of the wild type and mutant was labeled at the 3' end with biotin and phosphorylated at the 5' end . This primer anneals to nucleotides in the specific regions of the HBV pre-C/BCP gene immediately adjacent to the 3' end of both the mutant and wild type genomes. The tests were performed utilizing a reaction mixture containing 1x ligase buffer (100 mM MgCl2, 10 mM dithiothreitol, 10 mM NAD (Sigma, UK.), 80 Mm KCl (Sigma), 0.1% Triton X-100), 3 U of thermostable ligase (Epicentre Technologies, UK.) and 3 pmol of each ligation primer. Detection of mutant and wild type genotypes was achieved by the simultaneous addition of anti-digoxigenin-peroxidase (POD), (Boehringer Mannheim) and anti-fluorescein-alkaline phosphatase (AP), (Boehringer Mannheim) followed by sequential washing and addition of GIBCO ELISA amplification system (Invitrogen) and TMB substrate (Promega) to detect mutant and wild type sequences respectively. The optical densities were measured using a Multiscan Ascent Elisa plate reader (Thermo Labsystems, Finland) at wavelengths of 490 nm and 450 nm for mutant and wild type respectively. It was also possible to read the plates visually as this may be important for areas of poor resource settings where ELISA plate readers are not easily available (data not shown). Samples were considered mutant alone (magenta and positive OD490), wild type alone (yellow and positive OD450) or mixture of wild type and mutant (magenta and yellow plus positive OD490 and OD450). 2) Real-time PCR to quantify hepatitis B virus DNA in chronic carriers. DNA was extracted from 200 µL of serum using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was eluted into 100 µL nuclease-free water and 5?l added to a 25?l PCR reaction mixture. The reaction was carried out using a commercial SYBR-Green reaction mix (Qiagen, Hilden, Germany). The kit contains HotStarTaq polymerase which is included to avoid false positives in the quantitative PCR. The primer sequences were 5'-GTG TCT GCG GCG TTT TAT CA (sense) and 5' GAC AAA CGG GCA ACA TAC CTT (antisense) designed to amplify a 98 base pair product from positions 379 to 476 of the HBV genome. Thermal cycling was performed in an ABi 5700 sequence detection system (PE Applied Biosystems, Warrington, UK). Reaction conditions were: 95OC for 15 minutes followed by 40 cycles of 94OC for 15 seconds, 55OC for 30 seconds and 72OC for 30 seconds. A four point standard curve (1.5x108copies per millilitre (cpm), 1.5x106cpm, 1.5x104cpm, 1.5x102cpm) was generated from a high titre plasma donation quantified by end point dilution PCR. The calibration of this standard was confirmed by comparison with an International HBV DNA standard, (97/746) (NIBSC, Potters Bar, UK). Test samples falling above the top of the standard curve were re-assayed at a dilution of 1:100. Each test run included positive and negative controls. The performance of the assay was evaluated by comparison with a commercial assay (HBV Monitor, Roche Molecular Systems, Inc., Branchburg, NJ 08876 USA) performed according to the manufacturer's instructions. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Both tools are cheaper than conventional DNA sequencing and commercial assays. 
 
Description MRC show case 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Health professionals
Results and Impact The study was presented in a poster at MRC showcase in Manchester.

Generate discussion at the meeting
Year(s) Of Engagement Activity 2007