Blood Markers Of Hepatitis B Virus Infection In Patients With Hepatocellular Carcinoma Screening

Liver cancer is the sixth most common cancer and the third leading cause of cancer death worldwide.Most liver cancer patients are in developing countries,among whom,Chinese account more than a half.Most liver cancers are hepatocellular carcinomas(HCC),the major risk factors for which are chronic infections with the hepatitis virus B and C.As hepatitis B virus(HBV) is more prevalent in China,we have put much attention into the disease course of HBV infection.During the long-term disease progression from chronic HBV infection to liver cirrhosis and HCC,patients are lack of characteristic symptoms.It is often late stage when HCC is diagnosed.Treatment for such patients is limited with the five year survival rate lower than 5%.In light of the high burden of HBV chronic infection,it is significant to identify reliable,reproducible,and noninvasive biomarkers or a surveillance model for HCC targeting at the HBV infected population.The most widely used serum biomarker for HCC has been alpha fetoprotein(AFP).As up to 40%of HCC patients present with normal AFP levels and elevated AFP levels are observed in patients with chronic hepatitis or cirrhosis,the low sensitivity and specificity of AFP has always been called in question.However,there has been no other serum biomarkers which could be accepted as effective as AFP.Human blood contains a wide variety of chemically diverse molecular weight compounds ranging from volatile and nonvolatile low molecular ones such as organic acids and carbohydrates,to middle molecular ones such as polypeptides, and to large molecular ones such as proteins.These compounds differ against complex interactions between genetic factors,pathogen infection,toxicant exposure,and so on during disease process.They constructed a huge biomarker bank which could be applied for disease sceening,progression or prognosis evaluation.Techniques allowing high-flux data aquisition and analyzing are needed for researching in this huge biomarker bank.In the present,"omics" studies including genomics,proteomics and metabolomics are the newest in the biomedical field with the most rapid developed techniques such as chromatography or massspectrometry meeting the need of high-flux data aquisition and analyzing.The main purpose of this study is to explore new biomarkers for HCC from the HBV infected groups applying different techniques on the base of "omics" research.The contents are:the metabolomic research of HBV infected HCC patients based on Gas chromatography/Mass Spectrometry(GC/MS) and identification of HCC blood biomarkers applying the techniques of solid-phase microextraction and chemical derivatization;the serum metabolomic research in the HCC high risk groups:chronic hepatitis B patients and HBV infected cirrhosis patients based on GC/MS applying the technique of chemical derivatization;an exploration in the quantification of serum metabolomic biomarkers based on GC/MS applying the technique of microwave-assisted derivatization and isotope dilution;serum proteomic surveillance of hepatocellular carcinoma from hepatitis B virus infected groups with magnetic bead-based MALDI-TOF/MS;identification of new serum biomarkers for hepatocellular carcinoma using biotin-label-based antibody microarrays;a pilot study of identification and quantification of serum protein biomarkers in HCC based on the technique of isotope tagging for relative and absolute protein quantitation(iTRAQ).Part One The metabolomic research of HBV infected HCC patients based on Gas chromatography/Mass Spectrometry(GC/MS) and identification of HCC blood biomarkers applying the techniques of solid-phase microextraction and chemical derivatizationSection 1.Investigation of volatile biomarkers in HCC blood using solid-phase microextraction and gas chromatography-mass spectrometryObjective:To explore the technique of solid-phase microextraction(SPME) and gas chromatography-mass spectrometry(GC-MS) in the investigation of blood volatile biomarkers for HCC.Methods:The fiber,duration,temperature of extraction and the duration, temperature of desorption was tested and optimized to extact the blood volatile metabolites in 19 HCC patients and 18 normal controls.GC-MS was applied in separate,detect and identificate the extracted metabolites.The peak area values were gathered and compared between groups.Chi square test was used to screen the HCC biomarkers.Results:The best extraction fiber was CAR-PDMS.The optimal extraction duration,temperature was 15min and 60℃.The optimal extraction duration, temperature was 30s and 250℃.There were 47 volatile low molecular metabolites detected,19 of which were detected differently in the two groups(p＜0.05). Positive rates of and hexanal(sensitivity 97.4%,specificity 100%), 1-octen-3-ol(sensitivity 84.2%,specificity 100%) and octane(sensitivity 89.5%,specificity 100%)in HCC blood were found to be much higher than those in control blood.The three molecules of hexanal,1-octen-3-ol and octane were regarded as biomarkers of HCC with clinical diagnostic value.Conclusion:SPME/GC-MS is a simple,rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.Section 2.Investigation of nonvolatile biomarkers in HCC serum using chemical derivatization and gas chromatography-mass spectrometryObjective:To explore the technique of chemical derivatization and gas chromatography-mass spectrometry(GC-MS) in the investigation of serum nonvolatile biomarkers for HCC.Methods:Serum metabolome was detected through chemical derivatization followed by gas chromatography/mass spectrometry(GC/MS).The serum metabolic difference was compared between hepatocellular carcinoma(HCC,n=20) male patients and normal control male subjects(n=20).The acquired GC/MS data was analyzed by stepwise discriminant analysis(SDA) and support vector machine(SVM).Results:The metabolites including butanoic acid,ethanimidic acid, glycerol,L-isoleucine,L-valine,aminomalonic acid,D-erythrose, hexadecanoic acid,octadecanoic acid,and 9,12-octadecadienoic acid in combination with each other gave the strongest segregation between the HCC group and the control group.By applying these variables,our method provided a diagnostic model that could well discriminate between HCC and normal subjects. More important,the error count estimate for each group was 0%.The total classifying accuracy of the discriminant function tested by SVM 20-fold cross validation was 75%.Conclusion:This technique is different from traditional ones and appears to be a useful tool in the area of HCC diagnosis.Part Two Serum metabolomic study of two groups of cases with high risk of HCC:hepatitis B virus infected chronic hepatitis and cirrhosis patients by chemical derivatization and Gas chromatography/Mass spectrometryObjective:To investigate the serum metabolic profile of hepatitis B virus (HBV)infected chronic hepatitis and cirrhosis patients. Methods:HBV infected non-cirrhosis male subjects(n=20) and HBV infected cirrhosis male patients(n=20) entered this experiment.Serum metabolome was detected through chemical derivatization followed by GC/MS.The high-flux metabolomic data was analyzed by stepwise discriminant analysis(SDA).Results:From the 41 metabolites detected in serum,we selected metabolites including acetic acid,sorbitol,d-lactic acid,hexanoic acid, 1-naphthalenamine,butanoic acid,phosphoric acid,d-glucitol,glucose which in combination with each other could segregate the two groups.The error count was 0%for the non-cirrhosis group and 25%for the cirrhosis group.Conclusion:Chemical derivatization and GC/MS technique is applicable in metabolomic profiling in patients with HCC risk.Part Three An exploration for precise quatification of serum metabolomic biomarkers applying microwave-assisted derivatization and isotope dilution GC/MSObjective:To precisely quatificate serum glucose and isoleucine levels applying microwave-assisted derivatization and isotope dilution GC/MS in the investigation of the effect of pyrroloquinoline quinine(PQQ) on rats with alcohol assumption.Methods:Thirty male Sprague-Dawley rats divided into three groups.The first group was a normal control which received intragastric administration of saline for 8 weeks.Alcohol was administered intragastrically to the alcohol group and PQQ group for four weeks.PQQ was administered intragastrically to rats in the PQQ group at for another four weeks.Serum metabolites including glucose,isoleucine,the[U-¹³C₆]-glucose and[¹⁵N]-isoleucine were rapidly derivatized by N-Methyl-N-trimethylsilyltrifluoroacetamide(MSTFA) with microwave irradiation,and the trimethylsilyl derivatives were analyzed by GC/MS.T test was applied for comparison between the groups.Results:Derivatization of was completed within 3min.Serum glucose levels were higher in alcohol group than in normal group(10.89±0.93mmol/L vs 8.25±1.44 mmol/L,p＜0.01).Serum glucose levels were lower in PQQ group than in alcohol group(9.20±0.87 mmol/L vs 10.89±0.93 mmol/L,p＜0.01).Serum isoleucine levels were higher in alcohol group than in normal group (132.54±6.51 umol/L vs 107.83±11.72 umol/L,p＜0.001).There was no difference of serum isoleucine levels between PQQ group and alcohol group Conclusion:The combination of MAD and ID GC/MS has shown to be an accurate, rapid,simple and sensitive method for the quantification of glucose and isoleucine in serum samples.Serum glucose and isoleucine metabolism were affected by alcohol.PQQ could reverse alcohol exposure induced glucose elevation.It did not affect the metabolism of isoleucine whose level was elevated along with serum glucose.Part Four Serum proteomic surveillance of hepatocellular carcinoma from hepatitis B virus infected groups and identification of HCc biomarkers with magnetic bead-based MALDI-TOF/MSObjective:To determine whether serum proteomic profiling based on magnetic bead(MB) and matrix-assisted laser desorption/ionisation time-of-flight(MALDI-TOF) mass spectrometry(MS) can discriminate between hepatocellular carcinoma(HCC) and other HBV infected groups.To identify proteomic biomarkers for HCC.Methods:One hundred ninty-eight serum samples from 80 chronic hepatitis B,94 HBV infected HCC patients and 24 normal cases were analyzed by MALDI-TOF MS after peptides enrichment by MBs.Applying genetic algorithm,diagnotic models for HCC were built between 30 HCC patients and 24 normal cases/30 hepatitis patients.Validations were done with the left cases.Markers in the models were identified through LC/MS MS.Data from 22 HBV carriers and 23 HBV-infected cirrhosis patients was laterly gathered to be separated from additional 25 chronic hepatitis B patients,25 HCC patients and 25 normal controls.Results:The first three groups were well separated from each other.Two discrimination models were built for HCC.The overall recognition capability of the two models was 96.25%and 93.33%.The validation of them in the left patients showed misdiagnosis rates of 1.6%and 23.4%for HCC.Potential biomakers for HCC were identified as prothrombin precursor(fragment),isoform 1 of calcium-dependent activator protein for secretion,baculoviral IAP repeat-containing 6,et al.The laterly added two groups were also well separated from others.Conclusion:MB based MALDI-TOF MS approach is applicable in identifying serum proteomic biomarkers that can be used in the surveillance of HCC from HBV infected population. Part Five Identification of new serum biomarkers for hepatocellular carcinoma using biotin-label-based antibody microarraysObjective:Searching for hepatocellular carcinoma(HCC) biomarkers applying antibody arrays.Methods:In this study,serum cytokine detection using RayBio^? Biotin Label-based Human Antibody Arrayâ… (507 human proteins) was applied in normal cases(n=3),chronic hepatitis B patients(n=3) and HCC patients(n=3).Thirty proteins which were the most differently detected ones between HCC patients and normal/hepatitis cases were validated in additional 30 normal,30 hepatitis and 40 HCC cases.Biomarkers for HCC were selected through one-way ANOVA and diagonal linear discriminant analysis.Results:A diagnostic model constructed by two HCC biomarkers(MDC,MSPαchain)together with AFP had a sensitivity of 73.2%,specficity of 95%for HCC diagnosis.Conclusion:Antibody microarrays are applicable in searching for new biomarkers for HCC.Part Six Quantification of HCC serum proteomic biomarkers applying the methods of isobaric tags for relative and absolute quantitation(iTRAQ)and Liquid Chromatography/Mass SpectrometryObjective:To apply the methods of isobaric tags for relative and absolute quantitation(iTRAQ)and Liquid chromatography-Mass spectrometry to hepatocellular carcinoma(HCC) patients in the preliminary study of relative quantified detection of HCC proteomic biomarker.Methods:15 cases of hepatitis B virus infected HCC and 15 patients with chronic hepatitis B were recruited.Sera were collected and pooled within the same group.Multiple Affinity Removal System(MARS) was used for high-abundance serum proteins depletion and low-abundance proteins collection. SDS-PAGE was applied to assess the effect of MARS with the original sera and the low-abundance protein samples.After protein digestion by trypsin,iTRAQ reagents 114 and 117 were marked on peptides of HCC and hepatitis patients respectively.After being handled by Strong cation exchange chromatography (SCX),marked peptides were tested by LC-ESI-MS/MS.The software ProteinPilot was selected to make a comparison between the two different serum protein mixture. Results:With the depletion of high-abundance proteins by MARS system, low-abundance proteins have been well collected.There were 40 fractions after SCX handling,resulting in a total of more than 49,784 peptide fragments detected in the MS-MS system.Two hundred and fifty one proteins were identified through online spectra library search,twenty one of them presented quitely differently(more than 1.5 times the difference) in the two different serum pools.Conclusion:ITRAQ and liquid chromatography-mass spectrometry can be used for relative quantified detection of HCC proteomic biomarkers. There are significant difference of serum protein levels between patients with chronic hepatitis B and HCC.