Serum Proteomics Study Before And After Tace Therapy Of Liver Cancer

Transcatheter arterial chemoembolization (TACE) is widely recognized as the preferred method of non-surgical treatment of hepatic carcinoma and metastatic liver tumor. Evaluation of metastasis and recurrence after TACE for hepatic carcinoma currently is the integrated use of the means of imaging and serological methods such as periodic review of ultrasound, CT, MR, ECT, etc. serum AFP value is regularly reviewed for the AFP-positive patients. For many years Alpha-fetoprotein (AFP) has been a target of choice for screening diagnosis of hepatic carcinoma, but 20% to 40% of liver cancer patients with negative or low AFP.Emerging proteomics, is a subject studying all the proteins encoded by the genome. Through the help of Proteomics we can have a more detailed understanding of occurrence and development of tumor and able to find specific molecular markers to predict prognosis and treatment result.ClinProt technology (liquid-chip-time of flight mass spectrometry) is a promising new approach to proteome profiling of human serum based on magnetic bead separation and MALDI-TOF MS. It consists of bead separation system, MS system, analysis software and automatic processing system for fluid samples. It is a new application of proteomics technologies, a large number of groups have been using the technology in the study body fluids of patients and biomarkers for early diagnosis of cancer have been found. Purpose of this research is to search for molecular markers of the prognosis and efficacy of intervention therapy of hepatic carcinoma using the ClinProt technology and establish a model for diagnosis the disease and prediction of the efficacy, and identify the protein which has significant meaning for the therapy.Part OneThe establishment of the ClinProt platform and the preliminary study of serum markers for hepatic carcinoma with portal vein tumor thrombusPurpose To establish a stable ClinProt liquid platform for protein chip technology, choose the most optimal conditions, to evaluate the repeatability of the platform, then set up a screen potential protein biomarkers associated with portal vein thrombi (PVTT) formation in hepatocellular carcinorma(HCC) and build a predictive model.Materials and Methods Serum sample from healthy people was selected and purified by magnetic bead separation system, detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) system. Serum peptide spectrum was evaluated and explore the best technical parameters for assessment of serum sample and stability and reliability of the technology. The serum samples from 40 HCC patients (20 with PVTT and 20 free of PVTT) were enriched by the magnetic bead and analyze by MALDI-TOF-MS. Diagnostic model was established using genetic arithmetic (GA) combined with statistical neural networks (SNN).Result We summarize the best ratio of the sample injection volume: substrate= 1:5 (ul).When the temperature and humidity are controlled at 20℃-25℃and 30%-50% the crystallization effect is the best and the MS peaks are optimal. The replication of within-run and between-run tests were less than 23% and reaches the standard of proteomics experiments.39 peaks with mass charge ratio (m/z) were found (P<0.05). Among them, 8 protein peaks with them/z value of 1466.52,5823.1,8933.09,5353.95,4055.19,4420.43,5337.19 and 4091.46m/z were selected to establish predictive model of PVTT. Specificity and accuracy of this model and 92.86% and 92.86 in predicting PVTT. Negative predictive value is 100%, and positive predictive value is 83.3%.Conclusion Serum proteomic profiling using the ClinProt system is a promising good approach for proteomics study, ClinProt system has good operability and repeatability, the specificity of the discriminant model provides a new means for liver cancer research. The eight protein peaks may be potential biomarkers of PVTT in HCC patients and the predictive model may have great clinical significance in predicting the formation of PVTT.Part Two Preliminary study of serum markers for hepatic carcinoma patients before and after TACEPurpose Compare the serum proteomic patterns of a group of patients of hepatocellular carcinoma before and after TACE treatment. To explore the serum markers related to interventional radiolody.Materials and Methods Select 20 cases of hepatocellular carcinoma patients which the TNM stage wasⅠ,Ⅱ. Patients were all in the early morning fasting state, and we got 5ml sera from them before TACE(t0). Then we performed TACE by selectively introducing a microcatheter into the right or the left hepatic artery or a segmental branch of the hepatic artery and injecting a mixture of iodized oil (Lipiodol; Andre Guerbet, Aulnaysous-Bois,France) and epirubicin hydrochloride (30~50mg per body surface). This was followed by the introduction of a gelatin sponge (lmm*lmm*lmm). And after 45days (t1)we got another 5ml sera. And among them 8 patients got the third blood drawings 45 days after they were treated with TACE for the second time(t2). Clinical parameters (etiology, tumor grading, AFP levels) were recorded. The choice of TACE as treatment was based on the AASLD-EASL guidelines. The serum samples were purified using magnetic bead separation system and then tested with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and differences protein peak before and after the therapy were studied.Result The total of 20 patients in this group had undergone a total of 62times of TACE, an average of 3.1 times interventional therapy in 9.9 months, all patients survived, and 3 patients went for radical surgical resection,1 patient accepted liver transplantation,3 cases of PD,11 cases SD,2 cases of PR. One patients had arterioportal fistula, no patients had portal vein tumor thrombus or metastasis. After the establishment of serum protein spectrum, During m/z 800Da-10000Da range, a total of 107 protein peaks were detected,7 of which were statistically significant (P<0.05,3883,5634,6072,4645,8934,6091,6112 m/z).8 patients had a second TACE, use of 3883,5634 m/z further verification, the protein peaks have value of a certain distinction.Conclusion Comparing the serum proteomic patterns of patients of hepatocellular carcinoma before and after TACE treatment,3883, 5634,6072,4645,8934,6091,6112 m/z protein peaks were statistically significant and may have potential clinical value. We intend to identify the 3883Da peak in the next part.Part Three Identification of different protein concerning TACEPurpose To identify of the most significantly different protein peak(3883Da) in previous part of study and speculate the role it plays in liver cancer and its meaning before and after TACE.Materials and Methods Identification of the most significantly different protein peak 3883Da in previous part of study. Select ten high 3883Da peak expression cases for protein enrichment. Serum proteins were enriched and purified from the mixture of the sera of above ten using Clinprot magnetic beads.The purified protein was analyzed by using a LC-MS/MS system. Use data analysis software BioworksBrowser 3.3.1 SP1 for Sequest TM search. Database searching was performed with the program IPI Human (3.45). Parent ion error is set to 50ppm, fragment ion error set 1Da, non-endonuclease digestion, variable modification for the methionine oxidation.Result Through the identification of LC-MS/MS system, the amino acid sequence of 3883Da peak is "R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-".Searching the database, we confirm it as the peptide fragment of SERPINA5 (Plasma serine protease inhibitor) also Protein C inhibitor or Plasminogen activator inhibitor 3.Conclusion We identify the most significantly different protein peak (3883Da) using LC-MS/MS system successfully. The protein peak is Plasminogen activator inhibitor 3(PAI-3). Review related articles and concern with our study, we speculate PAI-3 plays important role in the occurrence and development liver cancer. Further investigation and validation the PAI-3 may help to in-depth the understanding of TACE for liver cancer will help to improve the effect of TACE treatment.