Study Of Novel Serum Liver Tumor Biomarkers And The Establishment Of Diagnostic Models For Liver Cancer

Objective:Liver cancer is a malignancy of the digestive system and has a high morbidity and mortality rate. However, the available diagnostic markers of liver cancer are unable to meet the demand of clinical diagnosis at present. The purposes of this study are to investigate the values of serum markers in the warning, early diagnosis and prognosis of malignant liver tumors, to establish the early diagnostic models and classification models for malignant liver tumors with serum markers and mass spectrometry technique, and to discover potential diagnostic polypeptide markers for malignant liver tumors.MethodsPart I The establishment of diagnostic models based on serum markers and evaluation of diagnostic values of serum markers for liver cancer(1) A retrospective cohort study was conducted and 109 cases of LC patients were followed up for 24 months. The relationship between the lifetime risk of developing HCC and levels of serum markers were analyzed. (2) The levels of serum markers of 142 HCC patients,93 SCHCC patients and 182 LC patients were examined. The early diagnostic moldels based on MLP, RBF and DA were constructed respectively to investigate the possibility of early diagnosis for the HCC by combining several serum markers. (3) A survival analysis was conducted with 36 HCC patients underwent surgery were followed up for 24 months. The relationship between the recurrence risk of HCC patients after surgery and levels of serum markers were analyzed.Part II Development of classification models for benign and malignant liver tumors using MALDI-TOF MS combined with MB-WCX and identification of novel peptide biomarkers.(1) Serum samples from 43 patients with malignant liver tumor and 52 patients with benign liver tumor were treated with MB-WCX kits and analyzed by the MALDI-TOF MS. Then we established GA, SNN and QC models to distinguish malignant from benign liver tumors. To confirm the clinical applicability of the models established, the blinded validation test was performed in 50 clinical serum samples. (2) Discriminatory peaks associated with malignant liver tumors were identified by a qTOF Synapt G2-S system.ResultsPart Ⅰ(1) 26.6 (29/109) percent of LC patients developed HCC during the study’s timeframe, statistical analysis showed that the serum levels of AFP, AFP-L3 and ALT in HCC patients were higher than those in non-HCC patients (t=-2.299,-2.384,-2.847; p<0.05); higher AFP and AFP-L3 levels are potential risk factors for malignization in LC patients (RR=2.41 and 2.26); serum GP73 level of LC patients decreased significantly after developed HCC (t=2.212; p=0.041). (2) The validation results of three models for training set and validation set are listed below:classification accuracy rate of MLP models were 66.7% and 63.6% respectively; RBF were 62.1% and 61.4%. DA models were 61.6% and 60.7%; (3) On univariate analysis, only GP73 was associated with tumor recurrence of HCC patients after surgery (x2=4.695, P=0.03). COX analysis showed that AFP-L3, Pt(s) and Pt(a) levels were independent risk factors for tumor recurrence (HR=1.003,3.465,1.137;p<0.05)Part Ⅱ(1) A total of 27 discriminant peaks in mass spectra of serum samples were found by ClinPro Tools Software. Recognition capability of models established were 100%(GA),89.38%(SNN) and 80.84%(QC), cross validation rates were 81.67%(GA), 81.11%(SNN) and 86.11%(QC). The accuracy rates of blinded validation test were 78%(GA),84%(SNN) and 84%(QC) respectively. (2) 3 peaks of m/z 2860.34, 2881.54 and 3155.67 were identified as a fragment of fibrinogen alpha chain, fibrinogen beta chain and Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) respectively.Conclusions:A 24 months flollow-up study confirmed that higher serum levels of AFP,AFP-L3,GP73,ALT were rik factors associated with the development of HCC. The detection of GP73,AFP,AFP-L3,Pt(s),Pt(a) before surgery has a certain guiding significance to predict the risk of tumor recurrence in HCC after surgery. We have established several diagnostic models for liver cancer based on serum tumor markers and peptide peaks. The identification results of mass spectrometry showed that FIBA, FIBB and ITIH4 peptides might be used as biomarkers for the diagnosis of malignant liver tumors.