| Objective:Hepatocellular carcinoma(HCC) is characterised with invisible early symptoms, high prevalence and poor prognosis, and is one of the most common malignant tumors worldwide. Hepatitis B virus(HBV) infection is considered as the most common risk factors for the development of HCC. The diagnosis of HCC depends on imaging examination, liver biopsy and serum levels of alpha feto-protein(AFP), so far the risk population of HCC is large and not all risk patients are able to receive routine imaging examination, while liver biopy, the considered golden standard, is a kind of invasive manipulation, the sampling accurancy and ocurrence of complications relies on the operating skills of doctors, and liver biopsy can not be performed on patients with significant liver cirrhosis. Serum markers for the diagnosis of HCC are quick, simple, convenient and affordable, but the diagnostic efficacy of AFP is not satisfied, the development of protein arrays facilitates the identification of new serum markers.Wnt signal pathway correlates with cell growth, differentiation, migration, embryo development and oncogenesis. Activation of Wnt pathway has been found in several tumors, and Wnt signal pathway is divided into canonical and non-canonical pathways. Glypican-3(GPC-3) is one of the heparan sulfate proteoglycans family members, and is able to upregulate the canonical and non-canonical Wnt pathways. several studies assessed the performance of GPC-3 on the diagnosis of HCC while provided different conclusions. Secreted frizzled related protein 4(s FRP-4) belongs to frizzled related protein family and have a potential Wnt binding site, and is considered as a regulator for Wnt signal pathway. s FRP-4 is an inhibitor in Wnt pathway, and the promoter area of s FRP-4 has been found with hypermethylation.We will first investigate the diagnostic value of GPC-3 for HCC using evidence-based medicine, then we will screen for candidate serum markers by protein arrays, and assess these potential serum markers in two independent populations.Methods:Part I:We performed a meta analysis of diagnostic accurancy. Studies that simultaneously assessed the serum levels of AFP and GPC-3 in HCC patients were enrolled. All citations were searched in EMBASE, Pubmed, Cochrane library and database from China. The name of the first author, country, publication time, baseline of patients, methods used for serum markers, cut-off values and other essential data were extracted by two independent authors. The quanlity of studies were assessed using QUADAS scale recommended by Cochrane, heterogeneity was discussed by threshold effect and non-threshold effect.Part II:16 patients dating 2013 to 2014 were enrolled from the outpatient department of infectious diseases, southwest hospital, 8 patients were diagnosed with HCC, 4 were with chronic hepatitis B(CHB) and 4 were with CHB related cirrhosis. After screening by protein arrays containing 507 cytokines, cytokines that were differentially expressed were obtained.Part III:All patients diagnosed with HCC and had not received anti-tumor therapy were enrolled from department of infectious diseases, southwest hospital from March 2013 to October 2014, and some controls were also enrolled. All subjects were divided into two populations(test group and validation group) according to the time enrolled, candidate serum markers with differential expression in part II were assessed in test group, those marker(s) with potential diagnostic values were validated in validation group, and compared with AFP.Part IV:A tissue array containing 30 patients and 60 samples(cancerous/paracancerous tissue each), the expression of candidate marker(s) were assessed in the tissue array by immunohistochemistry method.Results:Part I:A total of 543 citations were screened and 10 studies were finally obtained. Random effect model was used for data analysis. The poored sensitivity and specificity of GPC-3 were 59.2%(95%CI:55.1%-63.1%) and 84.8%(95%CI:82.0%-87.3%) in 10 studies, respectively, the DOR was 17.988(95%CI:5.361-60.353); the poored sensitivity and specificity of AFP were 51.9%(95%CI:47.7%-56.0%) and 94.0%(95%CI:92.1%-95.6%) with a DOR of 23.4(95%CI:10.291-53.207). the poored sensitivity and specificity of GPC-3 + AFP were 77.3%(95%CI:73.2%-81%) and 80.9%(95%CI:77.4%-84%)with a DOR of 21.255(95%CI:8.699-51.932) in seven studies.Part II:One downregulated cytokine CCR-7 and two upregulated cytokines FGF-23 and NT-3 in HCC were obtained from 507 cytokines. After deleted one patient, another 9 cytokines were obtained: s FRP-4, MIP-3 beta, IL-31 RA, PTX-3, insulysin/IDE, S100A10, MMP-2, P-selectin, IL-2 R alpha and RANK / TNFRSF11 A.Part III:The most promising cytokines CCR-7, FGF-23, NT-3 and s FRP-4 were assessed in test group, only s FRP-4 showed significant difference between HCC patients and non-HCC patients(P < 0.001). s FRP-4 also showed significant difference in validation group.In test group, the area under ROC(AUC) of s FRP-4 and AFP were 0.847(95%CI:0.788-0.906) and 0.857(95%CI:0.788-0.906), combined AUC was 0.941(95%CI:0.908-0.975). The sensitivity and specificity of s FRP-4 were 94% and 60.5% at the cut-off value of 46.37 ng/m L, the sensitivity and specificity of AFP were 75% and 84.9% at the cut-off value of 11.3 ng/m L, the combined sensitivity and specificity were 79.2% and 95.3%.In validation group, the AUC of s FRP-4 and AFP were 0.891(95%CI:0.839, 0.942) and 0.850(95%CI:0.781-0.919), combined AUC was 0.932(95%CI:0.890-0.975). The sensitivity and specificity of s FRP-4 were 78.6% and 84.4% at the cut-off value of 61.39 ng/m L, the sensitivity and specificity of AFP were 71.4% and 97.4% at the cut-off value of 13.5 ng/m L, the combined sensitivity and specificity were 79.2% and 95.3%.The s FRP-4 level did not correlate with AFP and blood sugar level(P > 0.05).Part IV:57 samples were positive and 3 samples were negtive in 60 samples of the tissue array. The stainings of s FRP-4 were stronger in paracancerous tissues than in cancerou tissues, and the stainings of s FRP-4 showed diluted trend as the decreased differentiation of tumor cells.Conclusion:Serum GPC-3 showed comparable diagnostic accurancy for HCC when compared with AFP, and the combination can increase the diagnostic sensitivity.Serum s FRP-4 levels were higher in HCC patients than in non-HCC patients and showed a comparative diagnostic value with AFP, their combination can increase the diagnostic efficacy of HCC.The expression of s FRP-4 were higher in paracancerous tissues than in cancerous tissues, and the expression level showed a positive correlation with the grade of differentiation. |
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