| ObjectiveThis study aimed to detect the expression of thrombospondin 4（THBS4）in patients with hepatocellular carcinoma,and evaluate the individual and combined diagnostic value of serum THBS4,alpha-fetoprotein（AFP）,AFP-L3 and golgi protein 73（GP73）in the detection of hepatocellular carcinoma,in order to improve the diagnosis of AFP negative hepatocellular carcinoma.Methods1.Tumor tissues and the corresponding adjacent tissues were collected from 16 AFP negative HCC patients.2.A total of 270 serum samples and clinicopathological data were collected from 110 patients with hepatocellular carcinoma（HCC）（including 48 patients with AFP<20ng/ml,21 patients with AFP 20-200ng/ml and 41 patients with AFP>200ng/ml）,24 patients with intrahepatic cholangiocarcinom（ICC）,32 patients with liver cirrhosis（LC）,32 patients with chronic hepatitis B（CHB）,26 patients with other gastrointestinal malignancies,33 healthy individuals and 13 HCC patients after treatment.3.Western blotting and q RT PCR assays were used to detect the expression of THBS4 on protein and m RNA level in AFP negative HCC tissues and the corresponding adjacent tissues.4.The serum levels of THBS4 and GP73 of patients with HCC,ICC,LC,CHB,other gastrointestinal malignancies and healthy individuals were detected by ELISA assays,and the serum levels of AFP and AFP-L3 of those were detected by electrochemiluminescence.Then,calculate the AFP-L3 ratio（AFP-L3R）.5.The individual and combined diagnostic value of serum THBS4,AFP,AFP-L3 and GP73 in the detection of HCC was compared through ROC curve analysis,and the area under curve（AUC）,sensitivity,specificity,accuracy,positive and negative predictive value were calculated simultaneously.The serum THBS4 levels between patients with HCC and benign liver disease in different AFP concentration groups（<20ng/ml,20-200ng/ml,>200ng/ml）were compared.AUC,sensitivity and specificity were calculated by ROC analyses to evaluate and compare the diagnostic value of THBS4,AFP-L3 and GP73 in HCC with different AFP concentrations.Logistic regression was used to analyze the diagnostic value of THBS4,AFP-L3 and GP73 in AFP negative HCC.The expression of serum THBS4 in patients with HCC before and after different treatments were compared.The relationship between serum THBS4 levels and the clinical characteristics as well as disease progression of HCC patients were analyzed.Results1.The expression of THBS4 in AFP negative HCC tissues was higher than that in the adjacent tissues.2.The expression levels of serum THBS4,AFP,AFP-L3 and GP73 in each group: the level of serum THBS4 in patients with HCC was significantly higher than that in patients with LC,CHB and healthy individuals.The level of serum THBS4 in patients with ICC was also significantly higher than that in patients with LC,CHB and healthy individuals.The expression levels of serum AFP and AFP-L3 in patients with HCC were significantly higher than those in patients with LC,CHB,ICC,other gastrointestinal malignancies and healthy individuals（P<0.01）.The level of serum GP73 in patients with HCC was significantly higher than that in patients with CHB,other gastrointestinal malignancies and healthy individuals,but there was no significant difference between HCC patients and patients with ICC or LC.3.The diagnostic value of serum THBS4,AFP,AFP-L3 and GP73 for HCC: the AUC of serum THBS4 in differentiating HCC from LC,CHB and healthy individuals were0.709,0.703 and 0.782 respectively（P<0.001）,which was slightly worse than AFP（AUCs were 0.736,0.758 and 0.89,respectively）.The value of serum THBS4 in differentiating HCC from LC and CHB was slightly lower than that of AFP-L3（AUCs were 0.73 and 0.739,respectively）.The value of serum THBS4 in differentiating HCC from LC and CHB was better than that of GP73（AUCs were 0.593 and 0.672,respectively）.4.The diagnostic value of combined detection of serum THBS4,AFP,AFP-L3 and GP73 for HCC: when AFP and GP73 were combined with THBS4 respectively in distinguishing HCC from non-tumors（LC and CHB and healthy individuals）,the AUC was higher than that when detected individually,but the increase was not significant.When AFP-L3 was combined with THBS4,the AUC reached to 0.828,which was significantly higher than that when detected imdividually,but the sensitivity was only55.45% shows a poor ability to recognize early HCC.The diagnostic performance when combined analysis of THBS4,AFP,AFP-L3 and GP73 was optimal with the AUC of 0.846,and the sensitivity and specificity of 79.09% and 76.29%,respectively.5.Comparison of serum THBS4 levels between patients with HCC and benign liver disease（BLD）with different AFP levels: in AFP<20ng/ml group,the serum levels of THBS4 in patients with HCC were significantly higher than that in BLD patients.In AFP 20-200ng/ml group,the serum levels of THBS4 in patients with HCC were also significantly higher than that in BLD patients.In AFP>200ng/ml group,there was no significant difference of serum THBS4 levels between HCC patients and BLD patients.6.Comparison of the diagnostic performance of serum THBS4,AFP-L3 R and GP73 in the detection of HCC patients with different AFP levels: in AFP<20ng/ml group,the AUCs of diagnostic value of THBS4 in distinguishing HCC from LC,CHB,healthy individuals and non-tumor were 0.632,0.604,0.744 and 0.670 respectively.The diagnostic value of GP73 in distinguishing HCC from LC,CHB,healthy individuals and non-tumor were 0.6,0.752,0.959 and 0.740 respectively.The diagnostic value of AFP-L3 in distinguishing HCC from LC,CHB,healthy individuals and non-tumor were 0.531,0.531,0.521 and 0.531.THBS4 has certain diagnostic value for AFP negative HCC,which was equivalent to GP73 and superior to AFP-L3.In AFP20-200ng/ml group,the AUCs of THBS4 in differentiating HCC from LC,CHB and non-tumor were 0.836,0.976 and 0.879 respectively,which was generally equivalent to that of AFP-L3（AUCs were 0.876,0.815 and 0.857 respectively）,while the diagnostic performance of GP73 was poor.7.The diagnostic value of serum THBS4,AFP-L3 and GP73 individually and combined in the detection of AFP negative HCC: The results showed that the AUC of THBS4 was 0.67 in distinguishing AFP negative HCC from non-tumor patients when detected individually,which was superior to AFP-L3（AUC 0.531）.When combined THBS4 with AFP-L3 and GP73,the AUC value reached to 0.754 with a sensitivity of83.33% at a specificity of 67.5%,significantly improved compared to that with AFP-L3 or THBS4 detected individually（P<0.05）.8.The post-treatment serum THBS4 levels in HCC patients were lower than those before treatment.9.The serum level of THBS4 in HCC patients was significantly correlated with tumor number,differentiation degree,microvascular invasion and BCLC stage（P < 0.05）.Conclusions1.Serum THBS4 has a diagnostic value for HCC with negative AFP and slightly elevated AFP.2.The combination of serum THBS4 with AFP,AFP-L3 and GP73 can improve the diagnostic accuracy of HCC.3.Serum THBS4 can be used for postoperative monitoring and indication of disease progression in HCC patients. |
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