iTRAQ–2DLC–ESI–MS/MS Based Identification of a New Set of Immunohistochemical Biomarkers for Classification of Dysplastic Nodules and Small Hepatocellular Carcinoma

The study aims to develop novel clinical immunohistochemical biomarkers for distinguishing small hepatocellular carcinoma (sHCC) from dysplastic nodules (DN). iTRAQ–2DLC–ESI–MS/MS technique was used to screen immunohistochemical biomarkers between precancerous lesions (liver cirrhosis and DN) and sHCC. A total of 1951 proteins were quantified, including 52 proteins upregulated in sHCC and 95 proteins downregulated in sHCC by at least 1.25- or 0.8-fold at p < 0.05. The selected biomarker candidates were further verified using Western blotting and immunohistochemistry. Furthermore, receiver operation characteristics (ROC) curves and logistic regression model were carried out to evaluate the diagnostic values of the biomarkers. Finally, aminoacylase-1 (ACY1) and sequestosome-1 (SQSTM1) were chosen as novel candidate biomarkers for distinction of sHCC from DN. A constructed logistic regression model included ACY1, SQSTM1, and CD34. The sensitivity and specificity of this model for distinguishing sHCC from DN was 96.1% and 96.7%. In conclusion, ACY1 and SQSTM1 were identified as novel immunohistochemical biomarkers distinguishing sHCC from DN. In conclusion, expression levels of CD34, ACY1, and SQSTM1 can be used to establish an accurate diagnostic model for distinction of sHCC from DN.

本研究旨在开发新型的临床免疫组化生物标志物，以区分小肝癌（sHCC）与不典型增生结节（DN）。研究采用了iTRAQ-2DLC-ESI-MS/MS技术，对癌前病变（肝硬化和DN）与小肝癌之间的免疫组化生物标志物进行筛选。共量化了1951种蛋白质，包括在sHCC中上调的52种蛋白质和至少以1.25-或0.8倍下调的95种蛋白质，p值均小于0.05。选定的生物标志物候选者进一步通过Western blotting和免疫组化进行验证。此外，通过受试者工作特征（ROC）曲线和逻辑回归模型评估了生物标志物的诊断价值。最终，氨基酸酰化酶-1（ACY1）和Sequestosome-1（SQSTM1）被选为区分sHCC与DN的新型候选生物标志物。构建的逻辑回归模型包括ACY1、SQSTM1和CD34。此模型在区分sHCC与DN方面的灵敏度和特异性分别为96.1%和96.7%。综上所述，ACY1和SQSTM1被鉴定为区分sHCC与DN的新型免疫组化生物标志物。总结而言，CD34、ACY1和SQSTM1的表达水平可用于建立准确区分sHCC与DN的诊断模型。