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.

本研究旨在开发用于区分小肝细胞癌（small hepatocellular carcinoma，sHCC）与发育异常结节（dysplastic nodules，DN）的新型临床免疫组织化学生物标志物。本研究采用同量异位素标记相对和绝对定量-二维液相色谱-电喷雾串联质谱（iTRAQ–2DLC–ESI–MS/MS）技术，对癌前病变（肝硬化与DN）及sHCC之间的潜在免疫组织化学生物标志物进行筛选。共计定量检测到1951种蛋白质，其中在sHCC中上调的蛋白质有52种、下调的有95种，差异倍数分别至少达1.25倍与0.8倍，且差异具有统计学意义（p < 0.05）。通过蛋白质印迹法（Western blotting）与免疫组织化学法对筛选得到的候选生物标志物进行进一步验证。此外，采用受试者工作特征（receiver operation characteristics，ROC）曲线与逻辑回归模型，评估该类生物标志物的诊断价值。最终选定氨基酰化酶-1（aminoacylase-1，ACY1）与Sequestosome-1（SQSTM1）作为区分sHCC与DN的新型候选生物标志物。本研究构建了包含ACY1、SQSTM1与CD34的逻辑回归诊断模型，该模型区分sHCC与DN的灵敏度达96.1%，特异度达96.7%。综上，ACY1与SQSTM1可作为区分sHCC与DN的新型免疫组织化学生物标志物；CD34、ACY1及SQSTM1的表达水平可用于构建精准的sHCC与DN鉴别诊断模型。