Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a tissue biomarker for detection of early hepatocellular carcinoma in patients with cirrhosis

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors occurring mainly in patients with chronic liver disease. Detection of early HCC is critically important for treatment of these patients.

Methods: We employed a proteomic profiling approach to identify potential biomarker for early HCC detection. Based on Barcelona Clinic Liver Cancer (BCLC) staging classification, 15 early HCC and 25 late HCC tissue samples from post-operative HCC patients and their clinicopathological data were used for the discovery of biomarkers specific for the detection of early HCC. Differential proteins among cirrhotic, early, and late tissue samples were separated by two-dimensional gel electrophoresis (2-DE) and subsequently identified by mass spectrometry (MS). Receiver operating characteristic (ROC) curves analysis were performed to find potential biomarkers associated with early HCC. Diagnosis performance of the biomarker was obtained from diagnosis test.

Results: Protein spot SSP2215 was found to be significantly overexpressed in HCC, particularly in early HCC, and identified as heterogeneous nuclear ribonucleoprotein K (hnRNP K) by tandem mass spectrometry (MALDI TOF/TOF). The overexpression in HCC was subsequently validated by western blot and immunohistochemistry. ROC curve analysis showed that hnRNP K intensity could detect early HCC at 66.67 % sensitivity and 84 % specificity, which was superior to serum α-fetoprotein (AFP) in detection of early HCC. Furthermore, the diagnosis test demonstrated, when combined with hnRNP K and serum AFP as biomarker panel to detect early HCC at different cut-off value, the sensitivity and specificity could be enhanced to 93.33 % and 96 %, respectively.

Conclusions: hnRNP K is a potential tissue biomarker, either alone or in combination with serum AFP, for detection of early HCC. High expression of hnRNP K could be helpful to discriminate early HCC from a nonmalignant nodule, especially for patients with liver cirrhosis.

Figure 1

Overexpression of hnRNP K in HCC tissues. (A) Representative protein profile of tissue sample from a late HCC patient is shown. The black rectangle entails the protein spot SSP2215. (B) Different expression of hnRNP K among cirrhosis(n = 40), early HCC(n = 15), and late HCC(n = 25) tissues in 2-DE gel. The protein spot marked with a square on two-dimensional gel electrophoresis image is protein SSP2215. (C) Histograms with relative protein intensities shows protein spot SSP2215 overexpressed in early and late HCC tissues compared with cirrhosis tissue. ^☆, p < 0.01; ^☆☆, p < 0.01.

Figure 2

Validation of hnRNP K overexpressed in HCC of different tumor stages. (A) Representative Western blot analysis demonstrates hnRNP K overexpressed in different stages of HCC tumors compared to cirrhosis samples. β-actin was used as internal loading control. The ratio of hnRNP K/β-actin protein was measured using Scion Image Beta 4.03 software. (B) Histograms showed the average densitometric ratio of hnRNP K to β-actin in early and late HCC tissue compared with cirrhosis control (^☆, p < 0.05). (Early HCC/cirrhosis = 3.48; Late HCC/cirrhosis = 3.30). (C) Representative Immunohistochemical staining showed the expression level and cellular localization of hnRNP K in early, late HCC tissues, and peri-tumor cirrhosis tissues. Stronger signal was found at the nucleus than that in cytoplasma in tumor cells, but limited nucleus staining in cirrhotic tissues (Original magnification × 400).

Figure 3

Diagnostic performance of hnRNP K and serum AFP in detection of HCC. The diagnostic accuracy of hnRNP K and AFP, in terms of sensitivity and specificity, are presented by receiver operating characteristic (ROC) curve analysis. In figures 3B and 3 C, corresponding to hnRNP K and AFP, the area under the curve for hnRNP K is markedly better than for the AFP in detecting early HCC. (A) ROC curve of hnRNP K in detection of HCC from cirrhosis (AUC = 0.89, Sen = 93.33%, Spe = 75%). (B) ROC curve of hnRNP K in detection of early HCC from late HCC (AUC = 0.75, Sen = 66.67%, Spe = 84%). (C) ROC curve of serum AFP in detection of early HCC from late HCC (AUC = 0.60). (Sen = 64.29%, Spe = 56%, cut-off value ≥ 100 ng/mL; Sen = 64.29, Spe = 40%, cut-off value ≥ 400 ng/mL). the two cut-off points of AFP are indicated with arrow.