EIF4EBP1 Overexpression Is Associated with Poor Survival and Disease Progression in Patients with Hepatocellular Carcinoma

<div><p>Objective</p><p>EIF4EBP1 acts as a crucial effector in mTOR signaling pathway. Studies have suggested that EIF4EBP1 plays a critical role in carcinogenesis. However, the clinical significance and biological role of EIF4EBP1 in hepatocellular carcinoma (HCC) have not been elucidated. Therefore, we aimed to investigate the clinical significance of EIF4EBP1 in HCC.</p><p>Methods</p><p>Total 128 cases of HCCs were included in this study. EIF4EBP1 expression in HCC tissues was detected by qRT-PCR, Western blot and immunohistochemistry, respectively. Then the relationships between EIF4EBP1 expression and clinical features as well as survival were analyzed.</p><p>Results</p><p>The expression level of EIF4EBP1 mRNA is significantly higher in 60% (24/40) of fresh HCC tissues than that in the matched adjacent nontumor liver (NCL) tissues (P = 0.044). Similarly, EIF4EBP1 protein is notably upregulated in 8 HCC tissues (randomly selected from the 40 HCCs) measured by Western blot and is significantly increased in another 88 paraffin-embedded HCCs (53%, 47/88) by immunohistochemistry compared with the matched NCLs (P < 0.001). EIF4EBP1 protein expression in HCC tissues is significantly correlated with serum AFP (P = 0.003) and marginally significantly associated with pathological grade (P = 0.085), tumor number (P = 0.084), tumor embolus (P = 0.084) and capsulation (P = 0.073). Patients with higher EIF4EBP1 protein expression have a much worse 5-year overall survival (40.3% vs 73.6%) and 5-year disease-free survival (33.0% vs 49.0%) than those with low expression. Furthermore, Cox regression analysis shows that EIF4EBP1 protein is an independent prognostic factor for overall survival (HR, 2.285; 95% CI, 1.154–4.527; P = 0.018) and disease-free survival (HR, 1.901; 95% CI, 1.067–3.386; P = 0.029) in HCC patients.</p><p>Conclusions</p><p>Our results demonstrate for the first time that EIF4EBP1 mRNA and protein are markedly up-regulated in HCC tissues, and the protein overexpression is significantly associated with poor survival and progression, which provide a potential new prognostic marker and therapeutic target for HCC patients.</p></div

The presence of CD66b+ neutrophils in colorectal and lymph node metastatic CRC tissues.

<p>(A) High intratumoral neutrophil was observed in a CRC (case 52), in which more than 60 intratumoral cells revealed positive immunostaining of CD66b (<i>upper panel</i>, ×100). (B) A CRC case (case 37) demonstrated low intratumoral neutrophil, in which less than 60 intratumoral cells showed immunoreactivity of CD66b (<i>upper panel</i>, ×100). (C) The corresponding adjacent mucosal tissue of case 52 showed low neutrophil, in which less than 60 cells revealed immunostaining of CD66b (<i>upper panel</i>, ×100). (D) High intratumoral neutrophil was observed in lymph node metastatic tissue, in which more than 60 intratumoral cells revealed positive immunostaining of CD66b (<i>upper panel</i>, ×100). The <i>lower panels</i> indicated the higher magnification (×400) from the area of the box in A, B and C, respectively.</p

Immunohistochemical staining of EIF4EBP1 protein in HCC and NCL.

<p>Eighty-eight archival formalin-fixed paraffin-embedded paired HCC samples were analyzed by immunohistochemistry with EIF4EBP1-specific antibody. Shown are representative results. The lower panel shows magnified images of the upper panel. <b>(A)</b> and <b>(F)</b>: Immunostaining of HCC tumor area and the adjacent non-tumor area. <b>(B)</b> and <b>(G)</b>: Noncancerous liver tissue was scored as no staining for EIF4EBP1 (Score 0). <b>(C)</b> and <b>(H)</b>: Well-differentiated HCC was scored as weak staining for EIF4EBP1 (Score 1). <b>(D)</b> and <b>(I)</b>: Moderately differentiated HCC was scored as moderate staining for EIF4EBP1 (Score 2). <b>(E)</b> and <b>(J)</b>: Poorly differentiated HCC was scored as strong staining for EIF4EBP1 (Score 3). Note: N: Noncancerous liver tissue; T: Tumor tissue; A–E with x200 magnification; F–J with x400 magnification.</p

Univariate and multivariate analysis of different prognostic factors in 229 patients with colorectal carcinoma.

<p>*Log-rank test;</p>†<p>Cox regression model;</p>‡<p>Mean age;</p><p>HR indicates hazards ratio; CI indicates confidence interval; WHO indicates World Health Organization.</p

ROC curve analysis was employed to determine the cutoff value for high intratumoral CD66b+ neutrophil in colorectal carcinoma.

<p>The sensitivity and specificity for each outcome were plotted: tumor grade (<i>P</i> = 0.575, A), T stage (<i>P</i> = 0.029, B), N stage (<i>P</i> = 0.263, C), M stage (<i>P</i> = 0.002, D), clinical stage (<i>P</i> = 0.005, E), and survival status (<i>P</i><0.001, F).</p

The expression levels of both EIF4EBP1 mRNA and protein in HCCs are significantly higher than in the matched adjacent NCLs.

<p><b>(A)</b> The relative EIF4EBP1 mRNA level was examined by RT-PCR. Shown is the relative expression level of EIF4EBP1 mRNA in HCC tissues compared with the matched NCLs (mean±SD; n = 40, Paired <i>t</i> test, <i>P</i> = 0.042); <b>(B)</b> EIF4EBP1 protein expression levels in 8 HCC tissues and the matched NCLs were detected by Western blot (representative image for 4 pairs of HCC samples), and lower panel shows the relative expression levels of EIF4EBP1 protein. <b>(C)</b> The relative expression level in each HCC tissue by immunohistochemistry is compared with that in its corresponding NCL tissue; <b>(D)</b> Relative expression level of EIF4EBP1 protein in 88 HCC tissues and the matched adjacent NCL tissues as measured by immunohistochemistry. (Y axis indicates the immunostaining score).</p

Kaplan–Meier survival curves of overall survival and disease-free survival in HCC patients with high- and low-expression of EIF4EBP1 protein.

<p>HCC patients with high expression of EIF4EBP1 have significantly lower overall survival rate <b>(A)</b> (log-rank, <i>P</i> = 0.0013) and disease-free survival rate <b>(B)</b> (log-rank, <i>P</i> = 0.0156) than those with low expression of EIF4EBP1.</p

Survival curves according to infiltration status of intratumoral neutrophils in subsets of CRC patients with different histological grade and pT/pN status (log-rank test).

<p>(A) <i>Grade 2</i>, probability of survival of grade 2 patients with CRC: low intratumoral neutrophil, n = 93; high intratumoral neutrophil, n = 81. (B) <i>Grade 3</i>, probability of survival of grade 3 patients with CRC: low intratumoral neutrophil, n = 20; high intratumoral neutrophil, n = 16. (C) <i>pT2</i>, probability of survival of pT2 patients with CRC: low intratumoral neutrophil, n = 45; high intratumoral neutrophil, n = 31. (D) <i>pT3</i>, probability of survival of pT2 patients with CRC: low intratumoral neutrophil, n = 66; high intratumoral neutrophil, n = 63. (E) <i>pN0</i>, probability of survival of pN0 patients with CRC: low intratumoral neutrophil, n = 96; high intratumoral neutrophil, n = 73. (F) <i>pN1</i>, probability of survival of pN1 patients with CRC: low intratumoral neutrophil, n = 29; high intratumoral neutrophil, n = 31.</p

Area under the receiver (AUC) operating characteristic curve for each clinicopathological feature.

<p>CI indicates confidence interval.</p

Correlation between the clinicopathologic variables and intratumoral CD66b+ neutrophil in colorectal carcinoma.

<p>*Chi-square test;</p>†<p>Mean age;</p><p>WHO indicated World Health Organization.</p