Reduced Lipid Peroxidation Predicts Unfavorable Prognosis in Hepatocellular Carcinoma, but Not Intrahepatic Cholangiocarcinoma

Abstract

Primary liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), remains a significant contributor to cancer-related mortality worldwide. Oxidative stress and lipid peroxidation play a key role in chronic liver diseases and have been shown to be pivotal for tumor initiation and progression. 4-hydroxy-nonenal (4-HNE), one of the major mediators of oxidative stress and a well-established biomarker for lipid peroxidation, can act as a signal transducer, inducing inflammation and exerting carcinogenic effects. However, the role of 4-HNE in primary liver cancer remains poorly explored. In this study, we investigated 4-HNE levels in 797 liver carcinomas, including 561 HCC and 236 iCCA, by immunohistochemistry. We then correlated 4-HNE levels with comprehensive clinical data and survival outcomes. In HCC, lower expression levels of 4-HNE were associated with vascular invasion, a high tumor grade, a macrotrabecular-massive HCC subtype, and poor overall survival. Concerning iCCA, large duct iCCA showed significantly higher 4-HNE levels when compared to small duct iCCA. Yet, in iCCA, 4-HNE levels did not correlate with known prognostic parameters or survival outcomes. To conclude, in HCC but not in iCCA, low amounts of 4-HNE predict unfavorable survival outcomes and are associated with aggressive tumor behavior. These findings provide insights into the role of 4-HNE in liver cancer progression and may enable novel therapeutic strategies.

Keywords: 4-HNE; biomarker; hepatocellular carcinoma; intrahepatic cholangiocarcinoma; liver cirrhosis; reactive oxygen species.

Figure 1

Low intratumoral amounts of 4-HNE predict unfavorable outcomes in HCC patients. (a) Representative immunohistochemistry images demonstrating 4-HNE staining levels categorized as none, weak, moderate, and strong. Bars: 50 µm. (b) Examples of representative 4-HNE staining patterns in normal hepatocytes, hepatocellular carcinoma (HCC), relapsed HCC, and HCC metastasis. Boxplot illustrating the reduction of the 4-HNE immunoreactive score (IRS) from (c) surrounding liver tissue to primary HCC, (d) primary HCC to HCC relapses, (e) HCC to lymph node metastasis, and (f) HCC to distant metastases. (g) Kaplan–Meier plot of overall survival in HCC patients stratified by 4-HNE IRS levels. If a p value is less than 0.05, it is flagged with one star (*). If a p value is less than 0.001, it is flagged with three stars (***). No significance is labeled as “ns”.

Figure 2

Boxplots comparing 4-HNE levels in different HCCs. 4-HNE levels in (a) patients with and without liver cirrhosis and, if present, in (b) patients with different Child–Pugh scores. (c) Distribution of 4-HNE levels in HCC patients with no (V0), microscopic (V1), or macroscopic (V2) vascular invasion. (d) HCC patients are categorized based on tumor differentiation (G1: well differentiated, G2: moderately differentiated, G3: poorly differentiated). (e) HCC patients with and without glutamine synthetase (GS) overexpression. (f) Illustration of 4-HNE levels among various HCC subtypes. If a p value is less than 0.05, it is flagged with one star (*). If a p value is less than 0.01, it is flagged with 2 stars (**). If a p value is less than 0.001, it is flagged with three stars (***). No significance is labeled as “ns”.

Figure 3

Multivariate Cox regression analysis of overall survival on prognostic factors in hepatocellular carcinoma (HCC) patients. The squares represent the hazard ratios, and the horizontal lines show the confidence intervals. The analysis includes vascular invasion (0: none, 1: microscopic, 2: macroscopic), Barcelona Clinic Liver Cancer (BCLC) stage (A, B, C, D), immunohistochemically detected 4-HNE levels (low, high), and serum alpha-fetoprotein (AFP) levels (low, high; cutoff: 240 ng/mL). Note the significantly decreased risk of adverse outcomes in patients with high 4-HNE levels.

Figure 4

4-HNE immunohistochemistry in intrahepatic cholangiocarcinomas (iCCAs) of small (a,a’) and large (c,c’) duct types and their respective lymph node metastases (b,b’,d,d’). iCCAs demonstrate an overall weak staining reaction to 4-HNE, which is contrasted by strong staining intensity in the adjacent non-neoplastic hepatocytes ((a’), top right), lymphocytes ((b’), right; (d’)), and stromal cells (a’,c’). Bars: 100 µm.

Figure 5

Semiquantitative analysis of 4-HNE in intrahepatic cholangiocarcinoma (iCCA) as analyzed by immunohistochemistry. (a) Boxplot analysis demonstrating significantly increased 4-HNE levels in large duct iCCA. (b–d) No significant differences in 4-HNE levels between primary iCCA and lymph node and distant metastases ((b): iCCA, all types; c: small duct iCCA; (d): large duct iCCA). (e) Kaplan–Meier plot shows no significant differences between iCCA with high and low 4-HNE expression. If a p value is less than 0.01, it is flagged with 2 stars (**). No significance is labeled as “ns”.