Ezrin Polarization as a Diagnostic Marker for Circulating Tumor Cells in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide, with no precise method for early detection. Circulating tumor cells (CTCs) expressing the dynamic polarity of the cytoskeletal membrane protein, ezrin, have been proposed to play a crucial role in tumor progression and metastasis. This study investigated the diagnostic and prognostic potential of polarized circulating tumor cells (p-CTCs) in HCC patients. CTCs were isolated from the peripheral blood of 20 HCC patients and 18 patients with nonmalignant liver disease (NMLD) via an OncoQuick^® kit and immunostained with Ezrin-Alexa Fluor 488^®, CD146-PE, and CD45-APC. A fluorescence microscopy was then performed for analysis. The HCC group exhibited significantly higher levels of p-CTCs, with median values of 0.56 p-CTCs/mL, compared to 0.02 p-CTCs/mL (p = 0.03) in the NMLD group. CTCs were detected in 95% of the HCC patients, with a sensitivity of 95% and specificity of 89%. p-CTCs were present in 75% of the HCC patients, with a sensitivity of 75% and a specificity of 94%. Higher p-CTC counts were associated with the significantly longer overall survival in HCC patients (p = 0.05). These findings suggest that p-CTCs could serve as valuable diagnostic and prognostic markers for HCC. The incorporation of p-CTCs into diagnostic strategies could enhance therapeutic decision-making and improve patient outcomes.

Keywords: cancer; circulating tumor cells; ezrin; hepatocellular carcinoma; liver surgery; personalized diagnosis and therapy; polarization; single-cell polarity.

Figure 1

Schematic representation of the workflow. Created in BioRender. Juratli, M. (2023) BioRender.com/k09p009.

Figure 2

Examples of polarized CTCs from individual patient samples (A–J). Anti-Ezrin-Alexa Fluor 488 (green), nuclear staining with DAPI (blue), CD146 (red), leukocyte/CD45 (violet) and merged images of all the fluorescence channels. Observed at 40× magnification.

Figure 3

Boxplots comparing (A) CTCs and (B) p-CTCs between patients with HCC and those with NMLD. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times of the IQRs from the first and third quartiles. p-values and ξ-effect sizes were determined using one-way ANOVA, and p < 0.05 was considered significant.

Figure 4

Kaplan–Meier analysis of HCC patients with and without p-CTCs. (A) Overall survival (OS) curve and (B) recurrence-free survival (RFS) curve. HCC patients with p-CTCs exhibited a longer mean overall survival (40 ± 8 months vs. 26 ± 22 months, p = 0.05). However, the difference in mean recurrence-free survival between HCC patients with and without p-CTCs was not statistically significant (36 ± 13 months vs. 25 ± 22 months, respectively; p = 0.20).

Figure 5

Forest plot of hazard ratios for age > 70 years (A), metastasis (B), p-CTC (C), recurrence (D) and tumor size >5 cm (E) in HCC patients. Hazard ratios were calculated via univariate Cox regression analysis.