Prognostic value of legumain in uveal melanoma

Abstract

The present study aimed to assess the expression of legumain in uveal melanoma (UM) cell lines and primary UM specimens, and to determine the possible association between legumain expression and clinical as well as pathological characteristics to reveal its impact on the prognosis of patients with UM. Records of primary UM cases treated at Beijing Tongren Hospital and Tianjin Eye Hospital between 1996 and 2005 were retrieved for analysis and a total of 82 patients with uveal melanoma were included in the study. The expression of legumain in the formalin‑fixed and paraffin‑embedded surgical specimens of these 82 patients was determined using immunohistochemical analysis. In addition, the expression of legumain was examined in two uveal melanoma cell lines using polymerase chain reaction and western blot analyses. The association of legumain expression with clinical/pathological characteristics was analyzed using the χ2 and Fisher's exact test. In addition, the impact of legumain on the prognosis of patients with uveal melanoma was examined. Upregulation of legumain was more predominant in the highly invasive uveal melanoma cell line MUM‑2B compared with that in the MUM‑2C with low invasiveness. Of 82 primary uveal melanoma tissues, 35 exhibited high expression of legumain, while the other 47 specimens exhibited low or negative expression of legumain. High legumain expression was primarily associated with local invasion of UM. Overall survival analysis revealed that the patients with high legumain expression exhibited poorer survival than patients with low/negative legumain expression. These findings suggested that upregulation of legumain is associated with malignant behavior of UM and that legumain may be used as an negative prognostic factor as well as a therapeutic target.

Figure 1

Immunofluorescence images of the MUM-2B and MUM-2C uveal melanoma cell lines (scale bar, 10 µm). Left panel, legumain was expressed in the two uveal melanoma cell lines; middle panel, nuclear staining with DAPI (blue); right panel, merged images.

Figure 2

Semi-quantitative analysis of legumain expression in the MUM-2B and MUM-2C uveal melanoma cell lines. (A) Gels showing the polymerase chain reaction amplification products of legumain and GAPDH mRNA in MUM-2B and MUM-2C cells. (B) Quantified expression of legumain mRNA relative to GAPDH in MUM-2B and MUM-2C cells (^**P<0.01; n=3 for each cell line). The mRNA levels in MUM-2B and MUM-2C cells were 0.89±0.03 and 0.37±0.11, respectively. (C) Western blot showing the expression of legumain protein in MUM-2B and MUM-2C cells. (D) Quantified expression levels of legumain protein relative to β-actin in MUM-2B and MUM-2C cells (^*P<0.05; n=3 for MUM-2B and n=5 for MUM-2C). The expression of legu-main protein in MUM-2B and MUM-2C cells was 0.82±0.02 and 0.48±0.09, respectively.

Figure 3

Expression of legumain in three UM cell types. Left panel, H&E staining showing the morphological classification of the three types of UM cell as (A) epitheloid, (B) mixed and (C) spindle-like. Representative immunohistochemical images of low legumain expression in (D) epithelioid, (E) mixed and (F) spindle-like UM cells. In mixed-cell UM, legumain was preferentially expressed in epithelioid cells (red arrows) rather than in spindle-like cells (green arrows). Representative immunohistochemical images of high legumain expression in (G) epithelioid, (H) mixed and (I) spindle-like UM cells (scale bar, 10 µm). UM, uveal melanoma; H&E, hematoxylin and eosin.

Figure 4

Analysis of the association between legumain expression and prognosis. (A) Kaplan-Meier survival curves of all 82 patients. The patients with high legumain expression exhibited poorer survival as compared to patients with low/negative legumain expression (log-rank test; P<0.001). (B) Kaplan-Meier survival curves of the 54 patients with local invasion. The patients with high legumain expression still exhibited poorer survival as compared to patients with low/negative legumain expression (log rank test, P<0.05).