J Pathol Transl Med.  2023 Mar;57(2):113-122. 10.4132/jptm.2023.02.01.

Clinicopathologic significance of the delta-like ligand 4, vascular endothelial growth factor, and hypoxia-inducible factor-2α in gallbladder cancer

• Park S
• Kim J
• Jang W
• Kim KM
• Jang KT

• KMID: 2540497
• DOI: http://doi.org/10.4132/jptm.2023.02.01

Abstract

Background
Gallbladder cancer (GBC) is usually detected in advanced stages with a low 5-year survival rate. Delta-like ligand 4 (DLL4), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-2alpha (HIF2α) have been studied for their role in tumorigenesis and potential for therapeutic target, and multiple clinical trials of the agents targeting them are ongoing. We investigated the expression of these markers in surgically resected GBC and tried to reveal their association with the clinicopathologic features, mutual correlation of their expression, and prognosis of the GBC patients by their expression.
Methods
We constructed the tissue microarray blocks of 99 surgically resected GBC specimens and performed immunohistochemistry of DLL4, VEGF, and HIF2α. We used the quantitative digital image analysis to evaluate DLL4 and VEGF expression, while the expression of HIF2α was scored manually.
Results
The expression of VEGF and HIF2α showed a significant trend with tumor differentiation (p= .028 and p= .006, respectively). We found that the high DLL4 and VEGF expression were significantly correlated with lymph node metastasis (p= .047, both). The expression of VEGF and HIF2α were significantly correlated (p < .001). The GBC patients with low HIF2α expression showed shorter recurrence-free survival than those with high HIF2α expression.
Conclusions
This study suggested the possibility of the usage of DLL4 and VEGF to predict the lymph node metastasis and the possibility of VEGF and HIF2α to predict the expression level mutually. Further studies may be needed to validate our study results and eventually accelerate the introduction of the targeted therapy in GBC.

Keyword

Gallbladder neoplasms; DLL4; Vascular endothelial growth factor; HIF2α; Targeted therapy

Figure

• 

  Fig. 1. Representative images of expression of anti-DLL4 and anti-VEGF antibodies in gallbladder cancer of each group, according to the Hscore calculated via Aperio ImageScope software. (A) High membranous expression of anti-DLL4 antibody (H-score 208.43). (B) Low membranous expression of anti-DLL4 antibody (H-score 40.28). (C) High cytoplasmic expression of anti-VEGF antibody (H-score 214.01). (D) Low cytoplasmic expression of anti-VEGF antibody (H-score 34.36). DLL4, delta-like ligand 4; VEGF, vascular endothelial growth factor.

• 

  Fig. 2. Images of hematoxylin and eosin stain (A) and expression of anti-DLL4 (B), anti-VEGF (C), and anti-HIF2α (D) antibodies in the corresponding area, showing tumor and surrounding soft tissue, including vessel, muscle, and nerve. Expression of anti-HIF2α antibody shows extensive background stain compared to other markers, which led to manual scoring of H-score. Digital image analysis would mistake the background staining and overestimate the H-score of such cases. DLL4, delta-like ligand 4; VEGF, vascular endothelial growth factor; HIF2α, hypoxia-inducible factor-2α.

• 

  Fig. 3. Representative images of HIF2α immunohistochemistry according to intensity score: (A) score 0, (B) score 1, (C) score 2, and (D) score 3. Background staining was intense in smooth muscles and lymphocytes. HIF2α, hypoxia-inducible factor-2α.

• 

  Fig. 4. The correlation matrix visualizing the correlation between the expression levels of DLL4, VEGF, and HIF2α (A). The Spearman correlation coefficients are recorded in the center of each box. The scatter plot showing the positive correlation between VEGF and HIF2α (B). DLL4, delta-like ligand 4; VEGF, vascular endothelial growth factor; HIF2α, hypoxia-inducible factor-2α.

• 

  Fig. 5. Kaplan-Meier survival curves according to HIF2α expression. (A, B) Recurrence-free survival curves for differently defined high/low expression groups. HIF2α, hypoxia-inducible factor-2α.

Reference

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