A novel definition of microvessel density in renal cell carcinoma: Angiogenesis plus vasculogenic mimicry

Abstract

The present study proposed the novel concept of total microvessel density (TMVD), which is the combination of the MVD and the vasculogenic mimicry (VM) status, and evaluated its clinical significance in patients with renal cell carcinoma (RCC). For that purpose, tumor samples from 183 patients with primary RCC were examined by CD34 single or periodic acid Schiff (PAS)/CD34 dual histology staining. MVD and VM were determined according to previous literature. Clinical information (tumor stage and grade, and duration of survival) was retrieved and analyzed. Survival information and VM-associated gene expression data of patients with RCC were also retrieved from The Cancer Genome Atlas (TCGA) database and the clinical significance of each individual gene was analyzed. The results indicated that MVD exhibited obvious differences among patients with RCC; however, it was not correlated with the stage/grade or length of survival in patients with RCC. In total, 81 patients (44.3%) were CD34(-)/PAS(+) and defined as VM(+), and they had a significantly shorter survival compared with that of VM(-) patients (P=0.0002). VM was not associated with MVD. TMVD was able to distinguish between patients with high and low MVD in terms of survival, thus TMVD was better compared with MVD alone at distinguishing between patients with different survival prognoses. TCGA data analysis revealed that among the VM-associated genes, nodal growth differentiation factor, caspase-3, matrix metalloproteinase-9 and galectin-3 had a statistically significant impact on the overall/disease-free survival of patients with RCC. In conclusion, the TMVD concept may be more appropriate and sensitive compared with the MVD or VM alone in predicting tumor aggressiveness and patient survival, particularly in RCC, which is a highly vascularized, VM-rich neoplasm, and certain VM formation-associated genes are negatively associated with the survival of patients with RCC.

Keywords: TCGA database; immunohistochemical staining; microvessel density; renal cell carcinoma; vasculogenic mimicry.

Figure 1.

MVD is not associated with the stage or grade in patients with RCC. (A-C) CD34 immunohistochemical staining of clear-cell RCC samples. MVD within hotspots was classified as (A) low (–30), (B) moderate (–50) and (C) high (60–80) (scale bar, 10 µm). Each condition is demonstrated with two representative images. (D and E) Comparison of the mean MVD between different (D) stages and (E) grades. RCC, renal cell carcinoma; MVD, microvessel density.

Figure 2.

VM tends to increase in patients with high stage or grade. (A-C) CD34/PAS dual staining in serial ccRCC sections. (A and B) As indicated in representative histology images, (A) VM(−) was defined as CD34(+)/PAS(+) and (B) VM(+) was defined as CD34(−)/PAS(+) (scale bar, 10 µm). Each condition is demonstrated with two representative images. (C) Comparison of VM status between patients with early and advanced stage. (D) Comparison of VM status between patients with low and high grade (E) Comparison of MVD between VM(−) and VM(+) cases. (F) Comparison of VM status between patients with low and high MVD. Black arrows indicate CD34(+) and red arrows PAS(+). VM, vasculogenic mimicry; MVD, microvessel density; PAS, periodic acid Schiff; ns, no significance.

Figure 3.

Survival analysis of genes closely associated with the formation of vasculogenic mimicry, which also shorten the overall survival and disease-free survival of patients with renal cell carcinoma, from The Cancer Genome Atlas database. The survival rate was expressed as the percentage. (A) Overall survival and (B) disease-free survival of patients with relatively high or low NODAL expression. Overall survival (C) Overall survival and (D) disease-free survival of patients with relatively high or low CASP3 expression. (E) Overall survival and (F) disease-free survival of patients with relatively high or low MMP9 expression. (G) Overall survival and (H) disease-free survival of patients with relatively high or low GAL3 expression. (I) Overall survival and (J) disease-free survival of patients with relatively high or low VE-cadherin expression. (K) Overall survival and (L) disease-free survival of patients with relatively high or low vimentin expression. EXP, expression; NODAL, nodal growth differentiation factor; CASP3, caspase 3; MMP9, matrix metalloproteinase 9; GAL3, galectin-3; VE-cadherin/CDH5, vascular endothelial cadherin; VIM, vimentin.

Figure 4.

VM rather than the MVD is able to distinguish patients with different survival prognoses, while the TMVD demonstrates superior discriminating capability compared with MVD. (A) Comparison of overall survival between patients with low and high MVD. The survival rate was expressed as the percentage. (B) Comparison of survival between VM(−) and VM(+) patients. (C) Comparison of survival between four different TMVD subgroups of patients. (D) Mechanistic diagram indicating the possible association between angiogenesis and VM via numerous associated genes. (E) Mechanistic scheme illustrating the function of angiogenesis and VM in supplying blood and promoting metastasis. VM, vasculogenic mimicry; TMVD, total microvessel density; VEGF, vascular endothelial growth factor; NODAL, nodal growth differentiation factor; CASP3, caspase 3; MMP9, matrix metalloproteinase 9; GAL3, galectin-3; VE-cadherin, vascular endothelial cadherin.