Expression of epidermal growth factor-like domain 7 correlates with clinicopathological features of osteosarcoma

Abstract

Epidermal growth factor-like domain 7 gene (EGFL7) encodes an angiogenesis related factor and plays a crucial role in many human cancers. Previous studies have suggestedthat EGFL7 acts as a facilitator for tumor angiogenesis. However, little is known as to its role in osteosarcoma. Our aim was to investigate the expression of EGFL7 and to explore its correlation with the clinicopathological features of osteosarcoma. Tumor tissues from 32 Chinese young patients (below age of 24) with osteosarcoma were collected and subjected to EGFL7 detection by immunohistochemistry. The tissues from 10 patients with osteochondroma were collected and analyzed as controls. The intratumoral microvessel density (MVD) was examined by immunohistochemical staining using CD34 antibody. The results showed that patients with osteosarcoma had higher levels of EGFL7 in the tumor tissues compared to patients with osteochondroma (p<0.001). The expression of EGFL7 was significantly higher in advanced osteosarcoma (Enneking IIB-III) than that in early tumor stage (Enneking IA-IIA) (p<0.01). There is also a significant correlation between increased expression of EGFL7 and the Enneking stage (R = 0.714, p<0.001). Moreover, we detected a higher level of EGFL7 expression in tumor tissues of patients with recurrent or metastatic (bone or lung) osteosarcoma than those without recurrence or metastasis after 3 years' follow-up (p<0.01). There is no detectable difference of EGFL7 expression between tumor tissues from different tumor location and sex. Finally, we showed that high level of EGFL7expression was significantly correlated with high MVD (R = 0.829, p<0.001). In conclusion, our study demonstrates for the first time that there was a tumor grade-dependent up-regulation of EGFL7 in osteosarcoma. Elevated EGFL7 expression correlated with poor clinical outcome: i.e. advanced tumor stage, recurrent and metastatic osteosarcoma. Our findings support EGFL7 as a potential prognostic marker, and may provide novel insights for the diagnostics and therapeutics of osteosarcoma.

Keywords: Osteosarcoma; clinicopathology; epidermal growth factor-like domain 7 (EGFL7); microvessel density (MVD); prognosis; tumor angiogenesis.

Figure 1

EGFL7 gene structure and its protein domains.The human EGFL7 gene is consisted four variants (V1, V2, V3 and V4) that encode three EGFL7 protein isoforms a-c. Compared to Variant 3, Variant 2 encodes a shorter isoform b. These variants encode three functional domains: an EMI domain (yellow box), a calcium-binding EGF-like domain (green box) and acoagulation Factor Xa inhibitory site domain (blue box). ATG: start codon; TGA: stop codon.

Figure 2

Varied EGFL7 expression in osteosarcoma patients and controls. (A, E) No expression of EGFL7 in controls, EGFL7 (-). (B-D, F-H) Expression of EGFL7 in different osteosarcoma tumor tissues: (B, F), EGFL7 (+); (C, G), EGFL7 (++); (D, H), EGFL7 (+++). (A-D, magnification ×400, E-H, magnification ×100).

Figure 3

EGFL7 expression in osteosarcoma and osteochondroma patients. Increased expression of EGFL7 in osteosarcoma tissues compare to osteochondroma, 93.8% osteosarcoma tissues had a positive EGFL7 expression.

Figure 4

EGFL7 expression in different stages of osteosarcoma. Increased expression of EGFL7 in advanced osteosarcoma (Enneking Stage), there was a positive correlation between EGFL7 expression in tissues and Enneking stage in patient with osteosarcoma (R = 0.714, p<0.001).

Figure 5

MVD staining in osteosarcoma patients and controls. CD34 staining was performed for MVD in tissues from osteosarcoma patients and osteochondroma control patients. The results showed that there were very low MVD staining in tissues from osteochondroma control patients (A, C), but significant higher MVD staining in tissues from osteosarcoma patients were observed (B, D, black arrows). (A, B, magnification 400×, C, D, magnification 100×).

Figure 6

EGFL7 expression and MVD staining in osteosarcoma. Relationship between EGFL7 and MVD in CD34 staining for MVD in tumor tissues was shown. Scattered plots showed the distribution of MVD in different levels of EGFL7 expression. Increased MVD were found in osteosarcoma tissues with higher EGFL7 expression (staining intensity ++ and +++). High MVD was found significantly correlated with the expression of EGFL7 in tumor tissues (R = 0.829, p<0.001).