Endostatin gene variation and protein levels in breast cancer susceptibility and severity

Abstract

Background: Endostatin is a potent endogenous anti-angiogenic agent which inhibits tumour growth. A non-synonymous coding polymorphism in the Endostatin gene is thought to affect Endostatin activity. We aimed to determine the role of this Endostatin polymorphism in breast cancer pathogenesis and any influence on serum Endostatin levels in healthy volunteers. Endostatin protein expression on a breast cancer micro array was also studied to determine any relationship to genotype and to breast cancer prognosis.

Methods: The 4349G > A (coding non-synonymous) polymorphism in exon 42 of the Endostatin gene was genotyped in approximately 846 breast cancer cases and 707 appropriate controls. In a separate healthy cohort of 57 individuals, in addition to genotyping, serum Endostatin levels were measured using enzyme linked immunosorbant assay (ELISA). A semi-quantitative assessment of Endostatin protein expression on immunostained tissue micro arrays (TMA) constructed from breast cancer samples of patients with genotype data was performed.

Results: The rare allele (A) was significantly associated with invasive breast cancers compared to non-invasive tumours (p = 0.03), but there was no association with tumour grade, nodal status, vascular invasion or overall survival. There was no association with breast cancer susceptibility. Serum Endostatin levels and Endostatin protein expression on the tissue micro array were not associated with genotype.

Conclusion: The Endostatin 4349A allele is associated with invasive breast cancer. The Endostatin 4349G > A polymorphism however does not appear to be associated with breast cancer susceptibility or severity in invasive disease. By studying circulating levels and tumour Endostatin protein expression, we have shown that any influence of this polymorphism is unlikely to be through an effect on the levels of protein produced.

Figure 1

Endostatin genotyping results obtained from the Taqman Sequence Detection System. Samples are categorised into common homozygous or 1:1 (red), rare homozygous or 2:2 (blue) and heterozygous or 1:2 (green) groups.

Figure 2

Negative (a), weak (b), moderate (c) and strong (d) Endostatin staining in invasive breast cancer. Magnification ×200.

Figure 3

Distribution of the Endostatin Rare Allele (4349A) in patients with non-invasive and invasive tumours. The Rare allele was present in 2 of 44 patients with non-invasive tumours (4.5%) and 130 of 784 patients with invasive tumours (16.6%). Chi square test for trend; p = 0.033.

Figure 4

Intensity of Endostatin staining on the breast cancer tissue micro array and overall survival in invasive breast cancer. There were 9 events in 71 patients with mild staining, 7 events in 91 patients with moderate staining and 1 event in 17 patients with intense staining. (Total number = 179; log rank test statistic-1.83; df = 2; p = 0.40)