Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast

Abstract

Introduction: Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1alpha and its downstream targets in phyllodes tumors and fibroadenomas of the breast.

Methods: The expression of HIF-1alpha, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium.

Results: Only two fibroadenomas displayed low-level stromal HIF-1alpha reactivity in the absence of CAIX expression. Stromal HIF-1alpha expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1alpha expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1alpha overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032).

Conclusion: These results indicate that HIF-1alpha expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1alpha most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1alpha and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1alpha and CAIX seem to be of minor relevance in breast fibroadenomas.

Figure 1

Examples of immunostaining for hypoxia-inducible factor 1 alpha (HIF-1α) and carbonic anhydrase IX (CAIX) in breast phyllodes tumors. (a) Malignant phyllodes tumor with stromal CAIX expression. (b) Same tumor as (a) with topographically overlapping HIF-1α overexpression. (c) Benign phyllodes tumor with CAIX-positive staining epithelium. (d) Borderline phyllodes tumor with HIF-1α overexpression in normal appearing epithelium and in subepithelial stroma.

Figure 2

Hot-spot microvessel counts in fibroadenomas and phyllodes tumors. Boxplot showing the large overlap in the numbers of microvessels between fibroadenomas (FA), benign phyllodes tumors (BePT) and borderline phyllodes tumors (BoPT) when counted in the hot-spot. MaPT, malignant phyllodes tumor.

Figure 3

Disease-free survival according to hypoxia-inducible factor 1 alpha (HIF-1α) status. The Kaplan–Meier survival curve illustrating disease-free survival for patients with breast phyllodes tumors with high expression (≥1%) versus low expression (<1%) of HIF-1α in the stromal component. Numbers of patients at risk at different time points are displayed below the horizontal axis.