Assessment of angiogenesis in the hyperplasia preinvasive, invasive breast carcinoma sequence

Tissue factor (TF), the primary initiator of coagulation, has been shown to stimulate angiogenesis, which is crucial for the development and metastasis of solid tumours, in part by upregulating vascular endothelial growth factor A (VEGF). Angiogenesis in invasive breast cancer is well documented, but little is known about the role of angiogenesis in premalignant breast disease, or when the angiogenic switch occurs during the development of breast malignancy. This study therefore quantifies angiogenesis, VEGF and TF in the hyperplasia, preinvasive, invasive breast carcinoma sequence.

One hundred and eighty-seven serial sections of normal human breast (n = 12), benign hyperplastic breast (usual ductal hyperplasia; n = 35), premalignant hyperplastic breast (atypical ductal hyperplasia; n = 31), preinvasive cancer (ductal carcinoma in situ; low/intermediate grade, n = 23; high grade, n = 43) and invasive breast cancer specimens (n = 43) were immunohistochemically stained for CD31 (pan endothelial cell (EC) marker), endoglin (proliferating EC marker), VEGF and TF. The microvessel density (MVD), a surrogate marker for angiogenesis, was quantified using Chalkley grid analysis. VEGF staining was assessed semi-quantitatively and TF expression was graded as present or absent.

CD31 staining was observed in ECs in all of the breast specimens observed. There was a significant increase in MVD between normal and hyperplastic/preinvasive breast cancer tissue (P < 0.005) and between preinvasive and invasive carcinomas (P < 0.0005), which was associated with a significant increase in VEGF expression in breast epithelial (P < 0.0005) and tumour cells, respectively (P < 0.0005). The significant increase in MVD observed between preinvasive and invasive cancers was also associated with a significant increase in TF expression in invasive tumour cells (P < 0.0005). In contrast to CD31 staining, endoglin was not expressed in normal breast, but was expressed by ECs in 11% of usual ductal hyperplasia cases, 13% of atypical ductal hyperplasia cases, 17% and 26% of ductal carcinoma in situ cases (low/intermediate grade and high grade, respectively) and 81% of invasive breast cancer specimens. A significant increase in the number of proliferating ECs was seen in invasive cancers compared with all the classes of breast tissue examined (P < 0.0005). Moreover, the significant increase in proliferating ECs seen between preinvasive and invasive carcinomas was associated with a significant increase in VEGF and TF expression in invasive tumour cells (P < 0.0005). There was evidence for a close association between VEGF and TF in tumour cells of invasive cancers (P = 0.007) and between VEGF and TF in ECs (P < 0.0005), suggesting a role for both in angiogenesis.

These data indicate that angiogenesis is initiated at the earliest stages of dysplasia and increases rapidly between preinvasive and invasive cancer. VEGF and TF expression patterns suggest these factors play a role in this process.

Affiliations

1. Microcirculation Research Group, Academic Unit of Surgical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, UK

   • JE Bluff
   • , NJ Brown
   • , MW Reed
   •  & CA Staton

2. Department of Pathology, School of Medicine and Biomedical Sciences, University of Sheffield, UK

   • SS Cross

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