Angiogenesis in ductal carcinoma in situ (DCIS) of the breast

Abstract

Background Up to 50% of recurrences of ductal carcinoma in situ (DCIS) of the breast are associated with invasive carcinoma but no pathological or molecular features have yet been found to predict for the development of invasive disease. For a tumour to invade, it requires the formation of new blood vessels. Previous studies have described a vascular rim around ducts involved by DCIS raising the possibility that the characteristics of periductal vascularisation may be important in determining transformation from in situ to invasive disease as well as risk of recurrence. Hypothesis I hypothesized that the risk of malignant tranformation (from normal breast to DCIS) and of invasive recurrence, following initial diagnosis and treatment of DCIS, is associated with the extent and pattern of periductal vascularity. It is likely that the periductal vessels are most important in this respect as incipient invasion is most likely to be associated with changes in vessels in the immediate vicinity of the tumour cells. Methodology I investigated to see whether changes in vasculature are related to the progression of in situ to invasive carcinoma and if so, which factors may be important in this change. I initially studied vascular density surrounding DCIS with and without invasive cancer and its relationship to specific angiogenic factors secreted by the tumour. Periductal vascular density and phenotype were determined using morphometry and a panel of anti-endothelial antibodies (von Willebrand factor [vWF], CDS31, CD141 and CDS34). These were related to the presence of invasive carcinoma at initial diagnosis, the histological features of DCIS and the risk of recurrence. For each histological section, the number of stained vessels and endothelial cells within 100 micrometers of foci of DCIS were counted. Up to 50 foci of DCIS on a single slide were scored and the microvessel density (MVD) and endothelial cell density (ED) were calculated for each focus. Normal lobules at least 2 mm away were used as controls. These studies suggested a change in phenotype and dual fluorescence immunostaining for CDS4+ and vWF+ vessels was carried out to confirm the change. The relationship between Thymidine Phophorylase (TP) expression on sections of pure DCIS and periductal vascularity was assessed as it had been previously shown to correlate with stromal vascular density. The relationship between stromal (hotspots) and periductal vascularity was also evaluated. Results Compared to normal lobules, pure DCIS exhibited a greater density of CD34+ and CDS1+ vessels but a decrease in those that were immunopositive for von Willebrand factor (vWF). DCIS associated with invasive carcinoma showed a profile of vascular immunostaining similar to that of pure DCIS but there were statistically significantly greater numbers of CDS4+ and CD141+ vessels and fewer staining for vWF. There was a significant negative correlation between vascular density and both the cross-sectional areas of the ducts involved and the extent of the necrosis of the tumour they contained. A correlation between vascular density and nuclear grade was also noted, being highest in the intermediate grade. On dual staining, the number of vessels stained only by CD34 was significantly higher around DCIS compared to adjacent normal lobules. For TP, although a relationship was seen between the H-score and MVD, a relationship with recurrence was not identified. Vascularity stained by anti-CD34 around DCIS which recurred was statistically significantly higher than those which did not. There was a significant and positive correlation between the two types of vascularity (p<0.001) in sections stained with anti-vWF antibody, but not anti-CD34. Conclusions Blood vessels surrounding DCIS appear to have a different immunophenotype when compared with blood vessels surrounding normal breast lobules. Increases in vascular density, as detected with the CD34 antibody, correlates with recurrence and the development of invasive carcinoma. Recurrent disease does not appear to be related to TP expression by DCIS. Periductal MVD appears to be more important than stromal MVD in predicting for recurrence in DCIS.