Examining the technique of angiogenesis assessment in invasive breast cancer

Abstract

Summary The intensity of angiogenesis as measured by the density of microvessels has been reported to be associated with a poor prognosis in invasive breast cancer in some, but not all, studies. The reasons for these discrepancies may be variations in the methodologies used. The monoclonal antibody used to identify the microvessels, the number of high-density areas or 'hotspots' counted and the type of value taken for statistical analysis (highest count or mean count) have varied between the different studies. We have assessed which of the three commonly used monoclonal antibodies provides the best visualization of microvessels in invasive breast cancer and have used methods that give reproducible data for the optimum number of 'hotspots' to count for each reagent. Thus, microvessels in formalin-fixed paraffin-embedded specimens from 174 primary breast cancers were immunohistochemically stained with monoclonal antibodies to FVIIIRAg, CD31 and CD34 and ten fields counted at 200 x magnification for each antibody. The highest count and the mean value of the highest of three, five and ten counts were used to examine the relationship between the density of microvessels and overall survival of patients with a median follow-up time of 7.1 years. Antibodies to CD31 and CD34 identified more vessels than antibodies to FVIIIRAg (median highest count per mm2: CD31 = 100, CD34 = 100, FVIIIRAg = 81). The monoclonal antibody to CD31, however, was the least reliable antibody, immunohistochemically staining only 87% of sections compared with 98% for the monoclonal to CD34 and 99% for the monoclonal to FVIIIRAg. There was a high degree of correlation between the number of vessels stained by the different antibodies, though there were some considerable differences in actual counts for serial sections of the same specimen stained by the different antibodies. Patients could be divided into two groups corresponding to those with high microvessel densities and those with low microvessel densities. Using Kaplan-Meier survival curves, there was a close association for all three antibodies between vessel density and survival whichever method of recording the highest vessel densities was used. Using log-rank tests and Cox's regression analysis, anti-CD34 gave the most significant results of the three antibodies, whereas a simple cut-off at the 75th percentile for the high and low groups produced the best association with patient survival. For anti-CD34 the highest microvessel density (P = 0.0014) and the mean value of the highest three microvessel densities (P= 0.004) showed a good correlation with patient death, whereas for anti-CD31 (P= 0.008) and anti-FVIIIRAg (P= 0.007) the highest count gave the best correlation using Cox's regression analysis.

Vascular density and phenotype around ductal carcinoma in situ (DCIS) of the breast

“…Both showed a significant increase in DCIS, with greater values in cases associated with invasive carcinoma. Our findings confirm those of Martin et al (1997), that CD34 antibodies give consistently higher vessel counts in breast carcinomas than antibodies against vWF or CD31.…”

von Willebrand factor expression in osteosarcoma metastasis

“…15 However, identification of vWF-producing osteosarcoma cells raises the possibility that other endothelial markers such as CD31 or CD34 may be more appropriate, especially for automated methods used to assess VD in tumors. 15,[36][37][38][39][40] Osteosarcomas with positive tumor cell staining tend to have high vWF mRNA expression (P ¼ 0.006), indicating that the osteosarcoma cells may contribute a significant proportion of the vWF mRNA expression seen in tumors. Furthermore, patients with paired tumor samples displayed higher vWF expression in the metastatic samples (P ¼ 0.005), suggesting that the metastatic process may select for tumor cells that express vWF more highly.…”

The role of microvessel density on the survival of patients with lung cancer: a systematic review of the literature with meta-analysis

“…Anti-CD34 antibody seems to be more reliable in terms of specificity and reproducibility than monoclonal antibodies generated against other endothelial cell antigens (Tanigawa et al, 1996). In invasive breast cancer, CD34 has been shown to yield higher microvessel values than CD31 or factor VIII (Martin et al, 1997) and does not stain any tumour or inflammatory cells as CD31 or factor VIII. CD31 is a transmembrane glycoprotein highly expressed in mature and immature endothelium and its localisation at the endothelial cell junctions suggests an important role in transendothelial migration.…”

“…An international consensus on the methodology and criteria of evaluation of microvessel density proposed that anti-CD31 monoclonal antibody should be the standard for microvessel assessment (Vermeulen et al, 1996) as it is superior on paraffin sections. But as CD34 has been shown to yield higher microvessel values than CD31 or factor VIII in breast cancer (Martin et al, 1997), it might be useful to combine CD34 and CD31 antibodies. In lung cancer, Offersen et al (2001) compared the staining with these three antibodies.…”