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.

Summary It has been suggested that the selective loss of E-cadherin expression can generate invasiveness in human carcinoma cells and might be a predictor of metastasis. Frozen sections of samples from 44 patients, 43 with suspected large bowel cancer and one with a liver recurrence were examined for E-cadherin expression using the antibody 6F9 specific for the human E-cadherin molecule. Twelve of the 40 patients with carcinoma already had lymph node involvement at the time of surgery. Samples from the primary carcinomas of only nine of these 12 patients showed reduced E-cadherin expression. However, the one lymph node with metastatic spread examined did show reduced E-cadherin expression. Four of the 40 carcinoma patients had liver involvement at the time of surgery. The primary carcinoma samples from only three of these four patients showed reduced E-cadherin expression. In addition only two out of the three liver metastases examined showed reduced expression. The primary carcinoma samples from seven patients with no evidence of tumour spread also exhibited reduced expression. Overall, analysis of the data suggests that there is no absolute correlation between reduced E-cadherin expression and tumour spread in carcinomas of the large bowel. E-cadherin (also known as Arc-1, uvomorulin and cell CAM 120/80) is one of a group of functionally related, integral membrane glycoproteins responsible for calcium-dependent cell-cell adhesion. Cadherins are responsible for the movement and rearrangement of cell collectives during embryogenesisi (Takeichi, 1988) and for the orderly structure of differentiated tissue. Cell transfection studies with E-cadherin cDNA in rodent systems have demonstrated directly that cadherin molecules are involved in cell-cell binding (Nagafuchi et al., 1987;Mege et al., 1988). Moreover, Behrens et al. (1989) demonstrated that epithelial cells deprived of their E-cadherin function by the addition of anti-E-cadherin antibodies, not only became less adhesive but became able to invade collagen gels and embryonal heart tissue. Shimoyama and co-workers (1989) also reported that colonies of cultured epithelial cells became dissociated and mobile after the addition of anti-E-cadherin antibody.Expression of E-cadherin has been studied in tissue sections from a variety of well and poorly differentiated human tumours, but not those arising in the large bowel, using immunohistochemical and immunofluorescence techniques (Eidelman et al., 1989;Shimoyama et al., 1989;Pfisterer et al., 1990; Shimoyama & Hirohashi, 1991a,b;Schipper et al., 1991). For the most part, E-cadherin expression has been shown to correlate with differentiation status, with lower levels of expression being observed in poorly differentiated tumours. This correlation with differentiation status has been observed for ovarian carcinomas (Pfisterer et al., 1990) and squamous carcinomas of the head and neck (Schipper et al., 1991 (Hashimoto et al., 1989) and the absence of expression in a grade IV hepatocellular carcinoma that went on to ...