Martin L, Green B, Renshaw C, Lowe D, Rudland P, Leinster S J, Winstanley J
Department of Surgery, University of Liverpool, UK.
Br J Cancer. 1997;76(8):1046-54. doi: 10.1038/bjc.1997.506.
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.
在一些（但并非所有）研究中，据报道，通过微血管密度测量的血管生成强度与浸润性乳腺癌的不良预后相关。这些差异的原因可能是所使用方法的不同。用于识别微血管的单克隆抗体、计数的高密度区域或“热点”数量以及用于统计分析的取值类型（最高计数或平均计数）在不同研究中有所不同。我们评估了三种常用单克隆抗体中哪一种能在浸润性乳腺癌中最佳显示微血管，并采用了能为每种试剂提供可重复数据的方法来确定最佳的“热点”计数数量。因此，对174例原发性乳腺癌福尔马林固定石蜡包埋标本中的微血管进行免疫组织化学染色，分别使用针对FVIIIRAg、CD31和CD34的单克隆抗体，每种抗体在200倍放大倍数下计数十个视野。使用最高计数以及三次、五次和十次计数中的最高值的平均值来检验微血管密度与中位随访时间为7.1年的患者总生存期之间的关系。与针对FVIIIRAg的抗体相比，针对CD31和CD34的抗体识别出更多的血管（每平方毫米最高计数中位数：CD31 = 100，CD34 = 100，FVIIIRAg = 81）。然而，针对CD31的单克隆抗体是最不可靠的抗体，免疫组织化学染色的切片仅占87%，而针对CD34的单克隆抗体为98%，针对FVIIIRAg的单克隆抗体为99%。不同抗体染色的血管数量之间存在高度相关性，尽管同一标本的连续切片用不同抗体实际计数存在一些显著差异。患者可分为微血管密度高和微血管密度低两组。使用Kaplan-Meier生存曲线，无论采用何种记录最高血管密度的方法，所有三种抗体的血管密度与生存率之间都密切相关。使用对数秩检验和Cox回归分析，抗CD34在三种抗体中给出的结果最显著，而在高低分组中以第75百分位数进行简单划分与患者生存率的相关性最佳。对于抗CD34，最高微血管密度（P = 0.0014）和最高三次微血管密度的平均值（P = 0.004）与患者死亡显示出良好的相关性，而对于抗CD31（P = 0.008）和抗FVIIIRAg（P = 0.007），使用Cox回归分析时最高计数的相关性最佳。
