Comparative Evaluation of Angiogenesis Assessment with Anti-Factor-Vill and Anti-CD31 Immunostaining in Non-Small Cell Lung Cancer

Anti-Factor VIII vessel immunostaining has been widely used in the detection of angiogenesis in non-small cell lung cancer and other tumors. Several new antibodies have shown a higher sensitivity, and anti-CD31 has recently been proposed to be the standard for microvessel study. In the present study, we comparatively evaluated the two antibodies in 134 cases of early operable non-small cell lung cancer. The F8186 (anti-Factor Vill-associated antigen) and JC7O (anti-CD31) MoAbs were used in paraffin-embedded material. Eye appraisal of vascular grade (VG) and microvessel score (MS) was performed by three experienced pathologists. Different cutoff points were used for the analysis of VG and MS correlation with nodal involvement, overall survival, and thymidine phosphorylase expression. Intraand interobserver variability was minimal for both antibodies. MS and VG were significantly correlated with each other. However, 54 and 22% of cases with high anti-CD31 VG or high MS, respectively, had low vascularization on anti-Factor VIII asseSsment. Anti-CD31 scoring was significantly associated with nodal involvement and overall survival for all cutoff points considered, which was not verified for antiFactor VIII staining. VG was the most significant indicator of nodal involvement and survival for both antibodies. Tumors with high VG by anti-CD31 but low or medium VG by anti-Factor VIII behaved as tumors of high neoangiogenesis, defining a poor prognosis (P = 0.005) despite the failure of anti-factor VIII antibody to highlight intense neoangiogenReceived 7/1 7/97; revised 8/26/97: accepted 9/4/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at Department of Radiotherapy and Oncology. University Hospital of Iraklion, Iraklion 71 1 10. P. 0. Box 1352, Crete, Greece. Phone: 30-81-392498; Fax: 30-81-392848. esis. Anti-CD31 MS significantly associated with thymidine phosphorylase overexpression (P 0.01), whereas no correlation was found for anti-Factor VIII counting. It was concluded that anti-CD31 microvessel immunostaining has several advantages over anti-Factor VIII, being a more sensitive method for highlighting small, immature microvessels or single endothelial cells. This could be of importance in revealing possible correlation of tumor angiogenesis with metastatic behavior, prognosis, or angiogenic factor overexpression. Vascular grading was the best method for neovascularization assessment, efficiently defining groups of tumors with aggressive clinical course. INTRODUCTION Tumor neoangiogenesis as a metastasis-defining mechanism and also as a prognostic marker is under intense investigation (1). A large body of studies on immunohistochemical staining of microvessels and subsequent assessment of microvessel density shows an association of the degree of intratumoral neovascularization with metastatic disease and poor prognosis in a variety of human cancers, although several studies disagree (2). This may be due to methodology differences. Anti-Factor VIII antibody was one of the first used for microvessel staining and the most popular in the published clinicopathological studies. However, new antibodies with a higher specificity, such as anti-CD31, anti-CD34, and PAL-E, have recently been introduced (3-5). The first study correlating microvessel density with prognosis in non-small cell lung cancer was reported by Macchiarini et al. in 1992 (6). The anti-Factor VIII immunostaining used in that study was subsequently followed by four other studies (7-10) confirming the prognostic significance of microvessel density. The first study of non-small cell lung cancer using the anti-CD3 1 antibody was reported by Giatromanolaki et al. (1 1), showing a strong correlation of VG with node involvement and overall survival. Anti-CD34 MoAb was used by Fontanini et al. ( 1 2) to show a significant increase of microvessel density during disease progression form hyperplasia to in situ and invasive carcinoma. An international consensus on the methodology and criteria of evaluation of microvessel density proposed that anti-CD3 1 MoAb2 immunostaining should be the standard for microvessel assessment (13), although it is stressed that unresolved technical aspects require a comparative confirmation. In the present study, 3 The abbreviations used are: MoAb, monoclonal antibody; PECAM. platelet/endothelial cell adhesion molecule; MS. microvessel score; Ci, confidence interval; VG, vascular grade: HVG, high VG; MVG, medium VG; LVG. low VG: HLI, high lymphocytic infiltration; LLI, low lymphocytic infiltration; TP, thymidine phosphorylase. Research. on September 21, 2017. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from ) 5 10 15 20 25 30 35 40 45 50 556065707580859095100 Microvessel Counts 2486 Angiogenesis Assessment in Non-Small Cell Lung Cancer