[CANCER RESEARCH 55, 5049-5053, November 1. 1995] Angiogenesis in Colorectal Tumors: Microvessel Quantitation in Adenomas and Carcinomas with Clinicopathological Correlations1 Paola Bossi, Giuseppe Viale, Arthur K. C. Lee, RosaMaria Alfano, Guido Coggi, and Silvano Bosari2 // Department of Pathology ¡P. B., G. C.I and Department of Pathology [G. V.j, University of Milan School of Medicine. European Institute of Oncology [G. V.J, and Department of Anatomic Pathology (lit, 1RCCS Ospedale Maggiore [R-M. A.I, Milan 20100. Italy; Department of Anatomic Pathology. Ltihey Clinic Medical Center. Burlington. Massachusetts OÃOE805 ¡A.K. C. L.j; Department of Pathology, Deaconess Hospital and Harvard Medical School, Boston, Massachusetts 02215 ¡A.K. C. L.J; and Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520 ¡S.B.] ABSTRACT Angiogenesis is a crucial step in tumor growth and progression. Its quantitation by microvessel counting is of prognostic value in several types of malignancies. Scarce data are available on angiogenesis in gas trointestinal tumors. We studied 36 adenomas and 178 large bowel car cinomas to evaluate the onset of angiogenesis in colorectal tumorigenesis and to assess the prognostic significance of microvessel quantitation. Endothelial cells were immunostained with an anti-CD31 mAb; in each case three microscopic fields ( x 200) with the highest number of microves- sels were counted: the average value of the three fields was used to evaluate the significance of microvessel density (MVD). MVD of normal mucosa (41 cases) served as controls. MVD was 42 ±10 in the normal mucosa, 64 ±10 in adenomas, and 115 ±39 in carcinomas (normal versus adenomas, P < 0.001; adenomas versus carcinomas, P < 0.0001). The transitional mucosa adjacent to carcinomas displayed intermediate levels of MVD (89 ±23; P < 0.001 versus adenomas; P < 0.001 versus carcino mas). High MVDs were not associated with métastases,disease stage, and patient survival. The data indicate that angiogenesis is an early, critical step in colorectal tumorigenesis. MVD, however, does not provide signif icant prognostic information in colorectal cancer patients. INTRODUCTION Angiogenesis, the physiological process of new blood vessel for mation, is essential in tissue development, reproduction, and wound healing (1). Unregulated angiogenesis, however, plays a critical role in several diseases, including diabetes, arthritis, and neoplasia (1). Tumor growth, after reaching the size of about 1-2 mm\ is strictly dependent on angiogenesis (2). Angiogenesis also contributes to the metastatic process, facilitating shedding of tumor cells into the blood vessels (3). In human tumors, the evidence that angiogenesis, quantitated by microvessel counting, could be related to métastasesand patient survival was initially reported for cutaneous melanomas (4), and subsequently for tumors of several other organs, including breast, lung, head and neck, prostate, and testis (5-9). Furthermore, the extent of neovascularization provides independent prognostic information in invasive breast carcinoma (10-13) and in brain tumors (14). The exact time when angiogenic clones appear within a tumor remains to be clearly defined. Experimental data suggest that angio genesis may occur as early as at the transition from hyperplasia to neoplasia (15). In human tumors, the presence of highly neovascular- ¡zedpremalignant, or in situ, lesions also suggests that angiogenesis Received 4/11/95; accepted 8/28/95. The cosls 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. ' This study was supported by: Ministero dell' Università ' e della Ricerca Scientifica e Tecnologica (Rome, Italy), PF ACRO-Consiglio Nazionale delle Ricerche (Rome), Grant 158 from Regione Lombardia (Milan, Italy). Italian Association for Cancer Re search (Milan), and the Eleanor Naylor Dana Charitable Foundation (New York, NY). Presented in part at the 84th Annual Meeting of the American and Canadian Academy of Pathology, Toronto. Ontario, Canada, 1995. 2 To whom requests for reprints should be addressed, at Yale University School of Medicine, Department of Pathology, 310 Cedar Street, Lauder Hall L108, New Haven, CT 06520-8020. may occur early in tumor progression. Indeed neovascularization has been observed in breast intraductal carcinoma (5, 10, 16) and in cervical intraepithelial neoplasia (17). Data on tumor angiogenesis and its clinical and prognostic impli cations have been obtained in several malignancies of parenchyma! organs, whereas tumors of the gastrointestinal tract have not been investigated extensively thus far. Invasiveness in the latter tumors occurs through the different layers of the intestinal wall, and it is not known whether this unidirectional growth pathway (at least for its clinically significant implications) is dependent on neovasculariza tion. In two studies focusing on gastric and rectal carcinoma, elevated microvessel counts have been correlated with the presence of métas tases and/or poor prognosis (18, 19). We investigated immunocytochemically a retrospective series of 36 adenomas and 178 carcinomas of the large bowel using an anti-CD31 mAb to visualize endothelial cells. We then quantitated MVD3 in the normal mucosa, benign and malignant tumors, and peritumoral tis sues. The purposes of our investigation were: (a) to determine whether angiogenesis can be documented in colorectal tumor progression and the time of its onset; and (b) to assess whether the quantitation of microvessels can be correlated to tumor aggressiveness and provide prognostic information. MATERIALS AND METHODS Thirty-six adenomas were obtained from the files of the Department of Pathology of the San Paolo Hospital (Milan, Italy), including endoscopically excised lesions and surgical resection specimens. For colorectal adenocarcinomas the study population included 178 patients who were treated surgically at the Lahey Clinic Medical Center (Burlington, MA) between 1982 and 1984. The 178 patients are a subpopulation of a previously published study of 206 patients in which we evaluated immunocy tochemically p53 accumulation (20). In brief, to be included in the study, patients had to meet the following criteria: (a) no history of previous malig nancies (excluding skin carcinomas); (b) surgical resection margins negative for tumor; (e) no preoperative treatment; and (d) no perforation of the bowel by the tumor. Among the 178 carcinomas, transitional mucosa, adjacent to but not infiltrated by the tumors, was available for evaluation in 94 cases. Normal colonie mucosa at a distance of at least 10 cm from the invasive tumors was investigated in 41 cases. The original slides and pathology reports were reviewed to confirm the pathological grading and staging according to the American Joint Committee for Staging of Cancer (21). Patient age, sex, and tumor location in the large bowel were also evaluated. Patients were followed up for at least 5 years, and their survival and clinical status were obtained from the tumor registry or contact with the patients' physician or both. Immunocytochemistry. For the immunolocalization of blood vessels, sec tions were stained with an anti-CD31 mAb (clone JC/70; Dakopatts, Glostrup, Denmark). Dewaxed sections were rehydrated and treated with 0.4% pepsin in 0.01 N HC1 for 30 min at 37°Cand with 5% normal horse serum for 20 min at room temperature before being subsequently incubated with the following: (a) JC/70 mAb diluted 1:15 in PBS overnight at 4°C;(b) biotinylated rabbit 3 The abbreviations used are: MVD, microvessel density; VEGF. vascular endothelial growth factor. 5049 Research. on January 9, 2016. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from