VASCULARITYAND PROGNOSIS OF METASTATIC MELANOMA Tatyana VLAYKOVA 1 , Timo MUHONEN 2 *, Marjo HAHKA-KEMPPINEN 2 , Seppo PYRHO ¨ NEN 2 and Antti JEKUNEN 2 1 Department of Chemistry and Biochemistry, Medical Faculty, Trakia University, Stara Zagora, Bulgaria 2 Department of Oncology, Helsinki University Central Hospital, Helsinki, Finland The clinical role of vascularity was examined in metastatic melanoma, analyzing the correlation of the blood vessel density and prognosis. Our study included 51 specimens of metastatic melanoma tissue samplesfrom 31 patientstreated with combined chemo-immunotherapy. PECAM-1 (CD31) was used for assessing vascularity by immunohistochemical staining. On the basis of blood vessel counts, patients were classified into 2 main groups: low and high vascularity. A higher blood vessel density was found to be associated with shorter survival, estimated from the primary diagnosis of the disease (38 months), compared with patients with low blood vessel counts (68 months). A similar tendency was observed when vascularity was correlated to the survival period after the detection of the first metastases (13 vs. 30 months) and with survival since the initiation of chemo-immunotherapy (8 vs. 16 months). W hen vascularity and some common prognos- tic factors, such as age, sex, DNA ploidy and W HO tumor response, were used for a Cox multivariate analysis, vascular- ity turned out to be the most significant independent prognos- tic factor. Our results suggest that counting the blood vessels identified by immunohistochemical staining for the endothe- lial cell–specific CD31 is a powerful predictor for prognosis in patientswith metastatic melanoma and should be considered when selecting patients for therapy. Int. J. Cancer 74:326–329, 1997. r 1997 Wiley-Liss, Inc. Experimental evidence suggests that tumor growth beyond a certain diameter (1–2 mm) is highly dependent on angiogenesis (Folkman, 1992). The further expansion of the tumor mass requires the induction of new capillaries that converge on the tumor. Thus, angiogenesis has been directly correlated with tumor growth and metastasis. Neo-vascularization also permits shedding of cells from the primary tumor, while decreased angiogenesis is associated with a decreased number of metastases. Many experimental studies have shown that neo-vascularization of a primary tumor increases the probability of metastasis. There- fore, tumor angiogenesis could serve as a significant prognostic factor for the progression of primary diseases. Blood vessel density has been shown to be a prognostic predictor in patients with lung cancer (Macchiarini et al., 1992), prostatic cancer (Weidner et al., 1993), malignant melanoma (Srivastava et al., 1988; Barnhill and Levy, 1993; Graham et al., 1994), head and neck squamous-cell carcinoma (Gasparini et al., 1993), gastric carcinoma (Maeda et al., 1995) and breast cancer (Horak et al., 1992; Ogawa et al., 1995). However, other studies failed to show any correlation between microvessel counts and disease-free or overall survival (Axelsson et al., 1995; Busam et al., 1995). A report on colorectel cancer even showed an opposite trend (Lindmark et al., 1996). All reports so far have studied primary tumors, while no information on metastasis is available. The present study was undertaken to investigate correlations, if any, between the vascularity in metastatic melanoma compared with the progression of disease and the survival of patients treated with chemo-immunotherapy. MATERIAL AND METHODS Patients and tissue samples The patient population consisted of 31 patients with metastatic melanoma, treated at the Department of Oncology, Helsinki University Central Hospital, Finland (Table I). All patients received therapy according to a protocol consisting of dacarbazine, vincris- tine, bleomycin and lomustine combined with natural leukocyte a-interferon (Pyrho ¨nen et al., 1992). Altogether, 51 samples of metastatic melanoma were obtained from these patients, collected between July 1989 and April 1995. After surgical excision, the tumor material was embedded in Tissue-tek and frozen in liquid nitrogen. All specimens were inspected by expert pathologists to confirm the diagnosis of melanoma. Of each specimen, 5-μm cryostat sections were cut and kept overnight at room temperature to evaporate the excess moisture. Sections were kept frozen at 270°C with no loss of immune reactivity. Antibody and reagents Mouse monoclonal antibody (MAb), clone WM59, which recog- nizes human platelet-endothelial-cell-adhesion-molecule-1 (PECAM-1, CD31), was used as primary antibody (purchased from Novocastra Newcastle, UK). The VectaStainABC Kit (Vector, Burlingame, CA) was employed for immunoperoxidase staining. Immunohistochemical staining The staining procedure was first optimized by a series of preliminary experiments. The final conditions were as follows: after rehydration in PBS, non-specific binding was blocked by incubation with normal horse serum diluted 1:66 in 0.3% BSA in PBS, according to the manufacturer’s directions. The primary MAb was applied at a dilution of 1:500 in 0.3% BSA on the slides and incubated at room temperature for 1 hr. After a wash in PBS, the secondary antibody was added and incubated at room temperature for 30 min. Slides were washed with PBS, and avidin-biotin- peroxidase complex was applied and incubated at room tempera- ture for 1 hr. Slides were washed again and peroxidase activity was developed in a 3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MO) solution with hydrogen peroxide according to the manufactur- er’s directions. Sections were counter-stained with Mayer’s haema- toxylin. Blood vessel quantitation This was performed with light microscopy by an observer who had no knowledge of the patients’ data. First, the areas with highest vascular density were identified by scanning at 403 magnification. Then, signals from 3–6 single fields of 0.16 mm 2 at 2503 magnification were counted and the mean and SD calculated. The presence of vessel lumen and of any red-stained endothelial cell that was clearly separated from adjacent vessels, tumor cells and the connective tissue elements were considered to be separate countable vessels. Contract grant sponsors: the Finnish Cancer Foundation and the HUCH Clinical Research Foundation. *Correspondence to: Department of Oncology, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland. Fax: 358 9 471 4201. E-mail: timo.muhonen@helsinki.fi Received 4 September 1996; accepted 31 January 1997 Int. J. Cancer (Pred. Oncol.): 74, 326–329 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer