ANGIOGENESIS AND SKIN CARCINOMAS WITH SKULL BASE INVASION: A CASE CONTROL STUDY

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1 ANGIOGENESIS AND SKIN CARCINOMAS WITH SKULL BASE INVASION: A CASE CONTROL STUDY Claudio R. Cernea, MD, 1 Alberto R. Ferraz, MD, 1 Inês V. de Castro, MD, 2 Miriam N. Sotto, MD, 2 Ângela F. Logullo, MD, 3 Carlos E. Bacchi, MD, 4 André S. Potenza, MD 1 1 Department of Head and Neck Surgery, University of São Paulo Medical School, São Paulo, Brazil cerneamd@uol.com.br 2 Department of Pathology, University of São Paulo Medical School, São Paulo, Brazil 3 Department of Pathology, Federal University of São Paulo, São Paulo, Brazil 4 Department of Pathology, São Paulo State University, Botucatu, Brazil Accepted 11 April 2003 Published online 20 April 2004 in Wiley InterScience ( DOI: /hed Abstract: Background. Some skin carcinomas may be very aggressive. Intensity of angiogenesis, measured by intratumoral vessel density using expression of CD34, has been associated with tumor aggressiveness. In this study, the expression of CD34 in basal cell carcinomas ( BCCs) and squamous cell carcinomas (SCCs) with skull base invasion was compared with that in tumors with good outcome. Methods. Expression of CD34 was graded as mild, moderate, and intense, in 24 BCCs and 11 SCCs with skull base invasion. The control group included 23 BCCs and 10 SCCs. Results. Intense expression of CD34 was noted in 25.00% of BCCs with skull base invasion, compared with 4.35% in the control group (p =.058). Regarding SCCs, intense expression of CD34 was found in 54.55% of aggressive tumors, compared with 10.00% in the control group (p =.133). Conclusions. A trend toward denser microvascular angiogenesis was observed in both BCCs and SCCs with skull base invasion compared with less aggressive controls. A 2004 Wiley Periodicals, Inc. Head Neck 26: , 2004 Correspondence to: Claudio R. Cernea Presented at the Sixth Research Workshop on the Biology, Prevention and Treatment of Head and Neck Cancer, October 9 13, 2002, McLean, Virginia. B 2004 Wiley Periodicals, Inc. Keywords: angiogenesis; basal cell carcinoma; squamous cell carcinoma; base of skull; immunohistochemistry INTRODUCTION Basal cell carcinoma (BCC) is the most common human malignant tumor. 1 Squamous cell carcinoma (SCC) is the second most frequent type. Both occur more frequently in whites after the sixth decade who had experienced prolonged exposure to sunlight. 2 Several factors have been associated with increased aggressiveness of these tumors: histopathologic subtype, differentiation, depth of invasion, and perineural invasion, among many others. 3 Sometimes these skin carcinomas are extremely aggressive, and despite adequate treatment, they may recur and invade fascia, muscle, and bone. Because of the associated deformity, some authors call them horrifying tumors. 4 6 In fact, skin carcinomas with skull base involvement are the main histologic types of tumors in some series of oncologic craniofacial operations. 7 9 The concept that tumor growth is dependent upon angiogenesis was initially established by 396 Angiogenesis in Aggressive Skin Carcinomas HEAD & NECK May 2004

2 Folkman et al 10 in 1971, and this relationship was confirmed thereafter There are several ways to evaluate the degree of angiogenesis, including expression of basic fibroblastic growth factor, epidermal growth factor, transforming growth factor, and vascular endothelial growth factor. 12 The intratumoral microvessel density(imd), which reflects the result of enhanced neoangiogenesis, shows close correlation with accelerated growth and propensity for metastatic spread in several cancers. 14 This IMD can be accurately graded, immunohistochemically measuring endothelialrelated antigens as factor VIII antigen, CD31, and CD34. According to some authors, CD34 is the more specific marker for neoformed vessels. 14 In 1975, Wolf and Hubler 15 developed an experimental model in hamsters to investigate what they believed was a tumor-angiogenic substance in BCCs and SCCs. Staibano et al 16 found an increased IMD in more invasive BCC, compared with a control group. However, Basarab et al l7 noted increased IMD in only 7% of 15 patients with BCC. Regarding SCC, Bolontrade et al, 18 using a murine experimental model, stated that angiogenesis was an initial event in carcinogenesis. On the contrary, Strieth et al 19 observed a significantly higher IMD in 16 advanced lesions, when compared with 10 biopsies of normal skin, 12 actinic keratosis, and nine early SCCs (with thickness < 2 mm). No report on angiogenesis in very advanced skin cancers was found in the literature. The objective of this study was to analyze the intensity of the angiogenesis, using immunohistochemical evaluation of IMD with antibody anti- CD34, in a consecutive series of very aggressive skin carcinomas with skull base invasion submitted to combined craniofacial oncologic operations. In addition, these findings were compared with those in skin carcinomas with good outcome treated in the same institution within the same time frame in a case-control study. MATERIALS AND METHODS Patients. A retrospective review of patients with BCC or SCC with skull base involvement treated at the Department of Head and Neck Surgery of the University of São Paulo Medical School was undertaken. Only cases with enough paraffinenbedded blocks to harvest slides for immunohistochemistry were included. Thirty-five patients constituted the two study groups: group 1, 24 BCCs (see Figure 1); and group 2, 11 SCCs. FIGURE 1. Patient with extensive BCC involving right frontoparietal region with a 12-year history. (A) Tumor. (B) Defect after craniofacial oncologic resection. (C) Postoperative result, after reconstruction with lateral thigh microvascular flap. Angiogenesis in Aggressive Skin Carcinomas HEAD & NECK May

3 FIGURE 2. Photomicrograph showing mild angiogenesis (ABC technique; counterstaining with Harris hematoxylin stain; original magnification, 400 ). FIGURE 3. Photomicrograph showing moderate angiogenesis (ABC technique; counterstaining with Harris hematoxylin stain; original magnification, 400 ). Seventeen patients (71%) in group 1 and five patients (50%) in group 2 had recurrent tumors after surgery, radiotherapy or both. One patient (4.17%) in group 1 and four patients (36.36%) in group 2 had lymph node metastasis. The two control groups were patients with BCCs and SCCs located on the head and neck area, treated at the Dermatology Department of the University of Sa o Paulo Medical School, with no recurrence for a minimum follow-up of 24 months: group 3, 23 BCCs; and group 4, 10 SCCs. for 12 hours at of 4jC. After washing with buffered saline solution, slides were incubated for 60 minutes with biotinylated antibodies anti-igg ( Vector Corp., USA). They were then incubated for 45 minutes with ABC Elite complex (Vector Corp., USA). Finally, slides were treated with 3,3Vdiaminodibenzidine (Sigma Chemical Company, USA) and with peroxide 0.1% (Sigma Chemical Company). Counter staining was performed with Harris hematoxylin stain for 5 minutes. The immunohistochemical built-in positive control was blood vessel normally present in this tissue. The procedure described by Hsu et al20 in 1981 was employed. Representative slides obtained from the tumors were washed with a buffered saline solution at ph 7.4. They were then incubated in a buffered citrate solution at ph 6.4 for 15 minutes (Gown et al21). Slides were then incubated with specific primary antibody against CD34, diluted 1:50, Immunohistochemical Analysis. Criteria for Interpretation of Immunohistochemical Staining for CD34. Immunoreactivity was considered positive when a membrane pattern in endothelial cells of tumor vessels was found. The intensity (number of vessels) was compared with that in normal skin and was graded as mild (Figure 2), moderate (Figure 3), and intense (Figure 4). All slides were blindly graded by two coauthors (AFL and CEB), with good correlation scores between them. Data were collected in a databank, using a software Excel (Microsoft Corp., USA), in a personal computer Pentium II 400 MHz Statistical Analysis. Table 1. Expression of CD34 in BCC. No. of patients (%) by angiogenic intensity Group FIGURE 4. Photomicrograph showing intense angiogenesis (ABC technique; counterstaining with Harris hematoxylin stain; original magnification, 400 ). 398 Angiogenesis in Aggressive Skin Carcinomas 1 3 Mild Moderate Intense 7 (29.17) 13 (56.52) 11 (45.83) 9 (39.13) 6 (25.00) 1 (4.35) p =.058 HEAD & NECK May 2004

4 (LG Electronics, South Korea). For the statistical analysis, either the chi-square test or the Fisher exact test were employed, to a significance level of RESULTS Angiogenesis in BCCs. Intense angiogenesis was observed in 25% of BCCs of group 1 (Table 1). Despite the fact that this prevalence was more than four times higher than in group 3 (4.35%), the difference was considered marginally statistically significant ( p =.058). Angiogenesis in SCCs. The majority of cancers in group 2 (54.55%) exhibited intense angiogenesis, compared with only 10.00% of tumors in group 4 (Table 2), but this difference was not statistically significant ( p =.133). DISCUSSION Table 2. Expression of CD34 in SCC. No. of patients (%) by angiogenic intensity Group Mild Moderate Intense 2 1 (9.09) 4 (36.36) 6 (54.55) 4 3 (30.00) 6 (60.00) 1 (10.00) p =.133 Extremely aggressive skin carcinomas are called by some authors horrifying tumors. 4 6 These skin carcinomas may show skull base involvement, and are the main histologic types of tumors in some series of oncologic craniofacial operations. 7 9 In these series, many BCCs and SCCs actually invaded duramater and brain. The IMD, which reflects the final result of enhanced neoangiogenesis, shows close correlation with accelerated growth and propensity for metastases in several cancers. 14 CD34 was chosen for this study because of its specificity for neoformed intratumoral vessels. Very few studies have analyzed the role of angiogenesis in aggressive skin cancer. 15 Its role remains controversial No reports on angiogenesis in very advanced skin cancers with skull base invasion were found in the literature. In group 1, 25.00% of BCCs exhibited intense angiogenesis, a much higher percentage than the 7% reported by Basarab et al. 17 In group 3, only 4.35% of the tumors showed this feature. The difference was considered to be marginally significant ( p =.058) but clearly demonstrated a tendency toward enhanced angiogenesis in aggressive lesions, already mentioned by Staibano et al. 16 They used immunohistochemical analysis of factor VIII and mentioned that it might be difficult to differentiate between preexistent and neoformed intratumoral vessels. In the present study, a different angiogenesis marker, anti-cd34, which is a more specific antibody against neoformed vessels, 14 was employed, supporting their statement. In group 2, more than half of SCCs (54.55%) showed intense angiogenesis. This percentage is almost six times greater than the 10% noted in the control group. This difference was not statistically significant, probably because of the size of the two groups. However, once again this accentuated difference cannot be ignored and may suggest a possible role for the angiogenesis process in these very aggressive SCCs. Evidently, the preliminary findings of this study need to be confirmed with larger series. Nevertheless, an increased prevalence of intense neoangiogenesis among BCCs and SCCs with skull base invasion was observed. Therefore, there might be a possible indication for some new antiangiogenic therapies that have been already reported in the literature, 16,22 24 in order to improve local control as well as survival in patients with these extremely advanced skin carcinomas. REFERENCES 1. Lang PG, Maize JC. Basal cell carcinoma. In: Friedman RJ, Rigel DS, Kopf AW, Harris MN, Baker D, editors. Cancer of the skin. Philadelphia: W.B. Saunders Company; p Freeman RG. Histopathologic considerations in the management of skin cancer. J Dermatol Surg 1976;2: Ruhoy SM, Flynn KJ, De Guzman MJ, et al. Pathology of selected skin lesions of the head and neck. In: Barnes L, editor. Surgical pathology of the head and neck, 2nd ed. New York: Marcel Dekker, Inc.; p Jackson R, Adams RH. Horrifying basal cell carcinoma: a study of 33 cases and a comparison with non-horror cases and a report on four metastatic cases. J Surg Oncol 1973; 5: Bianchini R, Wolter M. Fatal outcome in a metatypical, giant, horrifying basal cell carcinoma. J Dermatol Surg Oncol 1984;13: Horlock NM, Wilson GD, Daley FM. Cellular proliferation characteristics do not account for the behaviour of horrifying basal cell carcinoma: a comparison of the growth fraction of horrifying and non horrifying tumours. Br J Plast Surg 1998;51: Medina dos Santos LR, Cernea CR, Brandão LG. Results and prognostic factors in skull base surgery. Am J Surg 1994;168: Cernea CR, Teixeira GV, Medina dos Santos LR. Indica- Angiogenesis in Aggressive Skin Carcinomas HEAD & NECK May

5 tions for, contraindications to, and interruption of craniofacial procedures. Ann Otol Rhinol Laryngol 1997;106: Dias FL, Sá GM, Kligerman J. Prognostic factors and outcome in craniofacial surgery for malignant cutaneous tumors involving the anterior skull base. Arch Otolaryngol Head Neck Surg 1997;123: Folkman J, Merler E, Abernathy C. Isolation of a tumor factor responsible for angiogenesis. J Exp Med 1971;133: Folkman J. New perspectives in clinical oncology from angiogenesis research. Eur J Cancer 1996;32A: Petruzzelli G. Tumor angiogenesis. Head Neck 1996;18: McNamara A, Harmey JH, Walsh TN. Significance of angiogenesis in cancer therapy. Br J Surg 1998;85: Vermeulen PB, Gasparini G, Fox SB. Quantification of angiogenesis in nodular human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996;32A: Wolf JE Jr, Hubler WR Jr. Tumor angiogenic factor and human skin tumors. Arch Dermatol 1975;111: Staibano S, Boscaino A, Salvatore G. The prognostic significance of tumor angiogenesis in non-aggressive and aggressive basal cell carcinoma of the human skin. Hum Pathol 1996;27: Basarab T, Orchard G, Russel-Jones R. The use of immunostaining for bcl-2 and CD34 and the lectin peanut agglutinin in differentiating between basal cell carcinomas and trichoepitheliomas. Am J Dermatopathol 1998; 20: Bolontrade MF, Sterna MC, Binder RL. Angiogenesis is an early event in the development of chemically induced skin tumors. Carcinogenesis 1998;19: Strieth S, Hartschuh W, Pitz L. Angiogenic switch occurs late in squamous cell carcinomas of human skin. Br J Cancer 2000;82: Hsu SM, Raine L. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29: Gown AM, de Wever N, Battifora H. Microwave-based antigenic unmasking: a revolutionary new technique for routine immunohistochemistry. Appl Immunohistochemistry 1993;1: Streit M, Velasco P, Brown LF. Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am J Pathol 1999;155: Bielenberg DR, McCarty MF, Bucana CD. Expression of interferon-beta is associated with growth arrest of murine and human epidermal cells. J Invest Dermatol 1999;112: Bleuel K, Popp S, Fusenig NE. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci USA 1999;96: Angiogenesis in Aggressive Skin Carcinomas HEAD & NECK May 2004