Cytokine concentrations in basal cell carcinomas of different histological types and localization I. Elamin, R. D. Zecevic, D. Vojvodic, L. Medenica, and M. D. Pavlovic KEY WORDS basal cell carcinoma, cytokines, localization, aggressive histological subtypes, solar keratosis -A B S T R A C T Background: Basal cell carcinoma (BCC) is the most common malignant skin tumor. Cytokines as major mediators of the immune system have been shown to play an important role in biology of the neoplasm with the general predomination of Th2 cytokines, whereas IFN-y and other Th1 cytokines are prevalent in spontaneously regressing tumors. Objective: We were interested in comparing cytokine levels in BCC and cutaneous squamous cell tumors with BCC of different localization and histological subtypes. Material and methods: Explants from freshly excised BCC from 18 patients, and cutaneous squamous cell tumors (solar keratoses and Bowen's disease) from 9 patients were cultivated for 24 h. Cytokine (IL-2, IFN-y, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNFa, IL-1 p) concentrations in culture supernatants were determined by a sandwich immunoassay. Results: Tissue explants of BCC contained significantly higher concentrations of IL-1 p, IL-4, IL-5, and IL-6 compared to those of squamous cell tumors. Higher levels of TNF-a (p = 0.042), IL-4 (p = 0.028), and IL-5 (p = 0.012) were found in tumors localized to the head and neck compared to those on the trunk or extremities. Interleukin-6 concentrations were higher in aggressive BCC variants (infiltrative and micronodular), but the difference was not statistically significant (p = 0.068). Conclusions: Confirming the earlier findings that BCC is a tumor with a Th2 cytokine microenvironment, this study further shows that BCC situated on the head and neck produce even more of certain Th2 cytokines (IL-4 and IL-5) and TNF-a, a crucial immunosuppressive cytokine released upon UVB irradiation. Introduction Basal cell carcinoma (BCC) is the most common malignant tumor in whites and accounts for up to 80% of all malignant skin tumors (1, 2). Some authors put forward the hypothesis that BCC occurring at certain body sites or BCC of a particular histological subtype may define certain clinical behavior and may even have Figure 1. Distribution of histological subtypes of basal cell carcinomas in our patients. Figure 2. Concentration (mean ± SD) of TNF-a, IL-4, and IL-5 in BCC of different localizations (head and neck versus trunk and limbs). a different etiology (3-5). These studies have shown that the most common histological variety found on the trunk is the superficial type, thus linking this type of BCC with intense intermittent sun exposure. However, a recent Australian study seems to disprove these conclusions, showing a more equal distribution of superficial BCC on face, trunk, and limbs (6). Nevertheless, it is well known that head and neck BCC display a higher propensity for recurrence after treatment compared to tumors located on other body sites (1, 2). It is believed that the presence of hair follicles and the downward spread of the tumor along the follicles makes it less amenable to complete excision, thus increasing the likelihood for recurrence. The immune system has an important role in the biology of BCC (1). Cytokines, crucial players in the system, have been shown to exert important effects on the growth of BCC (7). We hypothesized that BCCs not only display different cytokine profiles compared to non-BCC skin tumors, but that tumors situated on the head and neck produce more immunosuppressive cytokines, which contribute to their more aggressive biological behavior. Patients and methods Upon approval of the Institutional Review Board, 18 successive patients (16 men; mean age 71.9 ± 9.2 years) with dermoscopically and histologically proven, previously untreated basal cell carcinoma were included in the study. The control group was comprised of 9 patients (7 men; mean age 72.7 ± 5.0 years) with histologi- cally confirmed, previously untreated squamous cell tumors (solar keratoses and Bowen's disease). The patients signed a written informed consent prior to inclusion in the study. The surgically-excised tumors were immediately halved, and one sample was routinely processed for pathohistology, whereas the other half was cultivated in the medium (5 mL of sterile RPMI, pH 7.4) at 37 °C with 5% CO2 for 24 h. After 24 h, the culture medium was collected, centrifuged at 2000 x g for 15 min, and the supernatant alliquoted and kept frozen at -70 °C until cytokine measurements were taken. Cytokines (IL-2, IFN-y, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNFa, IL-1b) were quantified by the commercial sandwich immunoassay FlowCytomix (Bender MedSystems, Burlingame, CA, USA). The obtained values were calculated per g of tissue and expressed as pg/mL. The results were analyzed with the two-sided Student i-tests. The p value was set at < 0.05. Results The excised tumors in patients and controls were separated into 3 groups according to their localization: trunk, head and neck, and extremities. BCC was localized to the trunk in 7 (39%) patients, to the head and neck in 6 (33%) patients, and to the extremities in 5 (28%) patients. Histologically, the most common form was superficial BCC (7/39), whereas micronodular and clear cell types were found in 2 and 1 patient, respectively (Fig. 1). Tissue explants of BCC contained significantly higher Table 1. Mean cytokine concentrations in tissue explants of basal cell carcinomas (Group 1) and squamous cell tumors (Group 2). Cytokine Group n Mean SD P 95% CI IL-6 1 18 1895.8889 2367.9993 0.024 203.95-2596.48 2 9 495.6667 353.4134 IL-8 1 18 284.2778 433.2373 > 0.05 -16.96-419.96 2 9 82.7778 60.8703 IL-10 1 18 12.8889 33.7027 > 0.05 -24.42-19.31 2 9 15.4444 20.9947 IL-2 1 18 0.2778 1.1785 > 0.05 -331.99-119.88 2 9 106.3333 293.9349 ifn-y 1 18 379.6667 1506.3704 > 0.05 -425.06-1082.17 2 9 51.1111 139.6159 IL-12 1 18 58.0000 140.4706 > 0.05 -29.83-118.06 2 9 13.8889 39.4412 TNF-a 1 18 72.7222 136.2095 > 0.05 -10.98-125.31 2 9 15.5556 12.7584 IL-1ß 1 18 197.8333 205.0111 0.004 57.66-262.89 2 9 37.5556 19.7301 IL-5 1 18 253.6667 319.9366 0.012 53.43-373.23 2 9 40.3333 27.0463 IL-4 1 18 148.6111 180.2172 0.015 26.03-208.51 2 9 31.3333 28.6836 concentrations of IL-1P, IL-4, IL-5, and IL-6 in comparison to those of squamous cell tumors (Table 1). When cytokine concentrations were analyzed in BCC located in different sites, we found higher levels of TNF-a (p = 0.042), IL-4 (p = 0.028), and IL-5 (p = 0.012) in tumors localized to the head and neck comparing to those on the trunk or extremities (Fig. 2). Then we compared cytokine concentrations in aggressive (micronodular and infiltrative) and non-aggressive (superficial and nodular) histologic types of BCC. There was no any significant difference in the cytokine levels. However, IL-6 concentrations were higher in aggressive BCC variants, although the difference did not reach statistical significance (p = 0.068). In addition, histologic subtypes of BCC did not differ with respect to cytokine concentrations in their tissue explants (not shown). Comment Basal cell carcinoma, under certain circumstances, undergoes spontaneous regression that is probably mediated by the immune system (7, 8). Islands of tumor cells are surrounded by regulatory CD4+, CD25+, Foxp3+ T (Treg) lymphocytes, and numerous immature dendritic cells. The microenvironment of BCC is dominated by Th2 cytokines (enhanced expression of IL-4, IL-10, and CCL22). A partial anti-tumor response is marked by the presence of CD8+ T cells and the activation of interferon and IL-12/23 genes (8). Gambichler et al. (9) have found higher levels of mRNK for IL-6 and IL-8 in the tissue of BCC in comparison to uninvolved skin or the skin of individuals without BCC (p < 0.05). It has been shown that IL-6 via basic fibroblast growth factor (bFGF) and cyclooxigenase 2 (COX-2) promotes angiogenesis in BCC (10). In addition, IL-6 enhances tumorigenic potential and anti-apoptotic activity in human BCC (11). Our findings are in line with the predominance of Th2 cytokines (IL-4, IL-5, IL-6) and IL-1P in BCC in comparison with solar keratoses and Bowen's disease (Table 1). IL-1p is produced by keratinocytes irradiated by ultraviolet (UV) light (12). A previous study on a BCC cell line has shown that it produced less IL-1p than normal keratinocytes (13). However, our results were obtained from freshly excised tumor tissue and thus may be more relevant for the in vivo situation. Together, it seems that microenvironment of BCC compared to healthy skin or squamous cell tumors displays a strong predominance of Th2 cytokines or, in other words, a specific state of immunosuppression. We have found that BCC situated on the head and neck express more Th2 cytokines (IL-4 and IL-5) and tumor necrosis factor (TNF)-a than tumors on the trunk and limbs (Fig. 2). Hence, the higher level of immunosuppression in tumors at these sites may be an additional explanation for their more aggressive biological behavior. It is known that TNF-a plays a central role in immunosuppression induced by UVB (14). 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Skov L, Allen MH, Bang B, Francis D, Barker JN, Baadsgaard O. Basal cell carcinoma is associated with high TNF-alpha release but not with TNF-alpha polymorphism at position "308. Exp Dermatol. 2003;12:772-6. AUTHORS' Issa Elamin, MD, Department of Dermatovenereology, Military Medical ADDRESSES Academy, Crnotravska 17, 11002 Belgrade, Serbia Rados D. Zečevic, MD, PhD, Professor of Dermatovenereology, same address Danilo Vojvodic, MD, PhD, Associated Professor of Clinical Immunology, Institute of Medical Research, Military Medical Academy, Crnotravska 17, 11002 Belgrade, Serbia Ljiljana Medenica, MD, PhD, Professor of Dermatovenereology, Institute of Dermatovenereology, Clinical Center of Serbia, Pasterova 2, 11000 Belgrade, Serbia Milos D. Pavlovic, MD, PhD, Professor of Dermatovenereology, Department of Dermatology and Venereology, University Medical Center Ljubljana, Zaloska 2, SI-1525 Ljubljana, Slovenia, E-mail: mdpavlovic2004@yahoo.com