Basal cell carcinoma (BCC) is the most frequent carcinoma in the. Superficial Radiotherapy for Patients with Basal Cell Carcinoma

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1 2708 Superficial Radiotherapy for Patients with Basal Cell Carcinoma Recurrence Rates, Histologic Subtypes, and Expression of p53 and Bcl-2 Beate Zagrodnik, M.D. 1 Werner Kempf, M.D. 1 Burkhardt Seifert, Ph.D. 2 Beatrix Müller, B.S. 1 Günter Burg, M.D. 1 Mirjana Urosevic, M.D. 1 Reinhard Dummer, M.D. 1 1 Department of Dermatology, University Hospital Zurich, Zurich, Switzerland. 2 Department of Biostatistics, University of Zurich, Zurich, Switzerland. BACKGROUND. The histologic subtype of a basal cell carcinoma (BCC) may be an important factor for the success of a certain treatment modality. In the current article, the authors report recurrence rates among patients with BCC after superficial radiotherapy as well as Bcl-2 and p53 expression levels stratified by BCC subtype. METHODS. The authors performed a retrospective study of 175 BCCs in 148 patients (64 female patients and 84 male patients; mean age, 69 years) who were treated with radiotherapy. According to their histologic patterns, BCCs were classified as nodular (n 103), superficial (n 25), and sclerosing (n 47). In addition, six patients with metatypic BCC were reviewed. Bcl-2 and p53 protein expression was examined on a tissue microarray of 60 BCC samples (18 nodular tumors, 12 superficial tumors, and 30 sclerosing tumors). RESULTS. The estimated 5-year recurrence rate for all patients with BCC was 15.8%: 8.2% for patients with the nodular subtype, 26.1% for patients with the superficial subtype, and 27.7% for patients with the sclerosing subtype (Kaplan Meier analysis: P 0.055). The median follow-up was 48 months. The mean time to recurrence was 20 months, and 86.4% of all recurrences occurred within 3 years after treatment. No gender-specific differences were observed. In addition, one of six metatypic BCCs recurred. Nuclear p53 immunoreactivity and low Bcl-2 expression were significantly correlated with the sclerosing subtype. Overall, 61.5% of patients developed additional neoplasms during follow-up (76 developed additional BCCs, 15 developed squamous cell carcinomas, and 6 developed Bowen disease). CONCLUSIONS. The sclerosing subtype of BCC was a risk factor for recurrence after radiotherapy. In contrast, excellent results were achieved for patients with predominant nodular subtype. Nevertheless, radiotherapy may be the therapy of choice for patients with all BCC subtypes, depending on the individual patient s characteristics. Expression analyses confirmed that p53 and Bcl-2 levels may be used as indicators for the aggressiveness of a BCC subtype. Due to the high incidence of additional skin malignancies, patients with BCC need careful followup. Cancer 2003;98: American Cancer Society. Address for reprints: Reinhard Dummer, M.D., Department of Dermatology, University Hospital of Zurich, Gloriastrasse 31, CH-8091 Zurich, Switzerland; Fax: (011) ; reinhard.dummer@usz.ch Received June 30, 2003; accepted August 25, KEYWORDS: basal cell carcinoma, histologic subtype, radiotherapy, recurrence rate, p53, Bcl-2. Basal cell carcinoma (BCC) is the most frequent carcinoma in the white human population. For Europe and Northern America, incidence rates between 20 and 300/100,000 have been reported. 1,2 Worldwide, these numbers still are increasing. 3 5 The etiology of BCC is multifactorial. The duration and intensity of sun exposure seem to play a major role. 6 Other risk factors include ionizing radiation, intensive photochemotherapy, and arsenic intoxication. 7 In addition, 2003 American Cancer Society DOI /cncr.11798

2 Recurrence of BCCs after Radiotherapy/Zagrodnik et al FIGURE 1. Inoperable nodular basal cell carcinoma on the neck of a 92-yearold patient (A) before and (B) 3 months after X-ray treatment. individuals with Fitzpatrick skin type 1 who carry gene variants of the melanocortin-1 receptor show a predisposition to BCC. 8,9 Additional genetic features associated with BCC development include polymorphism of the DNA repair gene XPD, 10 mutations in the tumor suppressor gene p53, 11 as well as loss of heterozygosity at the PTCH locus encoding the cell membrane receptor patched, which is involved in the Hedgehog signaling pathway. 12 BCC occurs predominantly in sun-exposed areas of the skin, such as the head, neck, and trunk. 13 Tumor growth is relatively slow, locally invasive, and essentially destructive. BCC exhibits a low metastatic potential. 14 According to the clinical and histologic characteristics, a number of BCC subtypes have been described: the superficial subtype, the nodular subtype, and the sclerosing (morpheaform) subtype. 13 The histologic subtype of BCC is an important factor for adequate treatment with regard to the age and health status of patient as well as the localization, size, and number of lesions. Current treatment modalities include excision, Mohs micrographic surgery, cryosurgery, radiotherapy, photodynamic therapy, 5-fluorouracil, and immunotherapy using interferon- or imiquimod. 15,16 Five-year recurrence rates ranged from 1% for patients who underwent Mohs micrographic surgery to 20% for patients who received photodynamic therapy or treatment with 5-fluorouracil. 15 However, each type of treatment has its advantages and disadvantages and may be the therapy of choice, depending on the individual patient. Radiotherapy is recommended for patients in poor general health, for elderly patients, and for patients with large facial lesions (Fig. 1), because the stress on the patient is low and because of its favorable cosmetic and functional results on the face. 17,18 Establishing predictive markers may help to improve the management of BCC. Candidates are proteins involved in apoptosis, such as p53, a tumor suppressor that frequently is mutated in human tumors, and Bcl-2, an antiapoptotic protein. A correlation between p53 expression and aggressive behavior of BCCs has been reported. 19 In contrast, high Bcl-2 levels appear to be associated with slow tumor growth. 20 In the current investigation, we report that BCCs of the sclerosing subtype have a high risk of recurring after radiotherapy. In addition, we detected a high frequency of p53 immunoreactivity and low Bcl-2 expression in this subtype on a tissue microarray. MATERIALS AND METHODS Patients We reviewed the medical records of 154 patients (65 females and 89 males) with a diagnosis of BCC who had been treated with radiotherapy between 1981 and 1991 at the Department of Dermatology, University Hospital Zurich (Zurich, Switzerland). We gathered information on patient gender and age, tumor location, and histologic subtypes of primary and recurrent tumors as well as additional new skin malignancies during follow-up. Multiple BCCs that occurred in a single patient were included. Patients were examined 1 month, 3 months, 6 months, and 12 months after treatment and then at yearly intervals for at least 5 years whenever possible. Sites affected by BCC were documented by photographs before and after therapy. Histologic Classification One hundred seventy-five BCC tissue samples were reclassified into 1 of the 3 major subtypes: nodular, superficial, and sclerosing (morpheaform) 13,21 (Fig. 2A C). In addition, six tumors of the metatypical type, which is a rare BCC variant with less peripheral palisading and nests of cells maturing into larger and paler cells, were observed. 22 Treatment Modalities All BCC diagnoses had been biopsy-proven before treatment. Patients were treated with radiotherapy using soft X-rays (20 50 kilovolts), as shown in Table 1.

3 2710 CANCER December 15, 2003 / Volume 98 / Number 12 FIGURE 2. (A) Nodular basal cell carcinoma (BCC). Large tumor nodules are shown with peripheral palisading of the cells that grow within the dermis. Surrounding retraction spaces may occur between the tumor islands. (B) Superficial BCC. There are small, focal nests of proliferating tumor cells that arise from the basal layer of the epidermis and extend into the dermis. (C) BCC of the sclerosing subtype is shown with elongated, irregular strands of cells within a dense, fibrous stroma. Further characteristics are irregular invasion into the surrounding tissue and poor peripheral palisading of nuclei. TABLE 1 Treatment Modalities Used in Radiotherapy of Basal Cell Carcinomas Lesion size (cm) Fractionation (grays) Daily Time interval (days) Treatment modalities were in line with the recommendations of Panizzon. 23 A safety margin of 10 mm was used. Tissue Microarray and Immunohistochemistry Tissue samples from 60 BCCs were used for immunohistochemistry. They comprised 30 high-risk tumors of the sclerosing subtype and 30 nonaggressive tumors (12 superficial BCCs and 18 nodular BCCs). Preparation of the tissue microarray and immunohistochemical staining was performed as described previously. 24,25 Monoclonal mouse antihuman Bcl-2 antibody (DAKO, Zug, Switzerland) was used at a dilution of 1:40, and mouse antihuman p53 antibody (DAKO) was used at a dilution of 1:2. Statistical Analyses Recurrence rates were calculated using Kaplan Meier analysis, and statistical differences were assessed using the Mantel Cox (log-rank) test. The chi-square test was used to determine possible significant differences between variables. P values 0.05 were considered statistically significant. Because of the small number of patients, BCCs of the metatypical type were not included in the statistical analyses. RESULTS Patients and Primary Tumors One hundred forty-eight patients (64 females and 84 males) had 1 of the major BCC subtypes. In 20 of those TABLE 2 Basal Cell Carcinoma Subtype-Specific Recurrences after Radiotherapy BCC subtype No. of patients No. of recurrences (%) Nodular (7.8) Superficial 25 4 (16.0) Sclerosing (21.3) BCC: basal cell carcinoma. patients, 2 lesions (up to 5 lesions) were treated with radiotherapy, yielding a total of 175 BCCs that were included in the analysis. One hundred three BCCs (58.9%) were of the nodular subtype, 25 BCCs (14.3%) were of the superficial subtype, and 47 BCCS (26.3%) were of the sclerosing subtype. The mean age standard deviation at diagnosis of the primary tumor was 69 years 11 years (range, years). Six patients (1 female and 5 males; mean age, 61 years) who had BCC of the metatypical type also were reviewed but were not included in the statistical evaluation. Follow-Up and Recurrences The median follow-up was 48 months (range, months). The recurrences rates for each subtype are shown in Table 2. Due to the variable follow-up duration, Kaplan Meier analysis was used to estimate the 5-year recurrence rates (Fig. 3). The overall 5-year recurrence rate (mean standard error of the mean) after radiotherapy was 15.8% 3.3% for all BCCs, 8.2% 2.8% for nodular BCCs, 26.1% 11.8% for superficial BCCs, and 27.7% 7.9% for sclerosing BCCs (P 0.055). The mean time to recurrence was 20 months (range, 5 51 months). Nineteen of 22 recurrences (86.4%) occurred within 3 years after treatment (Table 3). No significant gender-specific differences could be

4 Recurrence of BCCs after Radiotherapy/Zagrodnik et al TABLE 4 Anatomic Distribution for Each Subtype of Basal Cell Carcinoma Localization Nodular (%) Superficial (%) Sclerosing (%) Head Scalp Forehead/temple Chin Cheek Nose/nasolabial Eyelid Ear/periauricular Lip Trunk Upper limb Lower limb Genitalia FIGURE 3. Cumulative 5-year recurrence rates for different basal cell carcinoma subtypes (Kaplan Meier analysis). TABLE 3 Time to Recurrence by Histologic Subtype of Basal Cell Carcinoma BCC subtype No. of recurrences/effective no. of patients at risk (%) 0 12 mos mos mos mos mos Nodular 5/97 (5) 3/78 (4) 0/0 (0) 0/0 (0) 0/0 (0) Superficial 0/0 (0) 1/21 (5) 1/15 (7) 2/9.5 (21) 0/0 (0) Sclerosing 4/43.5 (9) 2/33 (6) 3/25 (12) 0/0 (0) 1/8.5 (12) BCC: basal cell carcinoma. observed. One BCC of the metatypical type recurred 44 months after X-ray therapy. Anatomic Distribution BCCs were located mainly on the head (135 tumors; 77.1%) and trunk (31 tumors; 17.7%). An opposite distribution was observed among superficial BCCs. The detailed anatomic distribution for the different subtypes is presented in Table 4. A similar result was obtained for recurrent tumors: Eighteen recurrent tumors (81.8%) were located on the head, and 4 recurrent tumors (18.2%) were located on the trunk. All BCCs of the metatypical type, including the recurrent tumor, occurred on the face. Multiple BCCs and Other Skin Malignancies Of 148 patients, 91 patients (61.5%) developed 1 or more additional skin tumors, including additional BCCs in 76 patients, squamous cell carcinomas in 15 patients, and Bowen disease in 6 patients. Bcl-2 and p53 Expression Bcl-2 and p53 protein expression was examined immunohistochemically using a tissue microarray of 60 BCC samples (Fig. 4) that were obtained from 30 lowrisk tumors (12 superficial BCCs and 18 nodular BCCs) and 30 high-risk tumors (30 sclerosing BCCs). The immunostaining results are provided in Table 5. All 9 p53-positive samples were of the sclerosing subtype, whereas moderate or strong Bcl-2 expression was observed in 21 low-risk BCCs and in 10 sclerosing BCCs. A significant correlation was found between high-risk tumors and p53 expression (P ) as well as negative or weak staining for Bcl-2 (P ). No statistically significant correlation was observed between p53 or Bcl-2 expression and recurring BCCs. From 10 patients, tissue samples of the primary tumor (2 superficial BCCs, 3 nodular BCCs, and 5 sclerosing BCCs) and of the recurrent tumor were available. In two tumors, substantial changes in the expression levels were observed. In one sclerosing BCC, p53 status was negative, and an intermediate Bcl-2 level was detected in the primary tumor; whereas the recurrent tumor expressed p53. A second recurrence of the sclerosing subtype switched on p53 expression. In this tumor, the histologic pattern changed as well, because the primary tumor was of the superficial subtype. DISCUSSION Several reports on treatment efficacy in patients with BCC have shown that the histologic subtype is an important risk factor for recurrence However, only limited data were available for radiotherapy. 32 Our large, retrospective study of 175 BCCs in 148 patients who were treated with superficial radiotherapy demonstrated that sclerosing BCCs have a high recurrence rate after radiotherapy.

5 2712 CANCER December 15, 2003 / Volume 98 / Number 12 TABLE 5 Bcl-2 and p53 Immunoreactivity in Basal Cell Carcinomas According to the Histologic Subtype Tumor no. BCC subtype Bcl-2 p53 Tumor no. BCC subtype Bcl-2 p53 FIGURE 4. Immunohistochemical staining of the basal cell carcinoma (BCC) tissue array. Part of the tissue array showing four BCC tissue samples stained with (A) hematoxylin and eosin and with antibodies against (B) p53 and (C) Bcl-2. Our Kaplan Meier estimates of the 5-year recurrence rates after radiotherapy ranged from 8.2% for patients with nodular BCC to 27.2% for patients with sclerosing BCC, resulting in an overall 5-year recurrence rate of 15.8%. Previous reports, which did not distinguish between the different histologic subtypes, yielded cumulative 5-year recurrence rates approximately 8% for patients with X-ray-treated BCC. The high recurrence rate in the current study may have been due to the large number of high-risk tumors, including large lesions in elderly patients who could not tolerate surgical excision and lesion of the more infiltrative and aggressive, sclerosing subtype ,33,35,36 In contrast to other studies that reported a frequency of 6% for sclerosing BCC, 13,37 this subtype was present in 27% of all tumors examined. This high value may be explained by the fact that radiotherapy was applied mainly to lesions of the face (especially in areas that are not recommended for surgery because of the less acceptable cosmetic and functional outcome, such as the nose, ears, eyelids, and lips), in which the largest proportion of the sclerosing subtype was found. 13,37 The estimated 5-year recurrence rate of 27.7% for patients with sclerosing BCC confirmed the high recurrence rates reported previously Superficial BCCs, which were located mainly on the trunk, 8,13,37,38 seem to have an increased tendency to recur due to their 1 s 31 sc 2 s 32 sc 3 s 33 sc 4 s 34 sc 5 s 35 sc 6 s 36 sc 7 s 37 sc 8 s 38 sc 9 s 39 sc 10 s 40 sc 11 (p) s 41 (p) sc 12 (p) s 42 (p) sc 13 n 43 (p) sc 14 n 44 (p) sc 15 n 45 (p) sc 16 n 46 (p) sc 17 n 47 (p) sc 18 n 48 (r) sc 19 n 49 (r) sc 20 n 50 (r) sc 21 n 51 (r) sc 22 (p) n 52 (r) sc 23 (p) n 53 (r) sc 24 (p) n 54 (r) sc 25 (p) n 55 (r) sc 26 (p) n o 56 (r) sc 27 (p) n 57 (r) sc 28 (r) n 58 (r) sc 29 (r) n 59 (r) sc 30 (r) n 60 (r) sc o BCC: basal cell carcinoma; s: superficial; sc: sclerosing; n: nodular; (p): recurring primary tumor; (r): recurrent tumor; : no staining; : weak staining; : intermediate staining; : strong staining; o: staining not performed (not enough tissue available). multifocal pattern. 39 Their relatively high estimated 5-year recurrence rate of 26% may be attributed to the large size of the tumors; in our department, smaller lesions on the trunk usually are treated by cryosurgery or excision. In addition, due to the small number of superficial BCCs, the statistical validity still has to be confirmed for this subtype. The nodular subtype, accounting for 70% of all BCCs, 13,30,37 constituted 59% of our tumors and, thus, was underrepresented. The estimated 5-year recurrence rate of 8.2% was comparable to the 5-year recurrence rates achieved with other treatment modalities. 15,40 The mean time to recurrence was 20 months, and 85% of all recurrences occurred within 3 years after treatment, which was in line with previous results. 34,40 However, 18% of recurrences reportedly appeared after more than 5 years and up to 10 years. 34 Because 61.5% of patients developed additional cutaneous

6 Recurrence of BCCs after Radiotherapy/Zagrodnik et al neoplasms, long-term follow-up is recommended for all patients with BCC Recently, it was shown that complex formation of p53 and Bcl proteins induced apoptosis by permeabilization of the outer mitochondrial membrane. 44 By testing p53 and Bcl-2 expression as predictive markers, we found a significant correlation between p53 immunoreactivity and the sclerosing subtype of BCC as well as between nonaggressive tumors and intermediate-to-strong Bcl-2 staining. In addition, p53 was identified exclusively in 32% of aggressive tumors. In contrast, Bcl-2 was found predominantly in nonaggressive tumors. Immunohistochemistry confirmed the results of two studies that were published recently, 19,20 suggesting that Bcl-2 and p53 protein expression are useful as predictive markers in BCC therapy. No statistically significant correlation was found between p53 or Bcl-2 immunoreactivity in the primary tumor and recurrence rates, which may have been due to the relatively small number of recurring tumors that were analyzed. After radiotherapy, p53 expression switched on in two recurrent tumors, which may have been due to a mutation in the p53 gene induced by therapeutic radiation exposure. In summary, the current retrospective study demonstrated that sclerosing BCCs have a greater potential to recur after radiotherapy. Therefore, we recommend increasing the radiation dose in patients with this tumor subtype. Radiotherapy is an excellent treatment option for patients with the predominant nodular subtype. However, it also may be the therapy of choice for patients with the other subtypes, depending on the age and health status of the patient as well as the localization and size of the lesion. 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