Treated with Radiation Therapy p53 Status and Local Recurrence of Cervical Carcinoma Manganese Superoxide Dismutase Expression Correlates with

Manganese Superoxide dismutase (Mn-SOD) inactivates the radiation effect by removal of radiation-induced toxic Superoxide radicals. The purpose of this study was to assess the correlation among Mn-SOD, radiation sensitivity, and prognosis following radiation therapy. The Mn-SOD, pS3 oncoprotein. and c-erbB-2 oncoprotein expressions in 52 specimens from patients with cervical cancer treated with radiation therapy were investigated immunohistochemically. The frozen sections were stained using antihuman Mn-SOD, anti-p53 monoclonal antibodies, and anti-c-ci blt-2 oncoprotein polyclonal antibody followed by the avidinbiotin peroxidase complex method. Correlations among Mn-SOD expres sion, prognosis, and failure patterns were analyzed. Additionally, corre lations between p53 and c-erbB-2 oncoproteins and Mn-SOD expression were investigated. Positive expression of Mn-SOD in cervical carcinoma was 48.1%. No significant difference in positivity of Mn-SOD expression was noted ac cording to stage and histolÃ3gica! subtypes. The 5-year survival rate of Mn-SOD-positive patients was 42.5%, significantly poorer than the 77.0% of Mn-SOD-negative patients i/' < 0.05). Analysis of the failure patterns revealed that patients with Mn-SOD expression showed a significantly higher incidence of local recurrence than those without. However, there was no difference in distant metastasis between them. Although both p53 and c-erbB-2 oncoprotein expressions were significantly associated with the prognosis of the same patients, Mn-SOD expression was associated with p53 oncoprotein expression but not with that of c-erbB-2 oncopro tein. Our results demonstrate that the Mn-SOD level of cancer cells is correlated with local control and is an important prognostic factor in radiation therapy for cervical cancer. The Mn-SOD level may help explain the intrinsic radiosensitivitv of cervical cancer cells. INTRODUCTION Mn-SOD2 is an enzyme that catalyzes the reaction O2~ + O2~ + 2H* —¿ » H-.O, + O-,. This enzyme is believed to protect against toxic by-products of oxygen metabolism (1). Radiation produces Superoxide radicals as a consequence of both radiolysis of water in tissues and the subsequent recombination of primary free radicals (2). Radiation-induced Superoxide radicals in the presence of oxygen increase the radiation response of tumors. This is known as the "oxygen effect" and is an important modification factor in radiation therapy (3). An increase in Mn-SOD activity after X-irradiation was reported in the mouse heart (1, 4). It is supposed that Mn-SOD removes toxic Superoxide radicals and protects against the damaging effects of ionizing radiation. Thus, under oxygenated conditions, Mn-SOD probably decreases the oxygen effect and may be involved in the induction of radiation resistance of normal tissues and malig nant neoplasms. The expression of Mn-SOD by transfection of human Mn-SOD cDNA into murine spontaneous fibrosarcoma, FSa-11, which Received 11/16/95; accepted 4/16/96. The costs 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 1To whom requests for reprints should be addressed. Phone: 043{ 251 )2111. ext. 512: Fax: 043(256)8301. " The abbreviations used are: Mn-SOD. manganese Superoxide dismutase; DSB. double strand break; SOD. superoxide dismutase. does not express Mn-SOD, was reported to increase radiation resist ance (5). Moreover, a decrease in Mn-SOD activity has been reported in radiation hypersensitivity syndromes such as xeroderma pigmentosum, Fanconi's anemia, and acute radiation syndrome (6-9). Nev ertheless, in animal experiments any association between radiation sensitivity and the SOD level has remained a matter of controversy (10, 11). In the clinical field, no reliable assessment of whether the SOD level of cancer cells is associated with radiation sensitivity or local control in radiation therapy has been reported. Superoxide has been proposed to play a role in carcinogenesis, cardiopulmonary disease, chemical toxicity, radiation injury, inflam mation, arthritis, and aging (12). Especially, the protective effects of SOD have been assumed to be related to the mechanisms of carcino genesis (13). Some evidence has been presented that the Mn-SOD gene is a potential tumor suppressor gene (14, 15). Increased Mn-SOD expression suppresses the malignant phenotype of human melanoma cells (15). Furthermore, the SOD level of various cancer cells was reported to be lower than that of normal cells (6, 16). In contrast, it was also reported that the Mn-SOD level varied in different cancers, and that some cancers showed substantial amounts of Mn-SOD (17). In ovarian cancer, the serum SOD level varied and was apparently associated with tumor progression (18). These findings indicate that the Mn-SOD activity in human cancers remains controversial and requires further study. Many oncogene expressions, including c-erbB-2, c-myc, H-ras, and p53. have been studied in terms of tumor progression, and their relationship with poor prognosis was reported (19-26). Moreover, these oncogenes are involved not only in cell cycle regulation mech anisms (26, 27) but also in intrinsic radiation sensitivity (28, 29). As tor Mn-SOD activity, it is known to vary according to the cell cycle (30). Therefore, the question arises as to whether Mn-SOD activity may be associated with these oncogenes. The present study was performed to analyze the correlation between the Mn-SOD level of malignant tumors and the outcome of patients with cervical cancer treated with radiation therapy. Additionally, the Mn-SOD expression was analyzed in relation to p53 and c-erbB-2 oncoprotein expressions. MATERIALS AND METHODS Fifty-two patients with invasive squamous cell carcinoma of the uterine cervix, who received radiation therapy alone at the National Institute of Radiological Sciences Hospital (Chiba. Japan) between 1988 and 1990, were analyzed. The minimum follow-up period was 4.5 years, and most of the patients were followed up tor more than 5 years. Clinical stages and histological subtypes are summarized in Table 1. The clinical staging and histolÃ3gica! classification were based on the criteria of the International Federation of Gynecology and Obstetrics (London. England) classification (31) and the World Health Organization (Geneva. Switzerland) classification (32). respec tively. The numbers of patients with stages I. II, III. and IV were 2, 13, 32. and 5, respectively. As tor histolÃ3gica! subtype, those with keratinizing. small cell nonkeratinizing and large cell nonkeratinizing types were 11, 12, and 29. respectively. Radiotherapeutic Protocol. Patients were treated with a combination of external and high-dose rate intracavitary irradiation. Details of the protocol have been reported elsewhere (33). External whole pelvis irradiation was performed with anterior-posterior and posterior-anterior parallel opposing