[CANCER RESEARCH 61, 3119 –3123, April 1, 2001] Unusual Profile and High Prevalence of p53 Mutations in Esophageal Squamous Cell Carcinomas from Northern Iran 1 Firouzeh Biramijamal, Abdolamir Allameh, Parvin Mirbod, Hermann-Josef Groene, Reet Koomagi, and Monica Hollstein 2 Departments of Genetic Alterations in Carcinogenesis [F. B., R. K., M. H.] and Cellular and Molecular Pathology [H-J. G.], German Cancer Research Center, D-69120 Heidelberg, Germany; Biochemistry Department, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran [A. A.]; and Tehran University of Medical Sciences and Health Services, Tehran, Iran [P. M.] ABSTRACT Over 15,000 human tumor p53 mutations have been recorded in the scientific literature, including over 700 mutations in esophageal tumors. There are no data on p53 mutations in esophageal cancer patients from Iran yet; however, this country experiences one of the highest cancer mortality rates in the world for esophageal squamous cell carcinomas (ESCCs). The causes of this high cancer burden in Iran remain obscure and do not appear to be related to tobacco and alcohol consumption, the two major risk factors identified in Europe and North America. Because molecular analysis of tumors can provide clues to endogenous or environ- mental factors contributing to high cancer risk, we examined 74 Iranian ESCCs for the presence of mutations in exons 5– 8 of the p53 gene by PCR and direct sequencing. Forty-eight of the 74 tumors (65%) had one or more p53 gene point mutations, including 5 patients with two or more mutations and one with a tandem mutation in codon 242. Surprisingly, over one-third of the 54 mutations we identified were transitions at CpG sites (20 of a total of 54 mutations, or 37%), a class of mutation that is significantly less common (16% of mutations) in the compilation of ESCC mutations from other countries ( 2 statistic, P < 0.0002), whereas trans- versions, which the literature shows to be common in ESCCs from non- Iranian patients, were infrequent in the tumors we examined here. Ele- vated levels of cyclooxygenase-2 and inducible nitric oxide synthase were observed in 74 and 91%, respectively, of tumors from Tehran as deter- mined by immunohistochemistry, and high COX-2 expression correlated significantly with the presence of a p53 mutation in the tumor. Mediators of the inflammatory response in esophageal mucosa, perhaps in conjunc- tion with specific dietary or cultural practices in Iran, may contribute importantly to the p53 mutation load in Iranian ESCC patients. INTRODUCTION p53 tumor mutations have been linked to specific carcinogen ex- posures, suggesting a molecular epidemiology approach to investiga- tion of cancers for which the causes have remained elusive. Although it is well-established that tobacco and alcohol, particularly in combi- nation, are the major causes of SCC in the United States and Europe, neither is thought to be important in the etiology of this disease in Iran, where both men and women are at elevated risk (1). Age- adjusted incidence figures of up to 171/100,000 have been reported in rural areas of northeastern Iran for ESCC, 3 the major histological subtype (2). The rates are 100-fold higher than in lowest incidence areas of the world, pointing to strong environmental and perhaps genetic influences. The precise risk factors responsible for the high prevalence of ESCC in northern Iran have remained a matter of conjecture. Cultural or environmental factors suggested to play a role include dietary vitamin and mineral deficiencies, mutagenic by-prod- ucts of opiates, and consumption of regional specialties or scalding beverages, which are strong irritants to the esophageal mucosa (2– 4). We initiated a study to examine the p53 status in tumors of 74 ESCC patients from northern Iran, 40 from the capital city Tehran, and 34 from the Caspian Littoral, because no p53 mutation data were available in the literature on tumors from this region. Additional tumor tissue from paraffin blocks was available for 23 of the patients from Tehran, allowing immunohistochemical assessment of expres- sion of COX-2 and iNOS, two enzymes known to be elevated in chronically inflamed tissues and in gastrointestinal tumors from other geographical areas. MATERIALS AND METHODS Tissue Collection and DNA Preparation. Formalin-fixed, paraffin- embedded esophageal tissue (surgically resected material or biopsy) from cancer patients diagnosed in Iran with ESCCs were collected for analysis. Informed consent was obtained from patients by participating scientists from Iran, and the study was approved by the Medical Ethics Committee, Ministry of Health, Iran as conforming to the ethical guidelines of the 1975 Declaration of Helsinki. Hospital records were used to verify age, permanent residence, smoking history, and ethnicity of individuals. Archived histology sections were examined by the collaborating pathologist in Tehran (P. M.), and serial sections of 10-m thickness were prepared for DNA extraction. Diagnosis of SCC was confirmed by the pathologist at the German Cancer Research Center, Heidelberg (H-J. G.), who designated an area of tissue material with 50% neoplastic cellularity for each specimen on H&E-stained slides, used to guide dissection for DNA extractions. Forty tumors were examined from patients who were long-term permanent residents of Tehran (most of whom were also born there) and for whom diagnosis of ESCC could be confirmed. Fixed specimens from 34 patients residing in the Caspian Littoral and who had undergone surgery in regional hospitals also were examined in the present study and subjected to the same scrutiny by the pathologists as the material from Tehran. Tissue areas with high (50%) neoplastic cellularity were dissected from dewaxed slides, and the material was digested by proteinase K in SDS-containing buffer for 3–5 days at 50°C to release DNA suitable for PCR. PCR and DNA Sequencing. Dewaxing, microdissection, DNA extraction, and PCR set-up were all performed in a special laboratory free of contamina- tion from PCR products, with reagents and equipment reserved for these purposes as we have described previously (5). Each exon (exons 5– 8) of the p53 gene was individually amplified by a single 40-cycle PCR, using intron- specific 20-mer primers as described by Lehman et al. (6), and 27–30-mer primers described by the Affymetrix GeneChip protocol (Affymetrix, Inc., Santa Clara, CA). The longer primers efficiently generated PCR product from template DNA of formalin-fixed specimens that had proved difficult to amplify with 20-mer primers. PCR products were purified with Microcon 100 (Milli- pore) filters and sequenced directly by BigDye fluorescent dye dideoxy sequencing and microcapillary electrophoresis with an ABI 310 Genetic Analyzer according to the supplier’s instructions (Applied Biosystems Inter- national). All samples with mutations were verified by two independent cycle sequencing PCR reactions and analysis of both DNA strands. In addition, of the 48 mutation-bearing tumor samples, 45 (94%) were reanalyzed by retriev- ing genomic DNA stocks, performing new PCR amplifications, and resequenc- Received 10/13/00; accepted 1/19/01. 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. 1 This work was supported by a Deutsche Akademischer Austauschdienst scholarship (to F. B.). 2 To whom requests for reprints should be addressed, at German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: 49-6221-42- 33-02; Fax: 49-6221-42-33-42; E-mail: m.hollstein@dkfz-heidelberg.de. 3 The abbreviations used are: ESCC, esophageal squamous cell carcinomas; ADC, adenocarcinoma; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; IHC, immunohistochemistry; PCR, polymerase chain reaction. 3119 on July 17, 2015. © 2001 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from