Anatomic Pathology / E-CADHERIN EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Hypermethylation in the E-cadherin promoter region and expression of the transcription factor Snail were analyzed in 41 cases of esophageal squamous cell carcinoma (ESCC) and paired normal squamous epithelium by methylation-specific polymerase chain reaction (PCR) and reverse transcription–polymerase chain reaction (RT-PCR) to clarify the mechanism regulating E-cadherin deletion; 93 cases of ESCC were analyzed immunohistochemically to determine the clinicopathologic impact of E-cadherin deletion. Hypermethylation of the E-cadherin promoter and Snail overexpression were detected in 25 cases (61%) by methylation-specific PCR and 34 cases (83%) by RT-PCR, respectively. Reduced E-cadherin expression, observed immunohistochemically in 55 cases (59%), correlated with hypermethylation (P = .0011) but not Snail overexpression (P = .685). Hypermethylation and Snail overexpression correlated significantly with E-cadherin deletion (P = .0018). Snail overexpression was unrelated to clinicopathologic factors. Reduced E-cadherin expression correlated with tumor invasion (P = .019) and vascular invasion (P = .052) but not other factors. E-cadherin deletion had prognostic impact in univariate (P = .023) and multivariate (P = .034) analyses. E-cadherin deletion was regulated by hypermethylation and Snail expression. Examination of reduced E-cadherin expression is important for assessing biologic behavior, including clinical outcome, in patients with ESCC. Esophageal cancer is a commonly occurring cancer that generally has an unfavorable prognosis despite the availability of multimodal therapies.1 In addition to the TNM classification, many genes have been evaluated as possible molecular markers for clarifying the biologic behavior of esophageal cancer or its clinical outcome, and several possible molecular markers have been reported.2-4 One of the potential molecular markers, E-cadherin, has been identified as a cell-adhesion molecule and is located at the 16q22 locus.5 To date, several reports have demonstrated the prognostic impact of reduced E-cadherin expression in various cancers of the digestive tract.6,7 In the esophagus, the impact of E-cadherin expression on cancer metastasis or progression also has been studied. Recently, the prognostic impact of reduced E-cadherin expression was reported in a multi-institutional study by the Japanese Society of Esophageal Disease.8 However, the mechanism regulating E-cadherin expression is still unclear. Cytosine methylation of the CpG island in the 5'promoter region is involved in the transcriptional inactivation of various genes and is a significant alternative to mutational inactivation in the development of cancer.9-11 Because the importance of cytosine methylation has become increasingly appreciated, many studies of methylation status have been conducted since Herman et al12 described a methylationspecific polymerase chain reaction (PCR) assay. Hypermethylation of the CpG island has been reported to be a major gene silencing mechanism for the E-cadherin gene in stomach and breast cancer and malignant melanoma.13-15 In contrast, the transcription factor Snail, located at 20q13.2, is a repressor of E-cadherin gene expression in epithelial tumor cells.16,17 The E-cadherin gene has an E-pal element containing 2 E-boxes including the sequence 5'-CACCTG in Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2004;122:78-84 79 79 DOI: 10.1309/WJL90JPEM17RBUHT 79 © American Society for Clinical Pathology the promoter. Snail has been reported to repress E-cadherin expression by binding to the E-boxes directly,17 although this function has been demonstrated in vitro but not in vivo. Therefore, study of Snail expression in surgically obtained tissue might clarify the regulatory mechanism controlling E-cadherin expression in the cancer tissue and reveal potential applicability to gene therapy. Although the incidence of adenocarcinoma of the esophagus is increasing in western countries, esophageal squamous cell carcinoma (ESCC) is the predominant histologic type globally, particularly in Japan and Asia in general.18 To our knowledge, the mechanism regulating E-cadherin expression in ESCC tissue has not been reported, and few studies have focused on adenocarcinoma of the esophagus. The present study is the first to clarify the regulation of E-cadherin expression in ESCC, and the results potentially might reveal a target for metastasis inhibition and provide treatment benefits to patients. In the present study, methylation-specific PCR assays of the E-cadherin promoter region, reverse transcription (RT)-PCR of Snail gene expression, and immunohistochemical staining for E-cadherin were conducted to clarify the regulation and role of E-cadherin expression in ESCC. Materials and Methods Patients and Tissue Samples We included 93 cases of esophageal squamous cell carcinoma (79 men and 14 women; mean age, 64.0 years; range, 36-84 years) in the present study. The patients had undergone esophagectomy and lymphadenectomy without preoperative supplemental therapy at our institute between January 1990 and December 2000. Resected specimens were classified in accordance with the TNM classification system of the International Union Against Cancer.19 In addition, invasion of the cancer into lymphatic and blood vessels was assessed microscopically. Genomic DNA and Total RNA Extraction For analysis of methylation status, fresh frozen tissue samples were obtained from a recent series that included 41 of the 93 ESCC cases. A section of the viable tumor was dissected macroscopically and stored at –80°C until use. Genomic DNA and total RNA were isolated from fresh frozen tissue samples using the QIAamp DNA Mini Kit and RNeasy Mini Kit (QIAGEN, Hilden, Germany), respectively, following the manufacturer’s protocol. Bisulfite Modification and Methylation-Specific PCR Aberrant methylation of the CpG island in the Ecadherin promoter region was studied via methylationspecific PCR. Bisulfite modification of genomic DNA, which converts unmethylated cytosines to uracil, was performed using the CpGenome DNA Modification Kit (Intergen, Purchase, NY) following the manufacturer’s protocol. A 1-μg sample of genomic DNA was used for bisulfite modification. CpGenome Universal Methylated DNA (Intergen) was used as a positive control for the methylated E-cadherin promoter region. PCR was carried out using 2 μL of bisulfite-modified genomic DNA under the conditions described in a previous report.20 Primer sequences for the methylated E-cadherin gene were (forward) 5'-TTAGGTTAGAGGGTTATCGCGT3' and (reverse) 5'-TAACTAAAAATTCACCTACCGAC-3', and those for the unmethylated E-cadherin gene were (forward) 5'-TAATTTTAGGTTAGAGGGTTATTGT-3' and (reverse) 5'-CACAACCAATCAACAACACA-3'. The annealing temperature was set at 57°C for methylated DNA and 53°C for unmethylated DNA.15 RT-PCR for Snail Expression Total RNA (1 μg), random primer (Roche Diagnostics, Indianapolis, IN), and a 10-mmol/L concentration of deoxynucleoside triphosphate and Super Script II (Invitrogen, Paisley, Scotland) were used for first-strand complementary DNA synthesis. Detailed conditions of reverse transcription were described in a previous report.21 Nested PCR was performed for Snail gene amplification as described in the literature. Primer sequences for the Snail gene were (forward) 5'-TGCGCGAATCGGCGACCC-3', (reverse) 5'-CCTAGAGAAGGCCTTCCCGCAG-3', (nested forward) 5'-ACTACAGCGAGCTGCAGG-3', and (nested reverse) 5'-GTGTGGCTTCGGATGTGC-3'. The annealing temperature was set at 60°C for the first and nested PCRs.15 The gene expression status of Snail was determined as overexpression when the PCR product was more amplified in tumor tissue compared with matched normal esophageal epithelium after visualization by electrophoresis. Visualization of PCR Products PCR products (10 μL) were analyzed by electrophoresis through a 2% agarose gel (High Strength Analytical Grade Agarose, BIO-RAD, Hercules, CA) containing ethidium bromide and visualized on a Mini-Transilluminator (Funakoshi, Tokyo, Japan). Immunohistochemical Analysis Immunohistochemical staining for E-cadherin was carried out using representative paraffin-embedded specimens from 93 patients. Sections (4-μm) were cut from resected specimens fixed in 10% buffered formalin and embedded in paraffin. After deparaffinization and rehydration, tissue sections were autoclaved at 121°C in citrate Takeno et al / E-CADHERIN EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA 80 Am J Clin Pathol 2004;122:78-84 80 DOI: 10.1309/WJL90JPEM17RBUHT © American Society for Clinical Pathology buffer (10-mmol/L concentration, pH 6.0) for 10 minutes for antigen retrieval. The specimens then were incubated with anti–E-cadherin monoclonal antibody (dilution 1:30; Novocastra, Newcastle upon Tyne, England) for 12 hours at 4°C. Immunohistochemical staining, following a standard avidinbiotin-peroxidase complex technique, was carried out using the Histofine SAB-PO (M) kit (Nichirei, Tokyo, Japan) and 3,3'-diaminobenzidine as the chromogen. Nuclei were counterstained with hematoxylin. Noncancerous epithelium was evaluated as a positive control sample, and a specimen analyzed without E-cadherin monoclonal antibody was used as a negative control sample. Immunohistochemical staining was evaluated by a combination of distribution and intensity in the cancer area. Concerning intensity, a positive reaction was determined as equal or strong intensity, and an unclear or weak reaction was determined as reduced intensity compared with matched normal squamous epithelium. Then, adding to intensity, specimens staining for E-cadherin were considered as positive when a preserved positive reaction could be observed in more than 50% of the cancer cells, whereas those with negative or unclear or weak reactions in more than 50% were characterized as reduced expression. The specimens were evaluated by 2 independent observers unaware of the clinical information (S.T. and Y.K.).