CDH1 Gene Promoter Hypermethylation in Gastric Cancer Relationship to Goseki Grading, Microsatellite Instability Status, and EBV Invasion Monika Zazula, PhD,* Ana Maria Ferreira, MSc,w Jacek P. Czopek, MD, PhD,* Piotr Kolodziejczyk, MD, PhD,z Anna Sinczak-Kuta, PhD,* Agnieszka Klimkowska, MSc,* Piotr Wojcik, MSc,* Krzysztof Okon, MD, PhD,* Magdalena Bialas, MD, PhD,* Jan Kulig, MD, PhD,z and Jerzy Stachura, MD, PhD* Abstract: Hypermethylation of the CDH1 promoter region seems to be the most common epigenetic mechanism in this gene silencing in gastric cancer. In this study, CDH1 promoter hypermethylation was observed in 54.8% (46/84) of the analyzed sporadic gastric carcinomas. We introduce a new relation: clustering of Goseki grading into 3 grade was determined by CDH1 promoter hypermethylation. The percen- tage of methylation in Goseki III cancers was significantly higher (83%) when compared with other grades; the lowest proportion was detected in IV (36%) and II (38%) groups, whereas grade I demonstrated typical percentage of promoter hypermethylation. A novel polymorphism R732R in exon 14 of the CDH1 gene was detected by mutational analysis. Addition- ally, all cases with the MSI-high phenotype revealed CDH1 promoter hypermethylation. In MSI-low and MSS gastric cancers, this percentage was lower, reaching 71% and 41%, respectively. Moreover, the methylation status was correlated with the LOH phenotype. We detected CDH1 promoter hypermethylation in all EBV-positive gastric cancers (5/5), whereas methylation in the EBV-negative group occurred in 58% of cases. We also report that ‘‘methylated’’ tumors were slightly larger than ‘‘nonmethylated,’’ whereas the second group revealed a higher probability of longer patient survival, though these relationships were not statistically significant. These results suggest that downregulation of E-cadherin, caused by promoter hypermethylation, in sporadic gastric carcinomas may be associated with a worse prognosis and specific tumor phenotype. Key Words: gastric cancer, CDH1 promoter hypermethylation, E-cadherin, Goseki grading (Diagn Mol Pathol 2006;15:24–29) T he calcium-dependent cell adhesion molecule E-cadherin is encoded by a tumor suppressor gene (CDH1) located on chromosome 16q22.1. This glycopro- tein plays a crucial role in cell-to-cell adhesion, main- taining cell polarity and the normal architecture of epithelium. Moreover, E-cadherin plays a role in cell signaling, cell differentiation, organogenesis, and meta- stasis formation. 1,2 In gastric carcinoma, 2 important mechanisms of E-cadherin downregulation have been described to date: mutations (germ-line and sporadic) 3,4 and methylation of the 5 0 CpG island in the promoter region, which may cause transcriptional repression of the CDH1 gene. 5 In normal tissues in embryogenesis, during cell differentia- tion and development process, the CpG methylation is present. 6 The abnormal activity of methyltransferases has been detected in several human neoplasms. 7 This results in aberrant CpG islands methylation and transcriptional silencing of many human tumor suppressor genes. In gastric carcinoma, hypermethylation of the E-cadherin promoter region seems to be the most common suppres- sion mechanism as the second-hit event in inactivation of the E-cadherin gene. 8,9 Helicobacter pylori infection may also affect this epigenetic mechanism of the gene silencing. 10 Analysis of microsatellite instability (MSI) and loss of heterozygosity (LOH) is recommended for screening patients with hereditary or sporadic tumors. The associa- tion of the CDH1 promoter methylation with the microsatellite instability phenotype in gastric carcinoma has been previously reported. 11,12 Microsatellites are DNA short tandem repeats (STR) of 1 to 6 nucleotides, dispersed throughout the human genome. The lengths of microsatellite sequences are highly polymorphic in human populations but appear to be stable during the life of an Copyright r 2006 by Lippincott Williams & Wilkins From the *Department of Pathomorphology, Collegium Medicum, Jagiellonian University, Krakow, Poland; wInstitute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal; and zDepartment of General Surgery, Collegium Medicum, Jagiellonian University, Krakow, Poland. Supported by grants from the Ministry of Science and Information Society Technologies of Poland (KBN 3 PO5A131 24, PBZ-KBZ- 091/PO5/24) and CELLION project, Marie Curie Actions, The Sixth Framework Programme. Reprints: Jerzy Stachura, Pathomorphology Dept, Medical College, Jagiellonian University, Grzegorzecka 16, 31-531 Krakow, Poland (e-mail: mpstachu@cyf-kr.edu.pl). ORIGINAL ARTICLE 24 Diagn Mol Pathol  Volume 15, Number 1, March 2006