COX-2 (PTGS2) gene methylation in epithelial, subepithelial lymphocyte and stromal tissue compartments in a spectrum of esophageal squamous neoplasia.
BACKGROUND
Previous studies have shown important effects of stromal elements in carcinogenesis. To explore the tumor-stromal relationship in esophageal neoplasia, we examined methylation of COX-2 (PTGS2), a gene etiologically associated with the development of gastrointestinal cancers, in adjacent foci of epithelium, subepithelial lymphocytes and non-lymphocytic stromal cells found in sections of normal squamous epithelium, squamous dysplasia and invasive esophageal squamous cell carcinoma.


METHODS
Adjacent foci of epithelium, subepithelial lymphocytic aggregates and non-lymphocytic stromal tissues were laser microdissected from six fully embedded, ethanol fixed, esophagectomy samples from Shanxi, China, a high-risk region for esophageal cancer. Promoter CpG site-specific hypermethylation status of COX-2 was determined using real-time methylation-specific PCR (qMS-PCR) based on Taqman Chemistry. The methylation status of a subset of samples was confirmed by pyrosequencing.


RESULTS
Forty-nine microdissected foci were analyzed. COX-2 gene methylation was significantly more common in subepithelial lymphocytes (12/16 (75% of all foci)) than in epithelial foci (3/16 (19%)) or foci of non-lymphocytic stromal tissues (3/17 (18%)) (Fisher's exact p=0.05). Two of three epithelial samples and all three stromal samples that showed COX-2 methylation were adjacent to foci of methylated subepithelial lymphocytes. Pyrosequencing confirmed the methylation status in a subset of samples.


CONCLUSIONS
In these esophageal cancer patients, COX-2 gene methylation was more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in both grades of dysplasia and in foci of invasive cancer. These findings raise the possibility that methylation of subepithelial lymphocytes may be important for tumorigenesis. Future studies of gene methylation should consider separate evaluation of epithelial and non-epithelial cell populations.
Dawsey
Sonja P
SP
Cancer Genetics Branch, Center for Cancer Research, National Cancer Institute, Rockville, MD 20892, USA.
Roth
Mark J
MJ
Adams
Lisa
L
Hu
Nan
N
Wang
Quan-Hong
QH
Taylor
Philip R
PR
Woodson
Karen
K
D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D051546:Cyclooxygenase 2 / Q000235:genetics*; D019175:DNA Methylation; D004847:Epithelial Cells / Q000502:physiology*; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D008214:Lymphocytes / Q000502:physiology*; D042282:Microdissection; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000502:physiology*
D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human