Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer

Abstract

Background: E-cadherin is an adhesion molecule involved in tumour invasion/metastasis. Silencing of E-cadherin by promoter CpG methylation has been shown in both familial and sporadic gastric cancers. Helicobacter pylori is a class I carcinogen in gastric cancer.

Aims: This study was undertaken to investigate the association between methylation of E-cadherin and H pylori in gastric mucosa from dyspeptic patients, and in intestinal metaplasia and primary and metastatic adenocarcinoma from surgical specimens of patients with gastric cancer.

Methods: E-cadherin methylation was studied using methylation specific polymerase chain reaction in microdissected tissue from biopsies or surgical resection specimens. E-cadherin expression was studied by immunohistochemistry.

Results: E-cadherin methylation was present in 31% (11/35) of gastric mucosae from dyspeptic patients, and was associated with H pylori infection (p=0.002), but was independent of the age of the patient or presence or absence of gastritis. E-cadherin methylation was present in 0% (0/8) of normal mucosa, 57% (12/21) of intestinal metaplasias, and 58% (15/26) of primary and 65% (21/32) of metastatic cancers. E-cadherin methylation status was concordant in 92% (11/12) of intestinal metaplasias and primary cancers, and in 85% (17/20) of primary and metastatic cancers from the same resected specimen. E-cadherin methylation in gastric cancer was associated with depth of tumour invasion (p=0.02) and regional nodal metastasis (p=0.05).

Conclusion: E-cadherin methylation is an early event in gastric carcinogenesis, and is initiated by H pylori infection.

Figure 1

(A) Confirmation of the specificity of methylation specific polymerase chain reaction (MSP). The wild-type E-cadherin gene sequence is shown together with the sequence of the methylated polymerase chain reaction (PCR) product. M1: PCR product (shown here from nucleotides 2280 to 2339, Genbank accession No D49685) with first primer set, as described by Herman and colleagues,²⁰ for the methylated E-cadherin allele (forward primer: 5′- TTA GGT TAG AGG GTT ATC GCG T -3′; reverse primer: 5′- TAA CTA AAA ATT CAC CTA CCG ACC -3′). Primers for the unmethylated E-cadherin allele were forward: 5′- TAA TTT TAG GTT AGA GGG TTA TTG T -3′; reverse: 5′- CAC AAC CAA TCA ACA ACA CA -3′. In the MSP product, methylated cytosine residues that are unchanged are underlined in blue, and the unmethylated cytosine residues that have become uracil/thymidine are underlined in red. (B) M2: PCR product from second primer set for methylated E-cadherin allele (shown here from nucleotides 2229 to 2283), as described by Graff and colleagues²¹, for the methylated E-cadherin allele (forward primer: 5′- GTG GGC GGG TCG TTA GTT TC -3′; reverse primer: 5′- CTC ACA AAT ACT TTA CAA TTC CGA CG -3′). Primers for the unmethylated E-cadherin allele were forward: 5′- GGT GGG TGG GTT GTT AGT TTT GT -3′; reverse: 5′- AAC TCA CAA ATC TTT ACA ATT CCA ACA -3′.

Figure 2

(A) Analysis of methylation of E-cadherin in gastric mucosae from patients with dyspepsia. Samples N3–N6 are methylated, and samples N1, N2, and N7 are unmethylated. Methylation specific polymerase chain reaction (MSP) was performed with the first primer set, resulting in a methylated polymerase chain reaction (PCR) product of 116 bp and an unmethylated PCR product of 97 bp. (B) Analysis of methylation of E-cadherin in gastric mucosae from patients with dyspepsia, as shown in (A), but with MSP performed with the second primer set. Samples N3–N6 are methylated, and samples N1, N2, and N7 are unmethylated. (C) Analysis of methylation of E-cadherin in intestinal metaplasia (IM), gastric adenocarcinomas (T), and metastatic lymph nodes (LN) from patients with gastric cancer. Samples IM2, T1, T2, LN1, and LN2 are methylated, and sample IM1 is unmethylated. MSP was performed with the first primer set.

Figure 3

Immunohistochemical staining for E-cadherin, showing (A) membranous distribution of staining in the normal gastric mucosa, (B) cytoplasmic staining in gastric cancer cells, and (C) cytoplasmic staining in neoplastic cells in a metastatic lymph node.