CD44 expression is regulated by translocation and promoter hypermethylation in gastric lymphoma

Abstract

One of the major genetic features of non-Hodgkin’s lymphomas (NHL) is the chromosomal translocation, most frequently involving the immunoglobulin heavy chain (IGH) gene at band 14q32. By employing the IgH dual color, break apart rearrangement probe (Vysis Inc.) for interphase FISH; followed by the long-distance inverse polymerase chain (LDI-PCR) strategy, we identified CD44 at chromosome 11p13 as a novel translocation partner gene of IGH in primary GL. The IGH/CD44 t(11;14)(p13;q32) was detected in 40% (4/10) mucosa-associated lymphoid tissue (MALT) lymphoma (3 positive cases are transformed MALT lymphoma cases) and 10% (4/40) diffuse large B-cell lymphoma (DLBCL) cases. This finding makes CD44 as the first cell adhesion molecule involving the IGH translocation and the third gene identified that involves the IGH translocation in MALT lymphoma. Immunohistochemistry showed that CD44 was expressed strongly in the membrane and cytoplasm of the tumor cells in 40% (4/10) MALT cases and 67.5% (27/40) DLBCL cases, including 8 IGH/CD44 translocation positive cases, while the remaining cases showed very weak or no CD44 expression. We next determined whether the hypermethylation of the promoter region of CD44 gene was responsible for the downregulation of CD44 in GL cases lacking CD44 expression. We first analyzed 10 mature B-cell lymphoma cell lines for the CD44 methylation status by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). Methylated alleles were detected in all the cell lines lacking CD44 expression but not in cell lines with CD44 expression. The treatment of the cell lines lacking CD44 with the demethylation agent 5-Aza-dC restored the CD44 expression. In the GL cases, methylated alleles were detected in 42% (21/50) cases and they significantly correlated with weak or no CD44 protein expression in the tumor cells. The most interesting observation was that the 3 CpG sites contained by E2F, Sp1 and EGR1 were nearly always methylated in cell lines lacking CD44 expression but not in the cell lines with CD44 expression. Similar results were also found in the GL cases. The correlation of CD44 expression and clincopathological parameters showed that strong CD44 expression was associated with poor prognosis in GL, and the cases with unmethylated CD44 were positively correlated with high stage (p=0.023). Overall, our results show that CD44 expression is regulated by different mechanisms operating simultaneously in GL, chromosomal translocation in cases with strong CD44 expression and gene silencing by promoter hypermethylation in cases lacking CD44.