Detection of hypermethylation of tumor suppressor genes in ocular adnexal lymphoma using multiplex ligation-dependent probe amplification

Abstract

Purpose: Ocular adnexal lymphomas (OAL) occur in the orbit, lacrimal drainage system, conjunctiva and eyelid. They comprise 8% of all extranodal non-Hodgkin lymphomas (NHL) with the extranodal marginal zone B-cell lymphoma (EMZL) being the commonest, accounting for 2/3 of them. Hypermethylation in CpG sites in promoter regions is an epigenetic change that could lead to silencing of a gene. Hypermethylation in tumor suppressor genes (TSGs) occurs in various tumors, including NHL. We examined the methylation status of multiple TSGs in OAL. Methods: Formalin-fixed paraffin-embedded (FFPE) OAL, including both EMZL and non-EMZL OALs, were examined. The majority of OAL were located in the conjunctiva. DNA was extracted and purified from FFPE, and only those with DNA of sufficient quantity and quality were selected for subsequent analysis using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). 33 EMZL and 27 non-EMZL were examined using two MS-MLPA kits, ME001B and ME002, in which methylation status of CpG sites in a total of 35 candidate TSGs were analyzed. Reactive lymphoid hyperplasia cases were used as controls. MLPA PCR products are separated by capillary electrophoresis, and sequencing data were statistically analyzed with specifically designed Excel spreadsheets (NGRL, Manchester, UK). CpG hypermethylation in patient samples was determined when statistical significance of standard error > 0.1 as compared to the reference samples. Results: Specific loci in eight TSGs including CDH13, WT1, MSH6, IGSF4, DAPK1, ESR1, p14-ARF and RAR-beta showed hypermethylation in at least 65% of the 33 EMZL OALs, as well as in majority of the 27 non-EMZL OALs. However, hypermethylation status varied among different subtypes of non-EMZLs, which includes follicular, diffuse-large-B-cell and mantle cell lymphomas. Validation of this data is currently in progress using pyrosequencing. Conclusions: Many OALs demonstrated hypermethylation. Correlation of methylation analysis data with clinical presentation and follow-up may reveal epigenetic markers of prognostic value in these tumors.