Prognostic impact of EZH2 expression, H3K27 trimethylation and DNA methylation in diffuse large B cell lymphoma

Purpose
International Prognostic Index (IPI) score is used to predict the prognosis in diffuse large B cell lymphoma (DLBCL) for around 20 years. However, few molecular prognostic factors were proposed.
Overexpression of Enhancer of zeste homolog 2 (EZH2) and decreased histone 3-lysine 27 tri-methylation (H3K27me3) are associated with poor prognosis in many cancers. Promoter CpG island hypermethylation of tumor-suppressor genes is a common hallmark of all human cancers. 5-methylcytosine (5-mC) levels in cancer cells can reflect the DNA hypermethylation status and measurement of 5-hydroxymethylcytosine (5-hmC) levels may estimate the DNA demethylation status in cancer cells. A unique EZH2 Tyr 641 somatic mutation was detected in DLBCL and follicular lymphoma. However, the clinical implications of DNA methylation status, DNA hydroxymethylation, EZH2 expression, the extent of H3K27me3 and EZH2 Tyr 641 somatic mutation in DLBCL patients have not been studied in a comprehensive or integrated way. We aim to see significant impact of DNA methylation, DNA hydroxymethylation, EZH2 expression levels and mutation status, and H3K27 trimethylation on the clinical and biological presentation of DLBCL.
Patients and Methods
We enrolled 107 consecutive patients with DLBCL in Far Eastern Memorial Hospital and 83 consecutive patients in Chi Mei Medical Center (diagnosed between 2002 and 2009). The demographic data, treatment regimens, and response of disease were reviewed retrospectively.
Immunohistochemistry (IHC) was used to examine 5-mC, 5-hmC, EZH2 expression and the extent of H3K27me3 in formalin-fixed, paraffin-embedded biopsy specimens of DLBCL. DNA extraction from the tissues was examined for EZH2 Tyr641 mutation. Statistical analysis was performed with the Stata statistical software (Small Stata, version 11.0, Stata Corp, College Station, TX).
Results
Statistical analysis was performed in 138 patients with Rituximab-CHOP and CHOP regimen. Totally we recruited 90 male (65%) and 48 female (35%) with a median age 57.2 years. Fifty-nine percent of the patients had stage I/II disease. According to the IPI, 70% of the patients were classified as low/low-intermediate risk (IPI=0-2) and 30% as intermediate-high/high risk (IPI=3-5). Thirty-seven patients received CHOP-like regimen and 101 patients received Rituximab-CHOP-like regimen as first-line chemotherapy. The median observation time for overall survival (OS) in the 138 patients was 42 mo. Male patients tended to have lower H3K27me3 expression (p=0.044). Certain clinical parameters such as IPI score, stage and response to treatment were correlated with OS. None of 48 patients harbored EZH2 mutation at Tyr641. Low expression 5-mC patients tended to have more complete response after standard chemotherapy (P=0.006). Low expression 5-mC tended to have lower H3K27me3 expression (P=0.014). And low expression 5-mC tended to have lower 5-hmC expression (P=0.001). There was no obvious relationship between the expression of EZH2 and degree of H3K27me3 in DLBCL tumor cells. Although there was no significant prognostic impact in univariate survival analysis, low 5-hmC expression seemed to have longer OS (P=0.061). When other strong prognostic factors were included in multivariate analysis, there was no significant prognostic impact in single epigenetic marker. Then we subdivide the patients by combination H3K27me3 with 5-hmC. The high H3K27me3/ low 5-hmC patients was associated with longer OS (P=0.0497) but there was no significant prognostic impact in multivariate survival analysis. In the subgroup analysis, we enrolled the 110 patients with complete remission (CR) after standard chemotherapy and subdivide the patients by combination H3K27me3 with 5-hmC. The high H3K27me3/ low 5-hmC patients was associated with longer OS in univariate (P=0.0131) and multivariate analysis (P=0.017).
Conclusion
High H3K27me3/ low 5-hmC expression was a possible favorable prognostic factor in DLBCL and it showed even more significant prognostic impact in CR patients. Theincidence of EZH2 mutation at Tyr641 in our cohort is much lower compared with western countries. Low expression 5-mC tended to have lower H3K27me3 expression and low expression 5-mC tended to have lower 5-hmC in DLBCL tumor cells. Our study suggests epigenetic markers may be an informative biomarker for prognosis prediction in DLBCL.