Low Expression and Promoter Hypermethylation of the Tumour Suppressor SLIT2, are Associated with Adverse Patient Outcomes in Diffuse Large B Cell Lymphoma
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SLIT2 has been classified as a major tumour suppressor gene due to its frequent inactivation in different cancer types. However, alterations of SLIT2 expression and relation to patient outcomes in diffuse large B cell lymphoma (DLBCL) remain undefined. The aim of this study was to investigate the expression and the methylation status of SLIT2 gene as well as its relation to patient outcomes in DLBCL. Immunohistochemical (IHC) staining was carried out to detect the expression of SLIT2 in a series of 108 DLBCL cases. Re-analysis of previously published dataset (GSE10846) that measured gene expression in DLBCL patients who had received CHOP or R-CHOP therapy was performed to identify associations between SLIT2 and patients survival. Laser capture microdissection was performed to isolate GC B cells and DLBCL primary tumor cells. Bisulfite treatment and methylation-specific PCR (MSP) analysis were done to assess SLIT2 promotor methylation status. We report that the expression of SLIT2 protein was reduced in a subset of DLBCL cases and this was significantly correlated with advanced clinical stage (p = 0.041) and was an independent predictor of worse overall survival (OS) (p = 0.012). Re-analysis of published gene expression data showed that reduced SLIT2 mRNA expression was significantly correlated with worse OS in R-CHOP-treated ABC DLBCL patients (p = <0.01). Hypermethylation of the SLIT2 promotor was significantly correlated with low SLIT2 expression (p = 0.009). Our results provide a novel evidence of reduced expression of SLIT2 that is associated with promoter hypermethylation and adverse outcomes in patients with DLBCL.
KeywordsSLIT2 DLBCL Hypermethylation Patient survival
This work was supported by Ministry of Higher Education fund support (#2012-13), Egypt and by Hong Kong HMRF grant (#16151042).
Ghada Mohamed undertook pathologic review of the cases, interpreted and analyzed the IHC results, performed laboratory work and co-wrote the manuscript.; Soha Talima undertook clinical data analysis and co-wrote the manuscript; Lili Li performed laboratory work and co-wrote the manuscript; Wenbin Wei undertook bioinformatic and statistical analyses, interpreted the bioinformatic data and co-wrote the manuscript; Zbigniew Rudzki Undertook pathologic review of the cases; Rasha Allam undertook statistical analysis and interpreted the data; William Simmons undertook cases’ data collection; Qian Tao supervised laboratory work and co-wrote the manuscript; Paul G Murray designed and oversaw the research and co-wrote the manuscript.
This work was supported by Ministry of higher education fund support (#2012–13), Egypt and by Hong Kong HMRF grant (#16151042). The funding sources had no influence on the study design, analysis, and interpretation of the data.
Compliance with Ethical Standards
Conflict of Interest
The authors declare no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the UK Health Research Ethics Committee (North West-Haydock; reference 15/NW/0079).
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