Pyrosequencing-based Quantitative Evaluation of p16 Methylation in Diffuse Large B-cell Lymphoma
Abstract
Introduction: Methylation of promoter region of p16 leading to gene silencing has been implicated in a wide range of malignancies including lymphomas. In diffuse large B cell lymphoma (DLBCL) particularly, a varying percentage of epigenetic inactivation of p16 promoter region was observed ranging from 16 - 54%. However, quantitative analysis of p16 promoter methylation in DLBCL has not been extensively studied in Malaysia. Objective: This study aims to quantitatively analyse p16 methylation in DLBCL samples pyrosequencing technique. Methods: Genomic DNA was extracted from 16 formalin-fixed paraffin-embedded lymphoma tissue blocks from patients diagnosed with DLBCL. Primers were designed to amplify bisulfite-treated DNA targeting p16 promoter region. Methylation status of 7 CpG sites was determined by pyrosequencing. Results: All the 16 samples studied showed promoter methylation of p16. The range of mean methylation percentage was 18 - 81%. Conclusion: The present study has successfully identified methylation of p16 in all 7 CpG sites though there is limitation in the sample size. Our experience shows that pyrosequencing is relatively inexpensive, is able to analyse up to 96 samples in a single assay that is reproducible, with simple data extraction from displayed pyrogram, and more importantly it helps overcome the complexities related DNA methylation research.References
Hunt K.E. and Reichard K.K. Diffuse large B-Cell Lymphoma. Arch. Pathol. Lab. Med. 2008; 132:pp.118-124.
Chan W.C. Pathogenesis of diffuse large B cell lymphoma. Int. J. Hematol. 2010; 92:pp.219-230.
Perry A.M., Mitrovic Z. and Chan W.C. Biological Prognostic Markers in Diffuse Large B-Cell Lymphoma. Cancer Control. 2012; 19:pp.214-226.
Choo, K.B. Epigenetics in disease and cancer. Malaysian Journal of Pathology. 2011; 33:pp.61-70.
Baylin S.B. DNA methylation and gene silencing in cancer. Nat. Clin.Prac. Oncol. 2005; 2:S4-S11.
Mulero-Navarro S. and Esteller M. Epigenetic biomarkers for human cancer: The time is now. Oncol. Hematol. 2008; 68:pp.1-11.
Bihl M.P., Foerster A., Lugli A. and Zlobec I. Characterization of CDKN2A(p16) methylation and impact in colorectal cancer: systemic analysis using pyrosequencing. J. Transl. Med. 2012; 10:pp.1-10.
Drexler H.G. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells. Leuk. 1998; 12:pp.845-859.
Li J., Poi M.J. and Tsai M.D. The Regulatory Mechanisms of Tumor Suppressor P16INK4A and Relevance to Cancer. Biochem. 2011; 50:pp.5566-5582.
Zainuddin N., Kanduri M., Berglund M., Lindell M., Amini R.M., et al. Quantitative evaluation of p16INK4a promoter methylation using pyrosequencing in de novo diffuse large B-cell lymphoma. Leuk. Res. 2011; 35:pp.438-443.
Herman J.G., Graff J.R., Myohanen S., Nelkin B.D. and Baylin S.B. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Med. Sci. 1996; 93: 9821-9826.
Mikeska T., Bock C., El-Maari O., Hubner A., Ehrentraut D., et al. Optimization of Quantitative MGMT Promoter Methylation Analysis Using Pyrosequencing and Combined Bisulfite Restriction Analysis. J. Mol. Diagn. 2007; 9:pp.368-381.
Mikeska T., Candiloro I.L.M. and Dobrovic A. The implications of heterogeneous DNA methylation for the accurate quantification of methylation. Epigenomics. 2010; 2:pp.561-573.
Colella S., Shen L., Baggerly K.A., Issa J.P.J. and Krahe R. Sensitive and quantitative universal PyrosequencingTM methylation analysis of CpG sites. BioTechniques. 2003; 35:pp.146-150.
England R. and Pettersson M. Quantitative analysis of methylation at multiple CpG sites by ProsequencingTM. Biotage AB, Uppsala, Sweden.
Tost J. and Gut I.G. DNA methylation analysis by pyrosequencing. Nat Protoc. 2007; 2:pp.2265-2275.
Havik A.B., Brandal P., Honne H., Dahlback H.S.S., Scheie D., et al. MGMT promoter methylation in gliomas assessment by pyrosequencing and quantitative methylation-specific PCR. J. Transl. Med. 2012; 10:pp.1-13.
Roessler J. and Lehmann U. Quantitative DNA Methylation Analysis by Pyrosequencing. In Pyrosequencing Methods and Protocols, Methods in Molecular Biology. pp 175-188. Springer Science+Business Media New York.2015.
Yoon S.O., Kim Y.A., Jeon Y.K., Kim J.E., Kang G.H., et al. Diffuse Large B Cell Lymphoma Shows Distinct Methylation Profiles of the Tumor Suppressor Genes among the Non-Hodgkin’s Lymphomas. Korean J. Pathol. 2008; 42:pp.16-20.
Amara K., Trimeche M., Ziadi S., Laatiri A., Hachana M., et al. Progonostic significance of aberrant promoter hypermethylation of CpG islands in patients with diffuse large B-cell lymphoma. Ann. Oncol. 2008; 19:pp.1774-1786.
Shaw R.J., Lilogluo T., Rogers S.N., Brown J.S., Vaughan E.D., et al. Promoter methylation of P16, RARβ, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br. J. Cancer. 2006; 94:pp. 561-568.
Delgado M., Piqueras J.F., Garcia M.J., Arranz E., Rivas C., et al. Hypermethylation of a 5’ CpG island of p16 is a frequent event in non-Hodgkin’s lymphoma. Leuk. 1997; 11:pp.425-428.
Baur A.S., Shaw P., Burri N., Delacretazz F., Bosman F.T., et al. Frequent Methylation Silencing of p15INK4b (MTS2) and p16INK4a (MTS1) in B-Cell and T-Cell Lymphoma. Blood. 1999; 94:pp1773-1781.
Pinyol M., Cobo F., Bea S., Jares P., Nayach I., et al. p16INK4a Gene Inactivation by Deletions, Mutations, and Hypermethylation Is Associated With Transformed and Aggressive Variants of Non-Hodgkin’s Lymphomas. Blood. 1998; 91:pp.2977-2984.
Villuendas R., Beato M.S., MartÃnez J.C., Saez A.I., Delgado B.M., et al. Loss of p16/INK4A Protein Expression in Non-Hodgkin's Lymphomas Is a Frequent Finding Associated with Tumor Progression. Am. J. Pathol. 1998; 153:pp.887-897.
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