Abstract 182: Direct bisulfite conversion from archived tumor samples for methylation detection
Authors: Liu Chunmei , Dong Shoulian , Stevens Junko 
Published: February 2011

DNA methylation is an important epigenetic mechanism of transcriptional control. DNA methylation plays an essential role in maintaining cellular function, and changes in methylation patterns may contribute to the development of cancer. Aberrant methylation of DNA is frequently found in tumor cells.

Formalin-fixed and paraffin-embedded (FFPE) tissue sections are used for routine histopathological diagnostics, but they have increasingly become material for molecular studies of genome using molecular biological techniques such as PCR, qPCR, sequencing, genotyping etc. A major limitation of FFPE samples is the significant degree of degradation of the nucleic acids recovered from fixed tissues especially for the aged, long term archived samples. Since FFPE samples provide such huge collections of clinical tissue bank, more and more efforts have been spent in recovering the high quality of DNA from these specimens.

Although the number of epigenetic cancer studies continues to grow, the wealth of FFPE samples available remains largely untapped. The value of FFPE samples and the potential they have to contribute to studies of the epigenome and the role of DNA methylation in numerous biological processes and disease, including cancer has been recognized more and more.

Couple methods have been wildly used for methylation detection nowadays. With all the methods available, The bisulfite conversion method remains to be the most commonly used technique today as it can provide single CpG site methylation detection. Currently available methods for genome study or epigenetic study require isolation of gDNA from FFPE samples. With that, all the protocols require deparaffinization of the specimen prior to DNA extraction. Deparaffinization is usually accomplished with 2 to 3 incubations in xylene and can be a nuisance, particularly if there are more than just a few specimens to deal with.

We, hereby, develop a new method to not only eliminate the deparaffinization but also eliminate the genomic DNA purification procedure. In our procedure, FFPE samples are directly threw in the denaturation/lysis reagent followed by bisulfite conversion and desulfonation procedure. The whole process can be completed within 2 hours. Even for long term archived samples, fair length of DNA fragments can be captured after the conversion. High quality of converted gDNA fragmented are produced which can be used for methylation detection at single CpG site level such as methylation profiling, deep sequencing etc.

Clinical archived breast cancer and adjacent normal tissues as well as paired lung cancer samples were directly bisulfite converted and both breast cancer and lung cancer related promoter regions such as APC, BCL2, PTEN, and SCGB3A1 were compared for the methylation pattern changes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 182.