Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer,Normal Colonic Mucosa,and Peripheral Blood Cells

Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.Genome-wide DNA hypomethylation is considered to play an important role in genomic instability and carcinogenic processes.^{1,2,3,4,5,6,7} DNA methylation measured in LINE-1 (long interspersed nucleotide element-1, or L1) has been correlated with global DNA methylation,^8,9,10,11 and linearly predicts survival of colon cancer patients.¹² In addition, LINE-1 methylation levels in synchronous colorectal cancer pairs (ie, two or more primary colorectal cancers) are significantly correlated, suggesting the presence of a nonstochastic component in a variation of LINE-1 methylation levels in cancer.¹³ Nonetheless, utility of measuring LINE-1 or global DNA methylation levels in peripheral blood leukocytes remains controversial. One study has reported no significant association between LINE-1 methylation levels in blood leukocytes and risk of colorectal adenoma,¹⁴ whereas another study has shown an inverse association between global DNA methylation level in blood leukocytes and colorectal adenoma risk.¹⁵ Additional studies using precise LINE-1 methylation measurements are needed. Moreover, LINE-1 methylation measurement in peripheral blood can provide a useful way to evaluate the activity of DNA methylation inhibitors in solid tumor patients.¹⁶Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics,¹⁷ typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no previous study has evaluated the precision of bisulfite conversion to measure LINE-1 methylation. In addition, although pyrosequencing appears to be superior to combined bisulfite and restriction analysis or bisulfite/real-time PCR (MethyLight) in terms of precision of LINE-1 methylation measurement on plasma DNA,¹⁶ no study has comprehensively evaluated performance of bisulfite-pyrosequencing assay to measure LINE-1 methylation in paraffin-embedded tissue.In this study, we assessed the precision of sodium bisulfite conversion and PCR-pyrosequencing assay using 10 paraffin-embedded colon cancer specimens, 5 matched normal colon specimens, and 5 unrelated blood specimens. We have shown that LINE-1 pyrosequencing assay has good precision and can reliably measure LINE-1 methylation in paraffin-embedded colon cancer, normal colon tissue, and peripheral blood cells.