Immunohistochemical evaluation of global DNA methylation: Comparison with in vitro radiolabeled methyl incorporation assay

The in vitro radiolabeled methyl incorporation assay, a commonly used techn ique to evaluate global methylation of DNA, has some disadvantages and limi tations. The purpose of the present study was to compare the results of glo bal DNA methylation evaluated by radiolabeled methyl incorporation (CPM/ mu g of DNA) with immunohistochemical staining of the same tissue sections wit h a monoclonal antibody developed against B-methylcytosine (5-mc). We used archival specimens of squamous cell cancer (SCC) of the human lung with a m atched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjec ts without lung cancer [noncancer specimens] to make this comparison. The i mmunostaining for 5-mc was reported as a percentage of cells positive for s taining as well as a weighted average of the intensity score. The results s uggested that both radiolabeled methyl incorporation assay and immunostaini ng for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissue s compared to matched uninvolved tissues. An advantage of immunostaining, h owever, is its ability to demonstrate hypomethylation of SCC compared to ad jacent bronchial mucosa on the same archival specimen, obviating the need t o use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically signi ficant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolab eled methyl incorporation assay and may be best suited for evaluation of gl obal DNA methylation when the methylation status of cancer cannot be normal ized by methyl incorporation of normal tissues or when the number of sample s available for evaluation is small.