Detection of Aberrant TERT Promoter Methylation by Combined Bisulfite Restriction Enzyme Analysis for Cancer Diagnosis

Aberrant CpG dinucleotide methylation in a specific region of the telomerase reverse transcriptase (TERT) promoter is associated with increased TERT mRNA levels and malignancy in several cancer types. However, routine screening of this region to aid cancer diagnosis can be challenging because i) several established methylation assays may inaccurately report on hypermethylation of this particular region, ii) interpreting the results of methylation assays can sometimes be difficult for clinical laboratories, and iii) use of high-throughput methylation assays for a few patient samples can be cost prohibitive. Herein, we describe the use of combined bisulfite restriction enzyme analysis (COBRA) as a diagnostic tool for detecting the hypermethylated TERT promoter using in vitro methylated and unmethylated genomic DNA as well as genomic DNA from four melanomas and two benign melanocytic lesions. We compare COBRA with MassARRAY, a more commonly used high-throughput approach, in screening for promoter hypermethylation in 28 formalin-fixed, paraffin-embedded neuroblastoma samples. COBRA sensitively and specifically detected samples with hypermethylated TERT promoter and was as effective as MassARRAY at differentiating high-risk from benign or Low-risk tumors. This study demonstrates the utility of this low-cost, technically straightforward, and easily interpretable assay for cancer diagnosis in tumors of an ambiguous nature.