Abstract 5329: Telomerase reverse transcriptase promoter methylation in metastatic melanoma
Authors: Borah Sumit , Lee Seung J. , Fan Yiping , Barnhill Raymond L. , Dummer Reinhard , Kirkwood John M. , Bahrami Armita 
Published: August 2018

Abstract
Aberrant methylation of a particular region of the Telomerase Reverse Transcriptase (TERT) promoter, termed the upstream of transcription start site (UTSS), is associated with increased TERT expression and may be useful for differentiating malignant tumors, such as childhood brain tumors, prostate cancer, gastric cancer and melanoma, from their benign/low-grade histologic mimics. However, it is unclear whether UTSS methylation is actually responsible for transcription or is only associated with it. The purpose of this study was to test the hypothesis that UTSS methylation is an activating mechanism for transcription of this gene in melanoma.
Since the majority of melanomas express TERT mRNA, we predicted that i) the majority of these samples would have some detectable aberration in the TERT promoter that might account for expression and ii) UTSS methylation would preferentially occur in samples which lacked other recurrent TERT promoter aberrations (point mutation or rearrangement). To test this prediction, we used a cohort of 57 adult melanoma samples to catalog the presence of UTSS methylation (by high-throughput bisulfite-sequencing), promoter mutation (by Sanger sequencing) and/or promoter rearrangement (by fluorescence in situ hybridization) in each case. 5 cases of atypical Spitz tumors and congenital nevi served as negative controls, since these do not express TERT mRNA and were therefore not expected to have such aberrations. As predicted, 9 of the 10 melanoma samples which lacked promoter mutations or rearrangement had a high level of UTSS methylation. Unexpectedly, UTSS methylation sometimes overlapped with the presence of point mutations (9 of 40 cases) or promoter rearrangement (3 of 5 cases). None of the negative control samples expressed TERT mRNA or harbored any promoter aberrations.
We next predicted that, in samples with methylated UTSS, the actively transcribed TERT allele(s) would harbor the methylated UTSS while the transcriptionally silent allele(s) would not. Since methylation analysis of clinical samples is complicated by the presence of unmethylated UTSS from contaminating normal tissue, we tested this prediction in two relatively pure cancer cell lines. Although only one TERT allele was transcribed in each cell line, as measured by digital droplet PCR, we found that in each case both the transcriptionally active and transcriptionally silent alleles had methylated UTSS.
These results suggest that UTSS methylation is not sufficient for TERT upregulation, although it may still be useful as a biomarker for diagnosing melanoma and other cancers. Future studies will be aimed to determine if methylation of other regions of the TERT promoter marks the active allele in samples lacking mutations or rearrangement.
Citation Format: Sumit Borah, Seung J. Lee, Yiping Fan, Raymond L. Barnhill, Reinhard Dummer, John M. Kirkwood, Armita Bahrami. Telomerase reverse transcriptase promoter methylation in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5329.