Poster Presentation 27th Lorne Cancer Conference 2015

Next generation amplicon based sequencing of TERT promoter mutations in melanoma cell lines and formalin fixed paraffin embedded clinical samples (#141)

Andrew J Colebatch 1 2 , Stephen Q Wong 2 , Paul M Waring 1 , Alexander Dobrovic 3 , Karen E Sheppard 2 4 , Grant A McArthur 2
  1. Department of Pathology, The University of Melbourne, Melbourne, VIC, Australia
  2. Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
  3. Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research , Melbourne, VIC, Australia
  4. Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, VIC, Australia

Telomerase reverse transcriptase (TERT) promoter mutations are the most common somatic mutations in melanoma. To systematically assess the clinical biology of melanomas, both in the research and routine clinical setting, we have included a TERT promoter amplicon into an amplicon based next generation cancer gene panel. The TERT assay uses Fluidigm index barcodes for pair-end sequencing using the Illumina Miseq system to evaluate the TERT promoter mutation status of melanoma samples. We then applied this assay to evaluate the TERT promoter mutation status, as well as BRAF and NRAS mutation status, across a panel of 61 melanoma cell lines and 118 formalin fixed paraffin embedded primary cutaneous melanoma samples derived from the Melbourne Melanoma Project cohort. The cell lines had typical TERT promoter mutations in 53/61 cell lines (87%) while the clinical samples had mutations in 80/107 (75%) with 11 samples failing to amplify. This difference showed a trend towards TERT mutations being more common in cell lines, but was not significant (p=0.08 Fisher's Exact test). We found that in all cell lines with TERT mutations the allelic fraction was near 100%. We also found two potentially novel recurrent promoter mutations, a GG>AA mutation at chr5:1295228-9 present in two cell lines and one clinical sample, and a G>A mutation at chr5:1295205 present in six clinical samples. In conclusion, this amplicon based sequencing method confirms the high proportion of melanoma cases that have TERT promoter mutations, identifies novel somatic promoter mutations and will allow TERT promoter mutations to be evaluated either as a stand alone assay or in next generation sequencing amplicon panels.