Abstract 1396: Validation of a MEK/MET-specific NGS panel for early phase trial interrogation
Authors: Maxwell Perry , Del Favero Jurgen , Fuchs Marc-Aurel , Tabernero Josep , Maughan Tim , Middleton Mark , Addams Richard , Rolfo Christian , Hennessey Brian , Laurent-Puig Pierre , Bardelli Alberto , Andre Thierry , Popovici Vlad , Johnstone Patrick , Wilson Richard , Lawler Mark , Van Schaeybroeck Sandra , Salto Tellez Manuel 
Published: July 2016

Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c-MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n = 3); BRAF (n = 4); KRAS (n = 8); NRAS (n = 1); MET (n = 8); PIK3CA (n = 6); and ERBB2 (n = 5).

Summary of Method

NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp.

Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing.

Results

The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100% coverage of all targets, a higher than 97% on target reads and higher than 99% of all amplicons within 20% of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5’ end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2% to 11.5% with an average variation of 5.2%. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways.

Conclusion

After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose.

Citation Format: Perry Maxwell, Jurgen Del Favero, Marc-Aurel Fuchs, Josep Tabernero, Tim Maughan, Mark Middleton, Richard Addams, Christian Rolfo, Brian Hennessey, Pierre Laurent-Puig, Alberto Bardelli, Thierry Andre, Vlad Popovici, Patrick Johnstone, Richard Wilson, Mark Lawler, Sandra Van Schaeybroeck, Manuel Salto Tellez. Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1396.