Genetic aberrations driving MET deregulation detected with anchored multiplex PCR and next-generation sequencing

Deregulation of the receptor tyrosine kinase, MET, is associated with aggressive phenotypes in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis. Several types of genetic aberrations can drive MET deregulation, including gene amplification, overexpression, single nucleotide variants (SNVs), exon 14 skipping and fusions. MET is a target of intensive drug development efforts, however the various mutated forms of MET exhibit unique drug sensitivities. Therefore, detection of these mutations has the potential to guide treatments for cancers driven by MET deregulation. Next-generation sequencing (NGS) enables comprehensive detection of all mutation types from whole genomes and transcriptomes. However, low detection sensitivity, high input requirement and high costs render these approaches impractical for routine detection of mutations from low-input clinical sample types. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that, by its scalable and quantitative nature, is well suited for detection of each of the modes deregulation of MET. We developed AMP-based VariantPlex™ and FusionPlex™ library preparation assays for NGS to detect mutations from DNA and RNA, respectively. We designed AMP probes covering the MET gene to detect copy numbers and SNVs from DNA, and fusions, exon skipping and expression levels from RNA. VariantPlex and FusionPlex kits enabled detection of MET amplifications, confirmed by FISH, and the resulting overexpression in FFPE samples. Exon 14 skipping was also detected and confirmed by RT-PCR in FFPE and in cells, with concomitant splice site mutations. Lastly, a GTF2I:MET gene fusion and a Y1253D activating point mutation were detected. These results show that AMP enables comprehensive and sensitive NGS-based detection of multiple mutation types from low-input clinical sample types.