Background: A 15-gene expression profiling (GEP) test is widely used for prognostication of metastatic risk in uveal melanoma (UM) patients. Because the amount of tumor tissue that can be safely obtained by biopsy from UM is limited, it is critical to obtain as much individualized genomic information as possible from each biopsy sample. Mutational profiling of UM tumors using next generation sequencing (NGS) in combination with GEP allows for analysis of both DNA and RNA from a single tumor sample, offers additional prognostic value, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of the seven genes known to be commonly mutated in primary UM.
Methods: 105 primary UM samples were analyzed, including 37 formalin-fixed paraffin embedded (FFPE) specimens and 68 fine needle aspiration biopsy (FNAB) specimens obtained with a 25- or 27-gauge needle. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of greater than 500X per region of interest in a clinical laboratory accredited by the College of American Pathologists (CAP) and certified under the Clinical Laboratory Improvement Amendments (CLIA).
Results: The 7-gene panel assay achieved a positive percent agreement (PPA) of 100% for detection of both single nucleotide variants (SNVs) and insertions/deletions (INDELs), with a technical positive predictive value (TPPV) of 99.4% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the reproducibility and repeatability of the assay. The limit of detection was determined to be 5% variant allele frequency (VAF) for both SNVs and INDELs, with a minimum DNA input requirement of 1.5ng for FNAB and 5ng for FFPE samples.
Conclusions: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small gauge needle biopsy sample.