Analytic Validation and Clinical Utilization of Comprehensive Genomic Profiling Test

Identification of genomic alterations by Next Generation Sequencing (NGS) has become an efficient clinical tool, particularly for oncology as molecular markers can guide personalized treatment. Currently available options for tumor profiling include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and, more recently, small panel next-generation sequencing (NGS).

Scientists at the Ashion Analytics, LLC, (Phoenix, AZ, USA) developed and analytically validated their comprehensive genomic profiling assay, GEM ExTra. The assay is for patients with advanced solid tumors that and uses Next Generation Sequencing to characterize whole exomes employing a paired tumor-normal subtraction methodology. The test was utilized in over 1,400 patient samples during a period of April 2018 and December 2019 across cancer centers to detect multiple actionable alterations in a variety of cancer types.

The assay detects single nucleotide variants (SNV), indels, focal copy number alterations (CNA), TERT promoter region, as well as tumor mutation burden (TMB) and microsatellite instability (MSI) status. Additionally, the assay incorporates whole transcriptome sequencing of the tumor sample that allows for the detection of gene fusions and selects special transcripts, including AR-V7, EGFR vIII, EGFRvIV, and MET exon 14 skipping events. The assay has a mean target coverage of 180× for the normal (germline) and 400× for tumor DNA including enhanced probe design to facilitate the sequencing of difficult regions.

The team found that found that 83.9% of tumor samples harbored at least one clinically actionable alteration (defined as positive) and the rest defined as negative, with a total of 1,267 positive and 242 negative reports (Detection Rate: 2018 = 76.4%, 2019 = 86.4%). Overall, 3,535 clinically actionable mutations were identified in the cohort (1,864 unique mutations), with a median of two clinically actionable alterations per tumor (mean = 2.93 ± 2.37) showing extensive variation across cancer types. Tumors with highest number of actionable mutations included skin (4.9 ± 2.2), endometrial (4.5 ± 3.9), and colorectal (4.1 ± 3.5). These results generally agree with previous estimates of driver events per patient in these tumor types. This is somewhat lower than previously reported in a pan-cancer study (4.6/tumor) of whole genomes, which also included driver copy number alterations which are not called out as actionable with GEM ExTra. Mean coding SNVs (i.e., missense, nonsense, stop codon) was 1.9 ± 1.4 per tumor which is within the range of predicted driver mutations in cancer.

The scientists concluded that they had developed and analytically validated a comprehensive genomic profiling assay with a 14-day turnaround time that can be adapted to all future tumor profiling needs due to combined DNA and RNA analysis. The GEM ExTra assay not only uses WES for tumor DNA profiling, but also identifies clinically actionable transcript variants and fusion genes through RNA sequencing, both of which ensure that GEM ExTra will be comprehensive in the future. The study was published on April 13, 2021 in the journal Oncotarget.

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