Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®

Abstract

We developed and analytically validated a comprehensive genomic profiling (CGP) assay, GEM ExTra, for patients with advanced solid tumors that uses Next Generation Sequencing (NGS) to characterize whole exomes employing a paired tumor-normal subtraction methodology. 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 select special transcripts, including AR-V7, EGFR vIII, EGFRvIV, and MET exon 14 skipping events. The assay has a mean target coverage of 180X for the normal (germline) and 400X for tumor DNA including enhanced probe design to facilitate the sequencing of difficult regions. Proprietary bioinformatics, paired with comprehensive clinical curation results in reporting that defines clinically actionable, FDA-approved, and clinical trial drug options for the management of the patient's cancer. GEM ExTra demonstrated analytic specificity (PPV) of > 99.9% and analytic sensitivity of 98.8%. Application of GEM ExTra to 1,435 patient samples revealed clinically actionable alterations in 83.9% of reports, including 31 (2.5%) where therapeutic recommendations were based on RNA fusion findings only.

Keywords: RNA sequencing; comprehensive genomic profiling; pan-cancer; precision medicine; whole exome sequencing.

Figure 1. Performance of SNV and Indel detection by GEM ExTra.

(A) Correlation of GEM ExTra SNV VAF to Horizon reference standard. (B) Correlation of GEM ExTra Indel VAF to Horizon reference standard. The black line is the regression line, and the gray area is 95% confidence interval. The dashed blue line indicates x = y. (C) Serial 1:1 dilution of 6 SNVs and their corresponding VAFs. Black line indicates the linear regression of the data. (D) Serial 1:1 dilution of 4 Indels and their corresponding VAFs. Black line indicates the linear regression of the data.

Figure 2. Biomarkers for immunotherapy by GEM ExTra.

(A). Frequency of high microsatellite instability in GEM ExTra. 7/30 tumor types harbored MSI-H tumors. (B) TMB landscape in GEM ExTra. Boxplots show distribution of TMB scores in mutation/Megabase. Black line within boxes shows median TMB score, the right edge of the box is the 75th percentile of interquartile of TMB scores, left edge of box is 25th percentile interquartile of TMB scores. Red, dashed lines indicate TMB threshold of 5 mut/MB for low TMB, and 20 mut/Mb for high TMB. Black dots or outliers are TMB scores outside the 1.5 interquartile range. Shaded are indicates TMB >= 20 mut/Mb.

Figure 3. Performance characteristic of GEM ExTra assay.

(A) Tumor specific positivity of GEM ExTra. (B) Violin plot of the number of clinically actionable events reported per tumor type. (C) Frequency of variant types across tumor types. (D) The thirty most common clinically actionable genes and number of variant types detected in each gene.

Figure 4. Mutation profiles of clinically actionable genes in GEM ExTra.

(A) The twelve most reported genes and their mutation distribution across tumor types. (B) Four selected clinically relevant genes with hotspot mutations and their frequency among various tumor types. Alterations colored by mutation types and their position with respect to domain structure is shown. RefSeq gene ID and count frequency shown on y axis.

Figure 5. Fusions detection in GEM ExTra.

(A) Fusions detected by tumor type. (B) Fusion’s detected in tumors types with RNA only findings. For those main fusion genes (e.g., BRAF) that were found to be fused with multiple partner genes (e.g., KIAA1549, or ARPC1A) the partner genes are separated from main fusion gene by a dashed line “-”, and the other partners listed consecutively, separated by a forward slash “/”.