Abstract LB-227: Guideline adherent clinical validation of a comprehensive DNA/RNA panel (523 genes-TruSight Oncology 500) for determination of single nucleotide variants (SNV’s), small insertions or deletions (Indels), copy number variations (CNV’s), splice variations (SV’s), gene fusions (GF’s), tumor mutation burden (TMB) and micro-satellite instability (MSI) on anext-generation sequencing (NGS)platform in a CLIA setting
Authors: Kolhe Ravindra , Ahluwalia Pankaj , Heneidi Saleh , Ananth Sudha , Kota Vamsi , Mondal Ashis 
Published: August 2019

Abstract
Introduction: Currently NGS techniques are being widely used as a tool in routine oncology workflows. Majority of labs use either DNA based small hotspots panels or two separate DNA and RNA panel. Unfortunately, this methodology can lead to an incomplete mutation profile because it lacks comprehensive tumor screening for theranostics. Many heterogeneous tumors carry multiple mutations and gene function can be altered by several types of variations including SNVs, CNV’s, Indels, SV’s and GF’s. Recently, it has been shown by various studies that TMB and MSI can correlate with a cancer patient’s responsiveness to checkpoint inhibitor immunotherapy. These findings have stimulated a widespread interest in the development of cost-effective assays that accurately measure TMB and MSI along with somatic variants. Here we assess whether TMB measured through NGS of a 1.9 MB-523-gene panel correlates with that determined through Foundation one CDx panel. We also assess and compare the accuracy of determining the MSI status of multiple cancers through sequencing a 523-gene targeted DNA panel vs MSI by PCR or MMR by immunohistochemistry. The assay is a targeted next-generation sequencing (NGS) assay designed to detect genetic alterations in 523 genes for SNVs, CNV’s, Indels, and SV’s & 55 genes for fusion and splice variant detection. Assessment of GF’s, SV’s, SNVs, CNV’s, TMB and MSI in one assay using DNA and RNA creates efficiencies in sample usage, time, and cost.
Methods: The validation was guided by the joint consensus recommendation for validation of NGS assays by the AMP, CAP & NYSDOH. The validation included evaluations of precision, analytic sensitivity, analytic specificity, accuracy, reportable range, and reference range. RNA and DNA from of 100 samples (mixture of known patient specimens, known CAP proficiency specimens as well as known synthetic reference standards {Accormetrix hotspot DNA panel Seracare CNV DNA panel, & Seracare fusion V2 panel}) was used. These clinical samples were earlier tested on gold standard orthogonal methods like PCR, FISH, CDx NGS, and karyotype ( n=100 clinical and reference materials formalin-fixed, paraffin-embedded (FFPE) tumor samples from lung, GI, skin, CNS, breast, kidney, uterus, ovarian, salivary, thyroid gland tumors, soft tissue & bone cancer etc. Libraries for were prepared using the Illumina TruSight Oncology 500 (TSO500+) kit and sequenced on NextSeq 550. Sequencing analysis, variant, TMB and MSI calling were performed using software provided by illumina.
Results: We were able to validate manufacture's analytical claims for all the variants groups including TMB and MSI in our validation.
Conclusions: DNA and RNA libraries are prepared in parallel, sequenced, and analyzed simultaneously for efficient assessment of numerous types of somatic variants. We anticipate that this approach of obtaining high-resolution data from an FFPE samples at reduced sequencing cost, will facilitate testing in different malignancies which was not previously possible especially for determination of TMB and MSI.
Note: This abstract was not presented at the meeting.
Citation Format: Ravindra Kolhe, Pankaj Ahluwalia, Saleh Heneidi, Sudha Ananth, Vamsi Kota, Ashis Mondal. Guideline adherent clinical validation of a comprehensive DNA/RNA panel (523 genes-TruSight Oncology 500) for determination of single nucleotide variants (SNV’s), small insertions or deletions (Indels), copy number variations (CNV’s), splice variations (SV’s), gene fusions (GF’s), tumor mutation burden (TMB) and micro-satellite instability (MSI) on anext-generation sequencing (NGS)platform in a CLIA setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-227.