Authors: Hestia S. Meller, Lesia Jackson, Gary Anthony Pestano; Biodesix, Boulder, CO
Date: Thursday, February 28th: 11:30 AM – 1:00 PM and 6:00 PM – 7:00 PM
Location: San Francisco Marriot Marquis, San Francisco, CA
Abstract 124: Development and correlative analysis of a TML assay in specimens with reference MSI and somatic variant results.
Background: Treatment of late-stage non-small cell lung cancer has been greatly impacted by the advances in immunotherapy. Molecular biomarkers such as tumor mutation load (TML or TMB) and micro-satellite instability (MSI) may identify patients with NSCLC that are more likely to respond to immune checkpoint inhibitors. In this report, we describe the development of an NGS-based assay for the robust detection of TNL and somatic variants simultaneously using a targeted assay that covers 1.7 Mb of the genome.
Methods: Initial feasibility included reference control cell-lines, HCC1143, and NIST8398, as well as FFPE (formalin-fixed, paraffin-embedded) samples from lung and colorectal tumors. In development, eight FFPE specimens from patients with late stage cancer and a reference MSI status were evaluated. FFPE slides were extracted using the MagMax FFPE DNA/RNA Ultra kit, and nucleic acid quality was assesses using qPCR. Extracted DNA was processed through the Oncomine TML Assay with a minimum input of 30 ng of DNA per specimen. Automated library and template preparation were performed followed by sequencing on the Ion GeneStudio S5 Plus system using replicate 540 chips. Eight samples with barcoded adapters were multiplexed per chip. Tumor mutation analysis and variant calling was performed using the Oncomie TML v2.0 workflow.
Results: The time from the FFPE extraction to result was 96 hours, and all samples passed QC metrics. TML scores ranged from 14.4-36.04 mutations/MB for all samples with MSI-high reference results. A very high level of inter-chip concordance was also observed (R2=0.990). Additionally, the variant caller function within the TML workflow was used to detect BRAF V600E mutations with 100% concordance to reference testing results.
Conclusions: The accurate quantification of somatic mutations paired with fast turn-around time and robust automated workflow is ideal for assessing the tumor mutation load as well as hotspot mutations from limited (FFPE) samples.