Validation and Clinical Applications of a Comprehensive Next Generation Sequencing System for Molecular Characterization of Solid Cancer Tissues.

Mehdi Dehghani,Lei Li,Robert J Amato,Kevin P Rosenblatt,Mrudula Rakhade

doi:10.3389/fmolb.2019.00082

Abstract

Identification of somatic molecular alterations in primary and metastatic solid tumor specimens can provide critical information regarding tumor biology and its heterogeneity, and enables the detection of molecular markers for clinical personalized treatment assignment. However, the optimal methods and target genes for clinical use are still being in development. Toward this end, we validated a targeted amplification-based NGS panel (Oncomine comprehensive assay v1) on a personal genome machine sequencer for molecular profiling of solid tumors. This panel covers 143 genes, and requires low amounts of DNA (20 ng) and RNA (10 ng). We used 27 FFPE tissue specimens, 10 cell lines, and 24 commercial reference materials to evaluate the performance characteristics of this assay. We also evaluated the performance of the assay on 26 OCT-embedded fresh frozen specimens (OEFF). The assay was found to be highly specific (>99%) and sensitive (>99%), with low false-positive and false-negative rates for single-nucleotide variants, indels, copy number alterations, and gene fusions. Our results indicate that this is a reliable method to determine molecular alterations in both fixed and fresh frozen solid tumor samples, including core needle biopsies.

Highlights

Cancers are products of micro-evolutionary processes; each cancer patient harbors a unique pattern of molecular alterations in oncogenes and tumor suppressors that together cause aberrant cell signaling that leads to cancer development and progression (Gerlinger et al, 2014; Martincorena and Campbell, 2015)
next-generation sequencing (NGS) enables the establishment of a tumor-agnostic program to study tumor heterogeneity among and within patients—for example, the dissimilar molecular alterations that are often found at different metastatic sites within the same patient
Tumor molecular profiling can be explored at different levels; DNA and/or RNA for detecting different types of molecular alteration such as single nucleotide variants (SNVs), insertion/deletions, copy number alterations (CNAs), and fusion transcripts (Hovelson et al, 2015; Luthra et al, 2017)

Summary

Introduction

Cancers are products of micro-evolutionary processes; each cancer patient harbors a unique pattern of molecular alterations in oncogenes and tumor suppressors that together cause aberrant cell signaling that leads to cancer development and progression (Gerlinger et al, 2014; Martincorena and Campbell, 2015). Recent advances in genotyping technologies such as high-throughput deep sequencing have led to the identification of frequent somatic molecular alterations in solid tumor cancers (Lawrence et al, 2013, 2014; Vogelstein et al, 2013; Hovelson et al, 2015). These advances have led to a paradigm shift in cancer companion diagnostics work-ups, from single gene–based tests to multiplexed next-generation sequencing (NGS)–based assays (Cronin and Ross, 2011; Coonrod et al, 2012; Koboldt et al, 2013; Aftimos et al, 2014). Tumor molecular profiling can be explored at different levels; DNA and/or RNA for detecting different types of molecular alteration such as single nucleotide variants (SNVs), insertion/deletions (indels), copy number alterations (CNAs), and fusion transcripts (Hovelson et al, 2015; Luthra et al, 2017)

Methods

Results

Conclusion