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

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

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.

Keywords: Ion AmpliSeq; analytical validation; molecular profiling; next-generation sequencing; solid tumor.

Copyright © 2019 Dehghani, Rosenblatt, Li, Rakhade and Amato.

Figures

Figure 1
Figure 1
OCAv1 test workflow. Hematoxylin and Eosin-stained slides were reviewed to ensure each specimen contained adequate tumor content. Laser microdissection was used to enrich tumor content to ≥50%. DNA and RNA were co-purified, then their quality and quantity were assessed. Libraries created from the corresponding DNA and RNA specimens were sequenced on the personal genome machine. Sequence data were analyzed and reviewed using the Ion Reporter software.
Figure 2
Figure 2
Lowest limits of detection for SNVs and Indels. The lowest limit of detection of OCAv1 for identifying SNVs and indels, with expected allele frequencies of ~2.5–50%, were evaluated using the AOHL member 1 to member 6 ladders and were sequenced. (A) Indicates the detection rates of the assay for SNVs and Indels with expected allele frequencies of ~5–30% (hotspot and non-hotspot variants); and, (B) shows the detection rate of the assay for variants listed in the hotspot bed file.
Figure 3
Figure 3
Heatmaps of somatic alterations detected. OCAv1 identified relevant somatic alterations in the 26 solid tumor registry specimens, including cases with RCC (A) and other types of cancers (B). All relevant high-priority molecular alterations, including SNVs, indels, CNAs, fusions, and variants of unknown significance, are shown in the heat map. Specific alterations and cancer types are shown at the bottom of the heat maps according to the keys.

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