Abstract
Abstract We previously developed a “concordance calculator” to quantify reproducibility of multi-variant calls among next generation sequencing (NGS) samples and replicates. This tool and a novel replicate approach have been also used to eliminate many different technical artifacts including post tissue collection modifications (PTCM) such as deamination and oxidation artifacts. Here we apply this approach to study the impact of tumor heterogeneity among consecutive FFPE tissue sections across entire tumor blocks and cannot detect any impact of heterogeneity among different sections/regions of tumors in terms of mutation profiles using the NGS AmpliSeq Cancer Panel, even though the tumor was visibly heterogeneous according to the H&E images and pathological review. RNA expression profiling using a NanoString Cancer Panel found significantly different expression patterns among different sections/ regions. Additional studies in a different tissue type also found no detectable discrepancies among different tissue sections in terms of their mutation profiles. If confirmed by further studies, these results using FFPE tissue sections would suggest that DNA mutation signatures as novel biomarkers for cancer diagnosis and prognosis might be less sensitive to tumor heterogeneity than RNA-based expression signatures, at least based on the performance and sensitivity of current DNA/RNA profiling technologies. Use of the concordance calculator to quantify reproducibility of multi-variant calls among Next Generation Sequencing replicates and to eliminate many different technical artifacts including PTCM also allowed us to develop an unconventional validation strategy. We call this validation approach “in situ analytical validation and evaluation (iSAVE).” As a proof of concept, we evaluated the RainDance ThunderBolts Cancer Panel and demonstrated analytical validation directly on each and every clinical sample. This strategy also comprises using a set of normal FFPE tissue samples in the validation process to eliminate platform-, panel-, amplicon-, library-preparation-, and mutation calling pipeline specific artifacts.
Highlights
Ever since the publication of “Intratumor Heterogeneity Revealed by Multiregion Sequencing” in New England Journal of Medicine (NEJM) in 2012 by Gerlinger et al questions such as, “How can we expect precision medicine to be effective if tumors are so heterogeneous?” are raised at almost every biomarker or precision medicine conference [1,2]
Colorectal cancer (CRC) FFPE tissue blocks that had been profiled with an next generation sequencing (NGS)-based cancer panel and their corresponding 5 μm sectioned slides were purchased from BioChain Institute, Inc. (Newark, CA)
Strategies or data analysis tools for developing reliable low false positive rate diagnostic NGS panels were adapted by scientists in the field for developing NGS mutation profiling prognostic panels even though in whole exome sequencing (WES) that both low false positive and negative rates are important
Summary
Ever since the publication of “Intratumor Heterogeneity Revealed by Multiregion Sequencing” in NEJM in 2012 by Gerlinger et al questions such as, “How can we expect precision medicine to be effective if tumors are so heterogeneous?” are raised at almost every biomarker or precision medicine conference [1,2]. Despite this concern, new biomarkers, genetic tests and gene signatures are discovered on a routine basis for both diagnostic and prognostic applications [3,4,5,6]. After trying to interpret the results obtained from Gerlinger’s publication, we initiated a study to investigate the impact of tumor heterogeneity on both RNA expression and DNA mutation profiling in different sections and regions of a tumor
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