br Single workflow testing of DNA and
Single-workflow testing of DNA and RNA also offers advantages for intra-assay confirmation. For example, we identified FGFR2 and MET amplification events by DNA analysis that exhibited concomitant RNA overexpression, thus providing further functional evidence for the amplification event. Detection of RNA 3′/5′ imbalances offers a means of affirming the presence of explicitly targeted gene fusions while also detecting the presence of fusions with rare, noncanonical breakpoints not represented by the set of RNA breakpoint-spanning amplicons. We
Translational Oncology Vol. 12, No. 6, 2019 An Integrated Next-Generation Sequencing System Haynes et al. 843
found that 4/7 gene fusions were supported by a corresponding 3′/5′ imbalance event. Of note, all of the ALK fusion events (N = 2) showed a supporting imbalance ratio, whereas none of the ROS1 fusions did (N = 2). This is because ROS1 is endogenously expressed in lung tissue, whereas ALK is not. Therefore, the ROS1 imbalance has a greater intrinsic background, which must be overcome in order to be detectable relative to ALK, and thus the sensitivity of imbalance markers inherently depends upon a gene's level of endogenous expression. Interpretation of histopathology is also enabled through integrative molecular analysis. We identified DNA mutations that were able to lend further insights to ambiguous histopathologies and an RNA 252917-06-9 signature that distinguished adenocarcinomas from squamous cell carcinomas.
Combined, single-pass testing of DNA and RNA increases the likelihood of identifying a therapeutically relevant oncogenic driving event. This is due to the observation that DNA and RNA drivers are often mutually exclusive molecular events. Thus, DNA driver mutation-negative cases have a higher likelihood of bearing an RNA driver and vice-versa. In our own study, we found DNA and RNA variants to be largely exclusive events with 9 of the 10 subjects positive for an RNA driver lacking any of the common DNA mutations covered by the panel. In fact, when considering the theranostic yield of the assay (the number of subjects for which a guideline-recommended or emerging targeted therapy was identified), the inclusion of RNA markers achieved a 41% increase over DNA markers alone. Yet there were exceptions to this exclusivity. For example, one case revealed both an EML-ALK fusion and a KRAS p. G12D mutation. The co-occurrence of these two events is rare but not unprecedented , and the low VAF of KRAS p.G12D in this specimen (6.9%) is suggestive of a subclonal or potentially distinct population of tumor cells. Mutations in KRAS have been established as a mechanism of innate and acquired crizotinib resistance [46–48], which further highlights the clinical research value of integrative profiling of DNA and RNA to identify and further characterize resistance mechanisms. Despite the encouraging response rates of ALK, RET, and ROS positive cases to TKI therapies, resistance and progression ultimately occur within 1 year of treatment for the vast majority of patients. Joint analysis of DNA and RNA can support the identification of intrinsic and acquired resistance and enable the informed selection of second- and third-line TKI therapies.
Despite the advantages of unified DNA/RNA analysis, current commercial offerings and molecular testing solutions are largely fragmented and cumbersome. The vast majority of published and commercialized workflows address either DNA or RNA analysis exclusively but not both [49–52]. Some workflows have attempted to combine both into a single protocol such as the Oncomine Dx Target Test, Focus, and Comprehensive NGS assays , but these assays lack the advantages of our approach, such as support for gene expression quantification and a single-source input of TNA which enables a single isolation and eliminates purification steps (such as DNase treatment) that risk material loss. Our method enables streamlined single-plate library preparation through shared master mixes and harmonized reaction conditions. Workflows such as the TruSight Tumor 170 offer single-plate RNA/DNA library prepara-tion but require sonication, ligation, hybridization, and clean-up steps that introduce workflow complexity and sample attrition. Our targeted amplicon sequencing workflow is free of these cumbersome steps and requires less material (20 ng TNA or 10 ng DNA and 10 ng RNA) than TruSight (minimum 40 ng DNA and 40 ng RNA). Finally, our incorporation of preanalytical QC measurements
to inform bioinformatics variant detection and interpretation enables the suppression of false-positive variant calls and flags samples at risk of false-negative calls, thus enabling accurate analysis of poorer-quality specimens. The analytical validity of our approach is supported by 100% confirmation rates of RNA fusions and MET exon 14 skipping events by qPCR or PCR/CE and high analytical concordance in our analysis of FNA smears from the BATTLE-2 trial with an independent NGS assay on matched surgical resections.