br Grant Support This work was supported in part by
1 Grant Support: This work was supported in part by Grant CP120017 from the Cancer Prevention and Research Institute of Texas to Asuragen (PI: G.J.L.), by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number R43ES024365 (PI: B.C.H.), and by the National Institute of General Medical Sciences under award number R44GM111062 (PI: B.C.H.).
Translational Oncology Vol. 12, No. 6, 2019 An Integrated Next-Generation Sequencing System Haynes et al. 837
of tumor DNA variation for established and emerging drug targets is now possible in clinical reference labs through validated NGS panels based on hybridization capture or targeted amplicon sequencing. While these targeted NGS technologies have largely addressed the challenge of clinical DNA-based testing, the analysis of other molecular modalities of diagnostic relevance remains unaddressed or requires disjointed workflows.
Gene fusions have emerged as an important class of markers for precision medicine in solid tumors. Transforming rearrangements of the anaplastic lymphoma kinase (ALK) gene are present in 3%-6% of lung adenocarcinomas (LUADs) , and these tumors are responsive to crizotinib . Rearrangements of ROS1 and RET have also been found in LUADs at a prevalence of 1%-3% [8–10] and are responsive to crizotinib and multikinase inhibitors cabozantinib and vandetanib, respectively [8,11]. In addition to ALK, RET, and ROS1, fusions involving NTRK1, FGFR1/2/3, and NRG1 genes have been reported in NSCLC among other cancers and represent emerging therapeutic targets . Gene fusions are detectable by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis of DNA, and this form of testing is routine in clinical reference labs.
Targeted RNA-Seq is an emerging form of testing for gene fusions with distinct advantages over IHC and FISH including sensitivity, specificity, and multiplexing density [12–15]. In UNC1999 to NGS assays developed for SNVs, INDELs, and CNVs, targeted NGS assays developed for gene fusion detection are predominately based on RNA-Seq. While NGS analysis of DNA can also detect chromosomal rearrangements and DNA mutations that lead to aberrant isoforms, RNA-based testing can be more sensitive, efficient, and functionally definitive considering that many DNA variants (e.g., multiple intronic breakpoints) give rise to the same oncogenic transcript. Unlike IHC, targeted RNA-Seq does not require overexpression of the 3′ fused gene, and unlike FISH, it verifies that the chimeric transcript is expressed and in-frame. In addition, targeted RNA-Seq is capable of detecting additional classes of clinically relevant RNA variation including aberrant splice variants such as the exon 14 skipped isoform of MET, which leads to a constitutively activated form of cMET that confers sensitivity to crizotinib . Targeted RNA-Seq enables the quantification of gene expression markers of therapeutic value such as signatures of response to immune checkpoint blockade. While IHC testing of PD-L1 remains the entrenched patient selection tool for checkpoint inhibitors, evidence is emerging for RNA signatures with predictive accuracy superior to PD-L1 .
Despite the advantages of NGS in the analysis of gene fusions and other classes of RNA markers, NGS workflows for DNA and RNA markers remain largely segregated, and adoption of NGS testing for RNA has lagged DNA-based testing. One cause for this disparity is that the majority of targeted RNA-Seq and DNA-Seq assays are developed without consideration for harmonizing assay inputs, workflow, or analysis. Consequently, separate tissue slides and isolations are often required for analysis of DNA and RNA through discrete NGS procedures. While some NGS workflows have sought to address the interassay harmonization challenge , gaps remain with respect to separation of DNA and RNA during isolation, rigorous preanalytical sample characterization, applicability to real-world clinical specimens, and the accuracy of bioinformatics pipelines [19,20]. All the while, clinical testing guidelines are increasingly recommending broad, multicategorical molecular testing to inform treatment decisions and disease management in multiple cancers.
Lung cancer represents a disease in which broad yet streamlined molecular testing capabilities are acutely needed given the vast array of targeted and immunotherapeutic options available.
Molecular profiling of NSCLC is recommended by National Comprehensive Cancer Network (NCCN) guidelines, but many patients are not fully tested [21,22]. Barriers to access include cost, the number of tests required (≥3 separate tests are currently required), suitability of specimens, and regional access [23,24]. Many clinical labs do not offer comprehensive testing due to a lack of resources, expertise, or time required to develop these assays. This study addresses the need for a comprehensive, streamlined, sample-to-answer NGS technology and accommodates the spectrum of clinical specimen quality and molecular markers required to satisfy evolving NSCLC biomarker testing recommendations. Our results demonstrate that an NGS workflow that unifies the analysis of DNA and RNA markers in NSCLC can be used with challenging formalin-fixed paraffin-embedded (FFPE) speci-mens to recapitulate well-characterized molecular profiles.