Background: DNA-based next-generation sequencing has been widely used in the selection of target therapies for patients with nonsmall cell lung cancer (NSCLC).RNA-based next-generation sequencing has been proven to be valuable in detecting fusion and exon-skipping mutations and is recommended by National Comprehensive Cancer Network guidelines for these mutation types. Methods:The authors developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for the detection of fusions, single-nucleotide variants (SNVs), and insertion/deletion (indels). In total, 1253 formalin-fixed, paraffinembedded samples from patients with NSCLC were analyzed by DNA and RNA panel sequencing in parallel to assess the performance of the RNA panel in detecting multiple types of mutations. Results:In analytical validation, the RNA panel achieved a limit of detection of 1.45-3.15 copies per nanogram for SNVs and 0.21-6.48 copies per nanogram for fusions. In 1253 formalin-fixed, paraffin-embedded NSCLC samples, the RNA panel identified a total of 124 fusion events and 26 MET exon 14-skipping events, in which 14 fusions and six MET exon 14-skipping mutations were missed by DNA panel sequencing. By using the DNA panel as the reference, the positive percent agreement and the positive predictive value of the RNA panel were 98.08% and 98.62%, respectively, for detecting targetable SNVs and 98.15% and 99.38%, respectively, for detecting targetable indels. Conclusions: Parallel DNA and RNA sequencing analyses demonstrated the accuracy and robustness of the RNA sequencing panel in detecting multiple types of clinically actionable mutations. The simplified experimental workflow and low sample consumption will make RNA panel sequencing a potentially effective method in clinical testing.See related editorial on pages 2294-6, this issue.
Currently, DNA and RNA are used separately to capture different types of gene mutations. DNA is commonly used for the detection of SNVs, indels and CNVs; RNA is used for analysis of gene fusion and gene expression. To perform both DNA sequencing (DNA-seq) and RNA-seq, material is divided into two copies, and two different proce-dures are required for sequencing. Due to overconsumption of samples and experimental process complexity, it is necessary to create an experimental method capable of analyzing SNVs, indels, fusions and expression. We developed an RNA-based hybridization capture panel targeting actionable driver oncogenes in solid tumors and corresponding sample preparation and bioinformatics workflows. Analytical validation with an RNA standard reference containing 16 known fusion mutations and 6 SNV mutations demonstrated a detection specificity of 100.0% [95% CI 88.7%~100.0%] for SNVs and 100.0% [95% CI 95.4%~100.0%] for fusions. The targeted RNA panel achieved a 0.73-2.63 copies/ng RNA lower limit of detection (LOD) for SNVs and 0.21-6.48 copies/ng RNA for fusions. Gene expression analysis revealed a correlation greater than 0.9 across all 15 cancer-related genes between the RNA-seq re-sults and targeted RNA panel. Among 1253 NSCLC FFPE tumor samples, multiple mutation types were called from DNA- and RNA-seq data and compared between the two assays. The DNA panel detected 103 fusions and 21 METex14 skipping events; 124 fusions and 26 METex14 skipping events were detected by the target RNA panel; 21 fusions and 4 METex14 skipping events were only detected by the target RNA panel. Among the 173 NSCLC samples negative for targetable mutations by DNA-seq, 15 (15/173, 8.67%) showed targetable gene fusions that may change clinical decisions with RNA-seq. In total, 226 tier I and tier II missense variants for NSCLC were analyzed at ge-nomic (DNA-seq) and transcriptomic (RNA-seq) levels. The positive percent agreement (PPA) was 97.8%, and the positive predictive value (PPV) was 98.6%. Interestingly, var-iant allele frequencies were generally higher at the RNA level than at the DNA level, suggesting relatively dominant expression of mutant alleles. PPA was 97.6% and PPV 99.38% for EGFR 19del and 20ins variants. We also explored the relationship of RNA expression with gene copy number and protein expression. The RPKM of EGFR transcripts assessed by the RNA panel showed a linear relationship with copy number quantified by the DNA panel, with an R of 0.8 in 1253 samples. In contrast, MET gene expression is regulated in a more complex manner. In IHC analysis, all 3+ samples exhibited higher RPKM levels; IHC level of 2+ and below showed lower RNA expression. Parallel DNA- and RNA-seq and systematic analysis demonstrated the accuracy and robustness of the RNA sequencing panel in identifying multiple types of variants for cancer therapy.
ABSTRACT:The effect of long-term exposure to tributyltin (TBT) on energy metabolism and adenosine triphosphatase (ATPase) enzymatic system in freshwater teleost was investigated. The impact on the parameters of energy metabolism (the content of glucose (Glu) and lactate (LA), and the activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase(LDH)) in the muscle tissue, as well as on the ATP enzymatic system (ATP content, Total-ATPase, Na -ATPase) in the gill tissue of common carp was evaluated. Fish were exposed to sublethal concentrations of TBT (75, 0.75, and 7.5 µg/l) for 60 days. Based on the results, long-term exposure to TBT could lead to obvious ATP enzymatic system responses, including the decreased ATP content and Na + -K + -ATPase activity, and the increased activities of Total-ATPase and Ca 2+ -Mg 2+ -ATPase. Moreover, the parameters of energy metabolism in muscle were also regulated, such as induced indices of the levels of Glu and LA, and the activities of HK and PK, and inhibited indices of LDH activity. Shortly, the measured physiological responses in fish could provide useful information to better understand the mechanisms of TBTinduced bio-toxicity, and could be used as potential biomarkers for monitoring the TBT pollution in the field.
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