Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration. Abbreviations: Becn1 /BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb /CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b /IL1B/Il-1β, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b /MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv /MEFV/TRIM20: Mediterranean fever; Nos2 /NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3 /NLRP3, NLR family, pyrin domain containing 3; Sqstm1 /SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf /TNF/TNF-α, tumor necrosis factor; Ulk1 /ULK1, unc-51 like kinase 1.
Introduction: Gene fusions, usually the result of chromosomal rearrangements, are frequently associated with many cancer types, and hence clinically actionable, making fusion detection an important part of cancer disease management. We developed a new, optimized version of the Seraseq® Fusion RNA Reference Material, and demonstrate its consistent performance across different NGS enrichment assays, sequencing coverage depths, bioinformatics pipelines, and RNA mass inputs from a multi-lab investigation (Sites A, B, C, D & E). Methods: Biosynthetic DNA was used for transcription of 18 RNA fusions, including more common fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. The in vitro transcribed RNAs were mixed with total RNA extracted from GM24385 reference cell line (The 1000 Genomes Project, Coriell). Digital PCR with TaqMan® chemistry was used to determine the target fusion RNA concentration and serve as the “truth” data set for comparison to NGS, which can be variable depending on input, assay, and bioinformatics. The fusion-total RNA mix was analyzed by five external laboratories; it was tested using the ArcherDx FusionPlex™ Solid Tumor Panel (Site A, Site B, Site C), the ArcherDx FusionPlex™ CTL Panel (Site C), the ArcherDx FusionPlex™ Lung Panel (Site B), a custom ArcherDx FusionPlex™ Panel (Site D), and the TruSight Tumor 170 Panel (Site E). Results: All eighteen (18) fusions in the new Seraseq Fusion RNA Mix v4 reference standard were detected as expected on each NGS platform with an average of greater than 85% of on-target reads across all assays. Even at inputs as low as 20 ng, all 18 fusions were typically detected above fusion-calling thresholds. In general, the results for individual fusions among the different NGS panels and among replicates were concordant, with observed variance in reads across some fusion junctions between assays and replicates. Within FusionPlex assay results, the average percent of reads supporting the fusion call across all fusions was about 63%, regardless of input (a range between 20 to 250 ng). Collectively, the multi-lab results confirm that the Seraseq® Fusion RNA Mix v4 reference standard is compatible with both amplicon and hybridization-capture based NGS assays. Conclusions: Seraseq RNA Fusion Mix v4 has broad NGS assay compatibility and allows for reliable and simultaneous detection of 18 clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this reference standard for targeted NGS assay development, assay validation, bioinformatics pipeline optimization, and as positive controls in clinical NGS RNA fusion assays. The biosynthetic manufacturing approach produces reference materials that provide consistent results for a wide variety of common and rare gene fusions. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella. Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1709.
Introduction: Genomic structural alterations are increasingly actionable for targeted therapeutics and personalized medicine. Molecular diagnostics are rapidly being introduced for detection of fusion RNAs by highly multiplexed next-generation sequencing assays. However, reference materials to aid in the development, optimization and validation of these assays are lacking. We developed Seraseq FFPE Fusion RNA Reference Material to fill this unmet need and show that this material is compatible across a wide range of NGS assay platforms. Methods: Biosynthetics were used for transcription of clinically actionable RNA fusions, including fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. Sixteen (16) in vitro transcribed RNAs were introduced into GM24385 reference cell line (The 1000 Genomes Project, Coriell). The cells were collected, fixed in formalin, and total RNA was isolated. Digital PCR with TaqMan® chemistry was used to determine the target Fusion RNA copies per microliter. Use of fusion-specific digital PCR provides an orthogonal method of verifying transcript levels and serves as the “ground truth” for the abundance of each RNA. NGS testing of the purified RNA used the ArcherDx FusionPlex™ CTL Panel, QIAGEN QIAseq Targeted RNAscan Panel, and the Thermo Fisher Oncomine Focus Assay. Results: All sixteen (16) fusions present in the prototype were detected as expected on each NGS platform. Results among the three different NGS platforms were generally concordant, although the reads across the fusion junctions did vary slightly among NGS assays and with digital PCR results. However, all methods indicated that the reference material gave low positive results, similar to a patient sample, and that the single reference material could serve as a positive control for detection of sixteen different fusions, which represent a variety of different solid tumor types. Conclusions: Seraseq FFPE RNA Fusion Reference Material allows simultaneous evaluation of detection for sixteen fusions observed in a variety of solid tumors, both common and rare. It provides a consistent, unlimited supply of QC materials particularly valuable for difficult to find rare fusions. The reference material generates low positive results on three leading assays, and this is important to truly challenge the assay system. This material is handled identically to a patient sample from extraction through analysis, and verifies performance at levels expected for patient samples. Citation Format: Catherine Huang, Yves Konigshofer, Lequan Nguyen, Rajeswari Vemula, Praveena Kamineni, Deepika Philkana, Ekta Jaiswal, Bharathi Anekella. Digital PCR-characterized, highly multiplexed, oncology RNA fusion reference materials: Performance on multiple NGS platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2017-3825
Tumor profiling often begins with a tissue biopsy that is formalin fixed and paraffin embedded (FFPE), a process that introduces various kinds of damage in the nucleic acids found in the tissue, however FFPE reference materials that closely mimic the damage profile of patient samples are lacking. Depurination, depyrimidination, deamination, oxidation, nicks, and double strand breaks may be found in DNA extracted from FFPE tissue, despite the use of extraction kits that attempt to repair some of this damage. We have developed a formalin-damaged, multiplexed biosynthetic reference material, Seraseq® FFPE Tumor DNA, to mimic this type of damage found in patient samples to create a more patient-like control. Biosynthetic DNA containing 24 cancer variants of interest were pooled in equivalent concentrations and introduced into the GM24385 reference cell line (Coriell) to simulate SNVs, indels, and structural rearrangements. Full length, allele-specific copies of ERBB2, MET, and MYC were also introduced to create copy number amplifications (CNV). Engineered cells were diluted to achieve desired allele frequencies and copy numbers as determined by digital PCR. The cells were processed with a proprietary FFPE protocol that mimics the damage seen in patient samples. Various FFPE extraction kits were used to isolate DNA and the amount of damage was assessed using the KAPA hgDNA Quantification & QC Kit. Allele frequencies and copy numbers were determined by digital PCR, the Archer VariantPlex Solid Tumor assay, and the Illumina TruSight Tumor 170 assay. DNA extractions of the FFPE reference material with the Qiagen QIAamp DNA FFPE Tissue Kit and the Promega Maxwell RSC FFPE DNA Kit yielded similar amounts (>200 ng per curl). All 24 variants were detected close to the 5-10% targeted allele frequency range and CNVs were within 5-10 total copies by dPCR. Similar results were observed with the TST170 assay. The quality as measured by Q120 bp/Q41 bp ratio using the KAPA hgDNA Quantification & QC Kit of the FFPE extracted damaged DNA was reduced by ~33% compared to non-damaged DNA extracted from FFPE, closer to the quality seen for some patient samples while still maintaining good amplifiability in downstream processes. The Seraseq FFPE Tumor DNA Reference Material performs as designed in digital PCR, amplicon-based, and hybrid capture-based NGS assays. It can be used in full process assay development and validation, bioinformatics pipeline optimization, and as a positive control in clinical research. The biosynthetic manufacturing approach allows for multiplexing of customizable variants and adjustable DNA damage. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Matthew G. Butler, Omoshile Clement, Bharathi Anekella. Development and performance of a formalin-damaged multiplexed DNA tumor mutation FFPE reference material [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1322.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.