Quantification Of Gene Expression Research Articles

Oxidative stress and NF-KB/iNOS inflammatory pathway as innovative biomarkers for diagnosis of drowning and differentiating it from postmortem submersion in both fresh and saltwater in rats.

Finding a dead body in water raises an issue concerning determining the cause of death as drowning because of the complex pathophysiology of drowning. In addition, the corpse may be submersed postmortem. Evaluate the role of oxidative stress markers and NF-KB/iNOS inflammatory pathway as diagnostic biomarkers in drowning and whether they could differentiate freshwater from saltwater drowning. This study included forty-five adult male albino rats classified into five groups: control group (C), Freshwater-drowned group (FD), Freshwater postmortem submersion group (FPS), saltwater-drowned group (SD), and saltwater postmortem submersion group (SPS).After the autopsy, the rats' lungs in each group were prepared for histological, immunohistochemical (caspase 3, TNF-α, NF-kB, COX-2 & iNOS), biochemical studies; MDA, NOx, SOD, GSH, VCAM-1, COX-2; and RT-PCR for the relative quantification of NF-kB and iNOS genes expression. Lung oxidative markers were significantly affected in drowned groups than in postmortem submersion groups. Inflammatory pathway markers were also significantly increased in the drowned groups, with concern that all markers were significantly affected more in saltwater than in freshwater drowned group. It is concluded that the tested markers can be used accurately in diagnosing drowning and differentiating it from postmortem submersion with a better understanding of the mechanism of death in drowning as both mechanisms, inflammatory and oxidative stress, were revealed and involved.

Characterising 24-h skeletal muscle gene expression alongside metabolic & endocrine responses under diurnal conditions.

Skeletal muscle plays a central role in the storage, synthesis, and breakdown of nutrients, yet little research has explored temporal responses of this human tissue, especially with concurrent measures of systemic biomarkers of metabolism. To characterise temporal profiles in skeletal muscle expression of genes involved in carbohydrate metabolism, lipid metabolism, circadian clocks, and autophagy and descriptively relate them to systemic metabolites and hormones during a controlled laboratory protocol. Ten healthy adults (9M/1F, mean ± SD: age: 30 ± 10 y; BMI: 24.1 ± 2.7 kg·m-2) rested in the laboratory for 37 hours with all data collected during the final 24 hours of this period (i.e., 0800-0800 h). Participants ingested hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 2200-0700 h. Blood samples were collected hourly for endocrine and metabolite analyses, with muscle biopsies occurring every 4 h from 1200 h to 0800 h the following day to quantify gene expression. Plasma insulin displayed diurnal rhythmicity peaking at 1804 h. Expression of skeletal muscle genes involved in carbohydrate metabolism (Name - Acrophase; GLUT4 - 1440 h; PPARGC1A -1613 h; HK2 - 1824 h) and lipid metabolism (FABP3 - 1237 h; PDK4 - 0530 h; CPT1B - 1258 h) displayed 24 h rhythmicity that reflected the temporal rhythm of insulin. Equally, circulating glucose (0019 h), NEFA (0456 h), glycerol (0432 h), triglyceride (2314 h), urea (0046 h), CTX (0507 h) and cortisol concentrations (2250 h) also all displayed diurnal rhythmicity. Diurnal rhythms were present in human skeletal muscle gene expression as well systemic metabolites and hormones under controlled diurnal conditions. The temporal patterns of genes relating to carbohydrate and lipid metabolism alongside circulating insulin are consistent with diurnal rhythms being driven in part by the diurnal influence of cyclic feeding and fasting.

An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

Influence of support materials on the electroactive behavior, structure and gene expression of wild type and GSU1771-deficient mutant of Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens DL1 is a metal-reducing dissimilatory bacterium frequently used to produce electricity in bioelectrochemical systems (BES). The biofilm formed on electrodes is one of the most important factors for efficient electron transfer; this is possible due to the production of type IV pili and c-type cytochromes that allow it to carry out extracellular electron transfer (EET) to final acceptors. In this study, we analyzed the biofilm formed on different support materials (glass, hematite (Fe2O3) on glass, fluorine-doped tin oxide (FTO) semiconductor glass, Fe2O3 on FTO, graphite, and stainless steel) by G. sulfurreducens DL1 (WT) and GSU1771-deficient strain mutant (Δgsu1771). GSU1771 is a transcriptional regulator that controls the expression of several genes involved in electron transfer. Different approaches and experimental tests were carried out with the biofilms grown on the different support materials including structure analysis by confocal laser scanning microscopy (CLSM), characterization of electrochemical activity, and quantification of relative gene expression by RT-qPCR. The gene expression of selected genes involved in EET was analyzed, observing an overexpression of pgcA, omcS, omcM, and omcF from Δgsu1771 biofilms compared to those from WT, also the overexpression of the epsH gene, which is involved in exopolysaccharide synthesis. Although we observed that for the Δgsu1771 mutant strain, the associated redox processes are similar to the WT strain, and more current is produced, we think that this could be associated with a higher relative expression of certain genes involved in EET and in the production of exopolysaccharides despite the chemical environment where the biofilm develops. This study supports that G. sulfurreducens is capable of adapting to the electrochemical environment where it grows.

Systematic evaluation of single-cell RNA-seq analyses performance based on long-read sequencing platforms

IntroductionThe rapid development of next-generation sequencing (NGS)-based single-cell RNA sequencing (scRNA-seq) allows for detecting and quantifying gene expression in a high-throughput manner, providing a powerful tool for comprehensively understanding cellular function in various biological processes. However, the NGS-based scRNA-seq only quantifies gene expression and cannot reveal the exact transcript structures (isoforms) of each gene due to the limited read length. On the other hand, the long read length of third-generation sequencing (TGS) technologies, including Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), enable direct reading of intact cDNA molecules. ObjectivesBoth ONT and PacBio have been used in conjunction with scRNA-seq, but their performance in single-cell analyses has not been systematically evaluated. MethodsTo address this, we generated ONT and PacBio data from the same single-cell cDNA libraries containing different amount of cells. ResultsUsing NGS as a control, we assessed the performance of each platform in cell type identification. Additionally, the reliability in identifying novel isoforms and allele-specific gene/isoform expression by both platforms was verified, providing a systematic evaluation to design the sequencing strategies in single-cell transcriptome studies. ConclusionBeyond gene expression analysis, which the NGS-based scRNA-seq only affords, TGS-based scRNA-seq achieved gene splicing analyses, identifying novel isoforms. Attribute to higher sequencing quality of PacBio, it outperforms ONT in accuracy of novel transcripts identification and allele-specific gene/isoform expression.

Lysophosphatidylcholine Acetyltransferase 2 (LPCAT2) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype.

Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor's cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7-a commonly used experimental murine macrophage model-to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts.

A rapid return to normal: temporal gene expression patterns following cold exposure in the bumble bee Bombus impatiens.

Bumble bees are common in cooler climates and many species likely experience periodic exposure to very cold temperatures, but little is known about the temporal dynamics of cold response mechanisms following chill exposure, especially how persistent effects of cold exposure may facilitate tolerance of future events. To investigate molecular processes involved in the temporal response by bumble bees to acute cold exposure, we compared mRNA transcript abundance in Bombus impatiens workers exposed to 0°C for 75 min (inducing chill coma) and control bees maintained at a constant ambient temperature (28°C). We sequenced the 3' end of mRNA transcripts (TagSeq) to quantify gene expression in thoracic tissue of bees at several time points (0, 10, 30, 120 and 720 min) following cold exposure. Significant differences from control bees were only detectable within 30 min after the treatment, with most occurring at the 10min recovery time point. Genes associated with gluconeogenesis and glycolysis were most notably upregulated, while genes related to lipid and purine metabolism were downregulated. The observed patterns of expression indicate a rapid recovery after chill coma, suggesting an acute differential transcriptional response during recovery from chill coma and return to baseline expression levels within an hour, with no long-term gene expression markers of this cold exposure. Our work highlights the functions and pathways important for acute cold recovery, provides an estimated time frame for recovery from cold exposure in bumble bees, and suggests that cold hardening may be less important for these heterothermic insects.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Searching for nature-based solutions: Molecular ecology of an endangered Himalayan medicinal herb shows roadmap for conservation and sustainable utilization

In recent times, to meet the surging demand of the herbal industry, overharvesting of medicinal herbs from their natural habitats has accelerated. This has posed severe extinction risks to plant species worldwide and therefore requires urgent conservation efforts and long-term sustainable solutions. By employing a molecular ecology approach, here we showcase a sustainable solution model for an endemic Himalayan medicinal herb, Trillium govanianum. The herb, being a rich source of diosgenin, is currently overharvested for its rhizome by extraction of the whole plant from the wild, leading to a drastic decline in its natural populations, and now declared Endangered. In the present study, we investigated whether there is an alternative sustainable source of diosgenin (i.e. leaves) other than rhizome in T. govanianum. We studied the spatial dynamics of diosgenin biosynthesis and accumulation using qRT-PCR-based targeted gene expression and High-Performance Liquid Chromatography-based diosgenin quantification in the leaf and rhizome samples of T. govanianum collected along the elevation gradients in Kashmir Himalaya. Our results revealed a higher diosgenin content in the leaf than in rhizome samples of T. govanianum. Expression analysis showed that, compared to the rhizome, the leaf samples exhibit relatively higher expression of all the seven genes of the diosgenin biosynthesis pathway. We also found a strong positive correlation between the expression of the targeted genes and diosgenin content in the leaf and rhizome samples. Overall, our findings offer a nature-based solution for the conservation and sustainable utilization of this imperiled endemic Himalayan herb, with lessons for elsewhere in the world.

The heterogeneous wound microbiome varies with wound care pain, dressing type, and inflammatory gene expression.

Wound dressing changes are essential procedures for wound management. However, ~50% of patients experience severe pain during these procedures despite the availability of analgesic medications, indicating a need for novel therapeutics that address underlying causes of pain. Along with other clinical factors, wound pathogens and inflammatory immune responses have previously been implicated in wound pain. To test whether these factors could contribute to severe pain during wound dressing changes, we conducted an exploratory, cross-sectional analysis of patient-reported pain, inflammatory immune responses, and wound microbiome composition in 445 wounds at the time of a study dressing change. We profiled the bacterial composition of 406 wounds using 16S ribosomal RNA amplicon sequencing and quantified gene expression of 13 inflammatory markers in wound fluid using quantitative real-time polymerase chain reaction (qPCR). Neither inflammatory gene expression nor clinically observed inflammation were associated with severe pain, but Corynebacterium and Streptococcus were of lower relative abundance in wounds of patients reporting severe pain than those reporting little or no pain. Wound microbiome composition differed by wound location, and correlated with six of the inflammatory markers, including complement receptor C5AR1, pro-inflammatory cytokine interleukin (IL)1β, chemokine IL-8, matrix metalloproteinase MMP2, and the antimicrobial peptide encoding cathelicidin antimicrobial peptide. Interestingly, we found a relationship between the wound microbiome and vacuum-assisted wound closure (VAC). These findings identify preliminary, associative relationships between wound microbiota and host factors which motivate future investigation into the directional relationships between wound care pain, wound closure technologies, and the wound microbiome.

OsCIPK2 mediated rice root microorganisms and metabolites to improve plant nitrogen uptake

Crop roots are colonized by large numbers of microorganisms, collectively known as the root-microbiome, which modulate plant growth, development and contribute to elemental nutrient uptake. In conditions of nitrogen limitation, the over-expressed Calcineurin B-like interacting protein kinase 2 (OsCIPK2) gene with root-specific promoter (RC) has been shown to enhance growth and nitrogen uptake in rice. Analysis of root-associated bacteria through high-throughput sequencing revealed that OsCIPK2 has a significant impact on the diversity of the root microbial community under low nitrogen stress. The quantification of nifH gene expression demonstrated a significant enhancement in nitrogen-fixing capabilities in the roots of RC transgenetic rice. Synthetic microbial communities (SynCom) consisting of six nitrogen-fixing bacterial strains were observed to be enriched in the roots of RC, leading to a substantial improvement in rice growth and nitrogen uptake in nitrogen-deficient soils. Forty and twenty-three metabolites exhibiting differential abundance were identified in the roots and rhizosphere soils of RC transgenic rice compared to wild-type (WT) rice. These findings suggest that OSCIPK2 plays a role in restructuring the microbial community in the roots through the regulation of metabolite synthesis and secretion. Further experiments involving the exogenous addition of citric acid revealed that an optimal concentration of this compound facilitated the growth of nitrogen-fixing bacteria and substantially augmented their population in the soil, highlighting the importance of citric acid in promoting nitrogen fixation under conditions of low nitrogen availability. These findings suggest that OsCIPK2 plays a role in enhancing nitrogen uptake by rice plants from the soil by influencing the assembly of root microbial communities, thereby offering valuable insights for enhancing nitrogen utilization in rice cultivation.

Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

FEVER: an interactive web-based resource for evolutionary transcriptomics across fishes.

Teleost fish represent one of the largest and most diverse clades of vertebrates, which makes them great models in various research areas such as ecology and evolution. Recent sequencing endeavors provided high-quality genomes for species covering the main fish evolutionary lineages, opening up large-scale comparative genomics studies. However, transcriptomic data across fish species and organs are heterogenous and have not been integrated with newly sequenced genomes making gene expression quantification and comparative analyses particularly challenging. Thus, resources integrating genomic and transcriptomic data across fish species and organs are still lacking. Here, we present FEVER, a web-based resource allowing evolutionary transcriptomics across species and tissues. First, based on query genes FEVER reconstructs gene trees providing orthologous and paralogous relationships as well as their evolutionary dynamics across 13 species covering the major fish lineages, and 4 model species as evolutionary outgroups. Second, it provides unbiased gene expression across 11 tissues using up-to-date fish genomes. Finally, genomic and transcriptomic data are combined together allowing the exploration of gene expression evolution following speciation and duplication events. FEVER is freely accessible at https://fever.sk8.inrae.fr/.

MiR-374a/b-5p Suppresses Cell Growth in Papillary Thyroid Carcinoma Through Blocking Exosomal ANXA1-Induced Macrophage M2 Polarization.

Papillary thyroid carcinoma (PTC), comprising 85% of all thyroid cancers, is an epithelial malignancy. The potential for malignant transformation in normal cells by thyroid cancer cells via exosomal Annexin A1 (ANXA1) delivery is investigated in this study. Our aim is to determine the impact of PTC cells on macrophage polarization through exosomal ANXA1 secretion and its implications for tumor progression.Exosomes in PTC cells were examined using transmission electron microscopy, exosome labeling, and nanoparticle tracking analysis. Real-time quantitative polymerase chain reaction was employed to quantify gene expression levels. Protein levels were determined through Western blot analysis. The interplay between genes was assessed using luciferase reporter and RNA pull-down assays. Functional experiments were conducted to investigate PTC cell proliferation and apoptosis.Our findings reveal that ANXA1 promotes PTC cell proliferation and inhibits apoptosis. Exosomes derived from PTC cells were found to promote macrophage M2 polarization. ANXA1 stimulates M2 polarization through the activation of the PI3K/AKT pathway. MicroRNA-374a-5p (miR-374a-5p) and microRNA-374b-5p (miR-374b-5p) were identified as inhibitors of ANXA1 expression and PI3K/AKT pathway activity, thereby inhibiting macrophage M2 polarization. Furthermore, miR-374a-5p and miR-374b-5p were observed to suppress PTC cell proliferation through their regulatory action on ANXA1.Our study suggests that miR-374a/b-5p inhibits PTC cell growth by blocking the macrophage M2 polarization induced by exosomal ANXA1.

The Expression Levels of TREX1 and IFN-α Are Associated with Immune Reconstitution in HIV-1-Infected Individuals.

TREX1 acts in the initial prevention of an autoimmune response, but it may contribute to the permissiveness of retrovirus infections. This study investigated the association between the levels of TREX1 gene expression with the polymorphisms TREX1 rs3135941 (T/C) and TREX1 rs3135945 (G/A), and the presence of antinuclear antibodies (ANA) in antiretroviral therapy (ART)-naïve individuals and after 1 year of treatment. Blood samples from 119 individuals with HIV-1 were subjected to genotyping of polymorphisms and quantification of TREX1 gene expression and HIV-1 viral load by qPCR. The concentration of IFN-α and the number of CD4+/CD8+ T lymphocytes were determined by ELISA and flow cytometry, respectively; ANA was investigated by immunofluorescence. A control group of 167 seronegative individuals was used for the comparison of genotypic frequencies. The frequency of the polymorphisms were not associated with HIV infection or with variations in the expression of TREX1 and IFN-α (p > 0.05). ART-naïve individuals exhibited higher TREX1 expression and lower IFN-α expression. After 1 year of ART, TREX1 levels were reduced, while IFN-α and CD4+ T lymphocytes were elevated (p < 0.05). Some individuals on ART presented ANA. These results suggest that ART-mediated restoration of immune competence is associated with a reduction in TREX1 expression, which may induce the development of ANA, regardless of the polymorphism investigated.

A comparative assessment of reference genes in mouse brown adipocyte differentiation and thermogenesis in vitro

ABSTRACT Adipogenic differentiation and thermogenesis in brown adipose tissue (BAT) undergo dynamic processes, altering phenotypes and gene expressions. Proper reference genes in gene expression analysis are crucial to mitigate experimental variances and ensure PCR efficacy. Unreliable reference genes can lead to erroneous gene expression quantification, resulting in data misinterpretation. This study focused on identifying suitable reference genes for mouse brown adipocyte research, utilizing brown adipocytes from the Ucp1-luciferase ThermoMouse model. Comparative analysis of gene expression data under adipogenesis and thermogenesis conditions was conducted, validating 13 housekeeping genes through various algorithms, including DeltaCq, BestKeeper, geNorm, Normfinder, and RefFinder. Tbp and Rer1 emerged as optimal references for Ucp1 and Pparg expression in brown adipogenesis, while Tbp and Ubc were ideal for the expression analysis of these target genes in thermogenesis. Conversely, certain conventional references, including Actb, Tubb5, and Gapdh, proved unstable as reference genes under both conditions. These findings stress the critical consideration of reference gene selection in gene expression analysis within specific biological systems to ensure accurate conclusions.

Abstract 6246: De novo mutation discovery in a mouse model and a human patient sample of non-Hodgkin's lymphoma

Abstract Cancer encompasses many different diseases, all typically involving an accumulation of genetic alterations. However, despite the genomic complexity of this group of diseases, cancer genomic studies typically rely on sequence alignment to a "reference genome" prior to analysis. Such approaches can provide gene expression quantification of known, annotated genes. However, they miss the detection of fusion genes, novel transcripts, virus insertions, and other genetic insults that may be present in cancer beyond an altered gene expression profile. Here, we develop a workflow for de novo identification and quantification of "outside of reference genome" genetic aberrations. This workflow leverages klue, a method we developed that is based on the compact de Bruijn graph, which can extract sequences uniquely found in a "tumor" sample but not in a "normal" control sample from genomic sequencing data. We first applied this method to an RNA-seq experiment from the EμSRα-tTA/TetO-MYC mouse model, which is a MYC-driven autochthonous transgenic mouse model of T-cell acute lymphoblastic lymphoma (T-ALL). We extracted k-mer contigs present in the mouse tumor samples but are present neither in the spleens of wild-type mice of the same genetic background nor in the standard mouse reference genome assembly. We then mapped RNA-seq reads from the T-ALL mouse tumor samples to these contigs. The contigs with the highest number of mapped reads (i.e. the highest counts) were run through BLAST to determine their identities. The mapped contigs with the highest counts were the tTA transgenic element sequence and the human MYC transgene. This result showcases this workflow's success in isolating and quantifying "reference-absent” tumor-unique sequences. Next, we applied klue to whole genome sequencing (WGS), whole exome sequencing (WES), and RNA-seq data from a patient with B-cell follicular lymphoma (with matched normal specimen) from the Texas Cancer Research Biobank, as this data is unrestricted open access. Tumor-unique contigs in sequencing reads were extracted and analyzed with BLAST and BLAT. In one case, a nonsynonymous point mutation in the SPI1 (also known as PU.1) oncogene was found. The sequencing reads from the tumor sample showed both the "normal variant" of SPI1 as well as the mutant SPI1:H268P, suggesting a heterozygous mutation. Altogether, these results suggest that the idea of first identifying tumor-unique sequences then doing post hoc analysis of those sequences is a viable approach for de novo mutation discovery in cancer genomics. Citation Format: Delaney K. Sullivan, Eissa Albinali, Mayuko Boffelli, Lior Pachter. De novo mutation discovery in a mouse model and a human patient sample of non-Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6246.

Abstract 2415: Resistance to immune checkpoint inhibitor (ICI) therapy in metastatic melanoma is revealed by deep circulating tumor DNA tracking in concert with tumor immune profiling

Abstract Monitoring clinically actionable and emerging mutations in circulating tumor DNA (ctDNA) may be crucial for guiding treatment, but adoption has been slow due to the narrow scope and reproducibility of current approaches. Here, we investigate melanoma patients receiving ICI over several years, beginning with extensive tumor mutational and immune profiling followed by deep, whole-exome ctDNA monitoring.Here, 66 plasma samples from 23 advanced stage melanoma patients that were collected throughout ICI treatment (up to 40 months) were profiled using the NeXT Liquid Biopsy® platform (NeXT LB). NeXT LB monitors exome-wide ctDNA by leveraging a patient’s tumor, PBMCs and plasma samples, detecting both tumor-derived and emerging mutations. In addition to ctDNA tracking, we also used the ImmunoID NeXT Platform® to immunologically profile tumor tissue; including gene expression quantification, HLA profiling (typing, mutation, and loss of heterozygosity), T-cell receptor and tumor microenvironment profiling, and neoantigen prediction. ctDNA dynamics and tumor immunity were correlated with clinical outcome. Patients who progressed had lower tumor mutational burden (TMB, Mann-Whitney (MW); p=0.03), and lower neoantigen burden (MW; p=0.04). Responding tumors were enriched for interferon gamma and inflammatory signaling (Wald; adjusted p&amp;lt;0.01) and myeloid dendritic cells (MW; p=0.01), as measured by baseline RNA expression profiling. With an average coverage of 2260X SD +/-157, plasma samples yielded from 4 to 4081 variants. The number of on-treatment tumor variants detected in plasma predicted increased OS hazard (HR=2.95, Wald p=0.03; Kaplan-Meier (KM) p=0.0003) and progression hazard (HR=1.74, Wald p=0.04; KM p=0.0003). Overall, 10% (4277) of all variants were novel mutations observed only in the plasma. Novel variants were less likely to emerge in genes conferring sensitivity to ICI therapy (mixed-model; p&amp;lt;0.001). Dynamic shifts in allelic fraction between baseline and the first available on-treatment timepoint predicted increased OS hazard (HR=3.02, Wald p=0.05) and progression hazard (HR=2.90, Wald p=0.02). In ctDNA clonal phylogenies, 100% of BRAF and NRAS mutations belonged to the dominant clone. By combining immune and ctDNA features, a multivariable model predicted resistance to ICI treatment (log-likelihood p=0.002; c-index=0.89). Using a single liquid biopsy platform, we profiled tumor immunity and tracked existing and emerging ctDNA variants over the course of ICI treatment. We demonstrate that, even though counts of on-treatment ctDNA variants were sufficient for significant survival models, deep profiling of both tumor immunity and ctDNA dynamics provided deeper understanding of ICI resistance. Subsequent studies will determine if ctDNA-guided intervention can improve clinical outcomes. Citation Format: Charles W. Abbott, Laura Keller, Isabel Heidrich, Julian Kött, Glenn Geidel, Daniel J. Smit, Ronald Simon, Stefan Schneider, Jason Pugh, Sean M. Boyle, Richard O. Chen, Klaus Pantel, Christoffer Gebhardt. Resistance to immune checkpoint inhibitor (ICI) therapy in metastatic melanoma is revealed by deep circulating tumor DNA tracking in concert with tumor immune profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2415.

Abstract 7408: Robust profiling of cancer-related gene fusions: Analytical validation of PANCARNA for multiple cancer types

Abstract The impact of examining gene fusion presence in cancer patients is often overlooked, and as such, no well-established workflow currently exists for robust profiling of tumor samples in this area. We established a targeted RNA sequencing panel (PANCARNA) for investigating gene fusions across multiple cancer types and performed analytical validation to confirm performance. Furthermore, we report on the applicability of using targeted RNA sequencing for the purposes of gene expression profiling. This panel covers 117 genes associated with solid tumors. Thirty-nine FFPE samples with gene fusion status validated using an orthogonal method were used to evaluate analytical performance. Of the 39 standard samples, 33 samples were positive for gene translocation events and 6 were negative controls. Samples were collected from across 10 cancer types. To examine the robustness of the panel, 12 samples with known fusions were selected to undergo testing. Each sample was tested in triplicate across two experimental batches. Seraseq Fusion RNA Mix V4 (SeraCare, Cat. 0710-0497) standard samples were used to test the limit of detection for PANCARNA. Additionally, we explored a cohort of 22 samples with over expressed genes and 4 samples without overexpressed genes to test the expression profiling capabilities of PANCARNA and compared the results to immunohistochemistry stains. Of the 39 samples tested using the orthogonal method and targeted RNA-Seq, 38 were found to have concordant fusion calls in both methods (Positive percent agreement = 97.0%, 32/33; Negative percent agreement = 100%, 6/6; Overall percent agreement = 97.4%, 38/39). In reproducibility studies, the assay produced consistent intrarun and interrun results in all samples (Repeatability = 100%, 12/12; Reproducibility = 100%, 12/12). The limit of detection for multiple fusions of interest, such as EML4-ALK, CD74-ROS1, TPM3-NTRK1, and SLC34A2-BRAF, is found to be 0.5-1 copies per ng of RNA. ETV6-NTRK1 can be reliably detected at 2-4 copies per ng of RNA. MET ex14 skipping events can also be detected in a few as 1-2 copies per ng of RNA. To validate the capability of PANCARNA to quantify gene expression, we tested 26 samples with known gene expression levels for three genes of interest (ERBB2, EGFR, MET). ERBB2, EGFR and MET amplification detection through PANCARNA was found to have a high concordance to IHC results (Sensitivity = 96.2%, 100%, 94.7%, respectively). In conclusion, PANCARNA demonstrated highly accurate and sensitive detection of clinically relevant gene fusions. This assay produces reliable results when detecting fusions and offers high concordance to results obtained through IHC/FISH, indicating that targeted RNA seq analysis can assist with profiling gene fusion events in a clinical setting. Citation Format: Haimeng Tang, Hua Bao, Rui Liu, Shuyu Wu, Sisi Liu, Xue Wu, Yang Shao. Robust profiling of cancer-related gene fusions: Analytical validation of PANCARNA for multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7408.

Abstract 6251: Validation and implementation of whole-transcriptome sequencing assay into cancer care: An experience from a cancer center

Abstract Introduction Transcriptome sequencing is a well-established diagnostic tool to characterize and quantify gene expression profiles and to detect fusion transcripts. The utilization of whole-transcriptome sequencing (WTS), in clinical workflows can tremendously help improve diagnosis, risk stratification, and therapy selection. However, there are multiple challenges to widespread clinical implementation of WTS including nucleic acid input, defining appropriate quality control (QC) criteria, tumor specific baseline controls and depth of sequencing. Here, we present a validation study to deploy routine whole transcriptome sequencing with automated analysis in a mid-size academic center. Methods Nucleic acid material was obtained through clinical residual samples in the Center for Genomics and Advanced Technology. RNA samples were extracted using the Purigen Ionic® FFPE to Pure RNA Kit. RNA input sample quantities ranged from 34-100ng. WTS libraries were prepared using the Illumina® RNA Prep with Enrichment (L) Tagmentation kit with the Illumina Exome Panel for FFPE tumor samples. The validation was performed on a set of patient samples with known clinically reported variants from the Illumina TruSight® Tumor 170 assay. A total of 84 true positives with reportable fusions, 5 splicing events, 16 negative samples, and 15 limit of detection (LOD) samples were used. Libraries were prepared in a three-plex format. Sequencing was performed on the Novaseq-6000 system using S2 and S4 flow cells with a target read count of 100 million. All data was processed through the AUGMET informatics platform for demultiplexing, quality control, fusion detection, transcript quantification and determination of alternatively spliced transcript. Results The WTS validation assay demonstrated 98% (n=49/50) sensitivity, with only one false negative, and 100% (n=68/68) specificity. A final review of our data was determined to develop stable QC standards for RNA sequenced libraries to be &amp;gt; 60M reads and &amp;gt; 130bp read length. When analyzing (LOD) for WTS, interestingly, read length was inversely proportional to input and as a result detection of the clinically relevant fusion. Based on our LOD experiments, a minimum input threshold of 20ng of RNA was determined to maintain the sensitivity and specificity. An LOD study for tumor purity was not performed due to the known lack of correlation between tumor purity and fusion transcript quantity. Discussion WTS can detect all clinically reported fusions and splicing events with comparable sensitivity to panel assays. Moreover, simultaneously available transcript quantification and variant calling provide the ability to implement several disease classifiers. This work lays the foundation for integration with our clinically available tumor exome sequencing assay to allow joint exome transcriptome analysis in a single informatics platform. Citation Format: Jillian A. Moran, Edward G. Hughes, Donald C. Green, Shrey Sukhadia, Gregory J. Tsongalis, Samantha F. Allen, Laura Tafe, Brianna Houde, Nevena B. Ognjenovic, Jennifer N. Barbuto, Ivy Riano, Parth Shah. Validation and implementation of whole-transcriptome sequencing assay into cancer care: An experience from a cancer center [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6251.