Whole-transcriptome RNA Sequencing Research Articles

Identification of novel therapeutic targets for contrast induced acute kidney injury (CI-AKI): alpha blockers as a therapeutic strategy for CI-AKI

Iodinated contrast is used for imaging and invasive procedures and it can cause contrast induced acute kidney injury (CI-AKI), which is the third leading hospital-acquired health problem. The purpose of the present study was to determine the effect of α-adrenergic receptor-1b (Adra1b) inhibition by using terazosin on change in kidney function, gene, and protein expression in C57BL/6J male mice, 6-8 weeks with chronic kidney disease (CKD). CKD was induced by surgical nephrectomy. Twenty eight days later, 100-µL of iodinated contrast (CI group) or saline (S group) was given via the carotid artery. Whole-transcriptome RNA-sequencing (RNA-Seq) analysis of the kidneys was performed at day 2. Mice received either 50-µL of saline ip or terazosin (2 mg/kg) in 50-µL of saline ip 1 hour before contrast administration which was continued every 12 hours until the animals were euthanized 2 and 7 days later. The kidneys were removed for gene expression, immunohistochemical analysis, and blood serum analyzed for kidney function. Differential gene expression analysis identified 21 upregulated and 436 downregulated genes (fold change >2; P < 0.05) that were common to all sample (n = 3 for both contrast and saline). We identified Adra1b using bioinformatic analysis. Mice treated with terazosin had a significant decrease in serum creatinine, urinary Kim-1 levels, HIF-1α, apoptosis, and downstream Adrab1 genes including Ece1, Edn1, pMAPK14 with increased cell proliferation. Contrast exposure upregulated Adra1b gene expression in HK-2 cells. Inhibition of Adra1b with terazosin abrogated Ece1, Edn1, and contrast-induced Fsp-1, Mmp-2, Mmp-9 expression, and caspase-3/7 activity in HK-2 cells.

Abstract SS1-04: Comprehensive genomic and transcriptomic profiling of molecular subtypes reveal ancestral differences in the activity of signaling pathways between patients with African and European ancestry

Abstract Background: Breast cancer demonstrates heterogeneity in biological features, and the therapeutic strategy depends on tumor subtype. African-Ancestry (AA) patients experience a disproportionally high rate of triple negative breast cancer (TNBC) and worse outcomes than European-Ancestry (EA) patients. However, the biological drivers causing this disparity between ancestral populations are not deeply understood. To address the issue, we performed genomic and transcriptomic sequencing of breast tumors for comparison between AA and EA patients according to breast cancer molecular subtypes. Materials and Methods: The study included 221 AA and 341 EA patients. Collected samples underwent the Tempus xT next-generation sequencing panel. Following DNA-panel and whole-transcriptome RNA-sequencing, we compared gene mutation rates, homologous recombination deficiency (HRD) scores, degree of immune infiltration and tumor mutational burden (TMB) between ethnicities and molecular subtypes. Additionally, differences between the activity of relevant signaling pathways were evaluated from RNA-sequencing data. Results: Relative to EA TNBC, AA TNBC tumors exhibited higher mutation rates in TP53 (94% vs 86%), KMT2C (17% vs 9%), APOB (19% vs 10%), BRCA2 (11% vs 5%), EP300 (8% vs 2%), NOTCH1 (12% vs 4%), and EGFR (11% vs 4%). Conversely, AA TNBC tumors had relatively lower rates of PIK3CA (10% vs 18%), RB1 (8% vs 15%), and NF1 (5% vs 11%) mutations. Among patients with HR+/HER2- breast cancer, AA tumors had higher mutation rates in CCND1 (23% vs 10%) and FGF3 (16% vs 10%) than EA tumors, but lower rates in TP53 (32% vs 39%). HRD scores were higher in TNBC and HR-/HER2+ tumors compared with the other subtypes (P&amp;lt;0.001). However, there was no significant difference between the HRD scores of AA and EA tumors within TNBC or HR-/HER2+ populations. The highest percentage of immune infiltration was observed in HR-/HER2+ tumors (P=0.036), with no difference between AA or EA groups. TMB did not differ across ancestries or subtypes. Although immune pathways were generally more active in TNBC compared to the other subtypes, there was no difference in pathway-specific immune activation between ethnicities. The G2M and E2F pathways were significantly more active in TNBC (P&amp;lt;2e-16 for both), in particular more active in AA than EA tumors (G2M, P=0.035; E2F, P=0.037). On the other hand, PI3K, ROS, and xenobiotic metabolism (XM) pathways were significantly less active in TNBC compared to the other subtypes (PI3K, P=2.4e-5; ROS, P=0.014; and XM, P&amp;lt;2e-16). Furthermore, these pathways were significantly less active in AA than EA tumors across all subtypes (PI3K, P=0.026; ROS, P=0.00035; and XM, P=0.00041) and within TNBC (PI3K P=0.012, ROS P=0.014, and XM P=0.00018). Conclusion: These data demonstrate significant differences in breast tumor heterogeneity and mutation spectrum in TNBC and HR+/HER2- breast cancers between AA and EA patients. Ancestral differences were also observed in the activity of relevant signaling pathways for TNBC. Overall, the results identify previously unexplored pathways and molecular phenotypes of aggressive disease, providing opportunities for development of more effective biomarker informed treatment of breast cancer in diverse populations. Citation Format: Minoru Miyashita, Joshua SK Bell, Yonglan Zheng, Toshio Yoshimatsu, Padma Sheila Rajagopal, Anna Woodard, Jean Baptiste Reynier, Elisabeth Sveen, Galina Khramtsova, Fang Liu, Abiola Ibraheem, Gini Fleming, Nora Jaskowiak, Rita Nanda, Benjamin Leibowitz, Nike Beaubier, Kevin White, Dezheng Huo, Olufunmilayo I Olopade. Comprehensive genomic and transcriptomic profiling of molecular subtypes reveal ancestral differences in the activity of signaling pathways between patients with African and European ancestry [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SS1-04.

Abstract PD7-06: Unstable mutational profile and heterogeneity of residual breast tumor following neoadjuvant therapy from comprehensive genomic and transcriptomic sequencing

Abstract Background: In patients with early breast cancer, neoadjuvant therapy is widely performed as standard of care. While the molecular targeting strategy makes progress in HER2 positive breast cancer, the optimal regimens for ER+/HER2- breast cancer (BC) and triple negative breast cancer (TNBC) remain undecided. Furthermore, there are few strategies for patients who do not achieve complete pathological response (pCR) who have worse prognosis. To address these unmet clinical needs, there is urgent need to examine the genomic alterations and immune microenvironment of residual tumors after neoadjuvant therapy. Here, we conducted a comprehensive analysis integrating genomic, transcriptomic, and clinical data to investigate the difference between primary breast cancer and residual disease. Materials and Methods: Using the large prospectively ascertained ethnically diverse Chicago Multi-Ethnic (ChiMEC) cohort of 562 participants with integrated genomic data, we identified 176 patients with breast cancer who underwent sequencing with Tempus xT next-generation sequencing panel including DNA- and whole-transcriptome RNA-sequencing. These included 131 primary breast tumors and 45 residual tumors after neoadjuvant therapy. We compared mutation rates between primary tumor and residual tumor in ER+/HER2- BC and TNBC. We also investigated homologous recombination deficiency (HRD) scores, tumor mutational burden (TMB), degree of immune infiltration, and microsatellite instability (MSI), and their association with survival. Results: Out of the 176 patients, there were 72 ER+/HER2- BC, 42 TNBC, and 44 HER2 positive cases. Among patients with HR+/HER2- BC, residual tumors had higher mutation rates in PIK3CA (61% vs 33%), CDH1 (33% vs 17%), CCND1 (39% vs 7%), FGF3 (28% vs 0%), FGF4 (33% vs 2%), FGF19 (33% vs 2%), and GATA3 (44% vs 7%) than primary tumors, but lower rates in TP53 (28% vs 48%), MAP3K1 (6% vs 40%), KMT2D (0% vs 33%), MCL1 (0% vs 30%), SPEN (6% vs 20%), ZFHX3 (0% vs 20%), ARID1A (11% vs 16%), KMT2C (0% vs 19%), LZTR1 (0% vs 19%), BCORL1 (6% vs 17%), NOTCH3 (6% vs 17%), FAT1 (0% vs 17%) and LPR1B (0% vs 17%). Relative to primary TNBC tumors, residual TNBC tumors exhibited higher mutation rates in PTEN (10% vs 3%), CCNE1 (10% vs 3%), CIC (10% vs 0%), and KMT2D (10% vs 3%). Conversely, residual TNBC tumors had relatively lower rates of MCL1 (0% vs 21%), RB1 (0% vs 12%), CDH1 (0% vs 12%), KMT2C (11% vs 18%), and PIK3CA (0% vs 15%), CDKN1B (0% vs 12%) and ETV6 (0% vs 12%). There was a significant trend of higher TMB (&amp;gt;5.0 mutations/megabase, m/MB) associated with improved disease-free survival among the 176 patients (P=0.031). TMB was not significantly different between primary tumors and residual tumors, or across subtypes. Microsatellite instability (MSI) status was high in 3 patients (1.6%) and equivocal in 5 patients (2.7%). TMB in MSI-high or MSI-equivocal tumors was significantly higher than TMB in tumors with microsatellite stability (37.7m/MB vs 5.3m/MB, P&amp;lt;0.001). HRD scores were highest in TNBC, and lowest in ER+/HER2- breast cancers (P=0.022). There was no significant association between the HRD scores and survival. To date, tumors from 106 patients have undergone immune profiling, with estimation of immune cell infiltration, macrophages, B cells, CD4, CD8, and NK cells. Initial analysis showed no association between immune profiling and survival. Conclusion: These comprehensive analyses demonstrated that mutation status in HR+/HER2- breast cancers and TNBC differs between primary and residual tumors after neoadjuvant therapy. We identified genomic alterations and pathways in residual tumors that could be further explored as potential targets in the adjuvant setting to improve long term outcomes for patients who do not achieve pCR. Citation Format: Minoru Miyashita, Masaya Hattori, Yonglan Zheng, Toshio Yoshimatsu, Joshua SK Bell, Padma Sheila Rajagopal, Anna Woodard, Jean Baptiste Reynier, Elisabeth Sveen, Galina Khramtsova, Fang Liu, Abiola Ibraheem, Gini Fleming, Nora Jaskowiak, Rita Nanda, Benjamin Leibowitz, Nike Beaubier, Kevin White, Dezheng Huo, Olufunmilayo I Olopade. Unstable mutational profile and heterogeneity of residual breast tumor following neoadjuvant therapy from comprehensive genomic and transcriptomic sequencing [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-06.

Effect of sex-specific differences on function of induced hepatocyte-like cells generated from male and female mouse embryonic fibroblasts

BackgroundThe liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male’s liver are good in alcohol clearance and lipid metabolism, while female’s liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently.MethodsMouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model.ResultsIn this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex.ConclusionIn conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

The intratumor microbiome predicts prognosis across gender and subtypes in papillary thyroid carcinoma

While the intratumor microbiome has become increasingly implicated in cancer development, the microbial landscape of papillary thyroid carcinoma (PTC) is essentially uninvestigated. PTC is characterized by varied prognosis between gender and cancer subtype, but the cause for gender and subtype-based dissimilarities is unclear. Women are more frequently diagnosed with PTC, while men suffer more advanced-staged PTC. In addition, tall cell variants are more aggressive than classical and follicular variants of PTC. We hypothesized that intratumor microbiome composition distinctly alters the immune landscape and predicts clinical outcome between PTC subtypes and between patient genders. Raw whole-transcriptome RNA-sequencing, Level 3 normalized mRNA expression read counts, and DNA methylation 450 k sequencing data for untreated, nonirradiated tumor, and adjacent normal tissue were downloaded from the Genomic Data Commons (GDC) legacy archive for 563 thyroid carcinoma patients. Microbe counts were extracted using Pathoscope 2.0 software. We correlated microbe abundance to clinical variables and immune-associated gene expression. Gene-set enrichment, mutation, and methylation analyses were conducted to correlate microbe abundance to characterize microbes’ roles. Overall, PTC tumor tissue significantly lacked microbes that are populated in adjacent normal tissue, which suggests presence of microbes may be critical in controlling immune cell expression and regulating immune and cancer pathways to mitigate cancer growth. In contrast, we also found that microbes distinctly abundant in tall cell and male patient cohorts were also correlated with higher mutation expression and methylation of tumor suppressors. Microbe dysbiosis in specific PTC types may explain observable differences in PTC progression and pathogenesis. These microbesprovide a basis for developing specialized prebiotic and probiotic treatments for varied PTC tumors.

Whole-Transcriptome RNA Sequencing Reveals the Global Molecular Responses and CeRNA Regulatory Network of mRNAs, lncRNAs, miRNAs and circRNAs in Response to Salt Stress in Sugar Beet (Beta vulgaris).

Sugar beet is an important sugar-yielding crop with some tolerance to salt, but the mechanistic basis of this tolerance is not known. In the present study, we have used whole-transcriptome RNA-seq and degradome sequencing in response to salt stress to uncover differentially expressed (DE) mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in both leaves and roots. A competitive endogenous RNA (ceRNA) network was constructed with the predicted DE pairs, which revealed regulatory roles under salt stress. A functional analysis suggests that ceRNAs are implicated in copper redistribution, plasma membrane permeability, glycometabolism and energy metabolism, NAC transcription factor and the phosphoinositol signaling system. Overall, we conducted for the first time a full transcriptomic analysis of sugar beet under salt stress that involves a potential ceRNA network, thus providing a basis to study the potential functions of lncRNAs/circRNAs.

Longitudinal transcriptome analyses show robust T cell immunity during recovery from COVID-19

Understanding the processes of immune regulation in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for improving treatment. Here, we performed longitudinal whole-transcriptome RNA sequencing on peripheral blood mononuclear cell (PBMC) samples from 18 patients with coronavirus disease 2019 (COVID-19) during their treatment, convalescence, and rehabilitation. After analyzing the regulatory networks of differentially expressed messenger RNAs (mRNAs), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) between the different clinical stages, we found that humoral immunity and type I interferon response were significantly downregulated, while robust T-cell activation and differentiation at the whole transcriptome level constituted the main events that occurred during recovery from COVID-19. The formation of this T cell immune response might be driven by the activation of activating protein-1 (AP-1) related signaling pathway and was weakly affected by other clinical features. These findings uncovered the dynamic pattern of immune responses and indicated the key role of T cell immunity in the creation of immune protection against this disease.

Expression Profiling of Long Noncoding RNA and Messenger RNA in a Cecal Ligation and Puncture-Induced Colon Injury Mouse Model.

Background Emerging evidence reveals that long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of sepsis. However, the detailed regulatory mechanisms of lncRNAs or whether certain lncRNA could serve as a biomarker in the septic colon remains unclear. The aim of this study was to investigate the profiles of lncRNAs and mRNAs in the septic colon through whole-transcriptome RNA sequencing and to reveal the associated regulatory mechanism. Method and Result We established a mouse model of sepsis by cecal ligation and puncture (CLP). Colon samples were collected upon CLP or sham surgery after 24 h. Whole-transcriptome RNA sequencing was performed to profile the relative expressions of lncRNAs and mRNAs. 808 lncRNAs and 1509 mRNAs were differentially found in the septic group compared with the sham group. Bioinformatics analysis including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG) was performed to predict the potential functions of these RNAs. GO analysis showed that the altered lncRNAs were enriched and involved in multiple immune responses, which may be a response to sepsis stress. KEGG analysis indicated that upregulated lncRNAs were significantly enriched in the p53 signaling pathway, NF-κB signaling pathway, and HIF-1 signaling pathway. Downregulated lncRNAs were mostly found to be involved in tight junction, leukocyte transendothelial migration, and HIF-1 signaling pathway. Conclusion Our results indicate that these altered lncRNAs and mRNAs may have crucial roles in the pathogenesis of sepsis. This study could contribute to extending the understanding of the function of lncRNAs in sepsis, which may help in searching for new diagnostic biomarkers and therapeutic targets to treat sepsis.

Two-step mixed model approach to analyzing differential alternative RNA splicing.

Changes in gene expression can correlate with poor disease outcomes in two ways: through changes in relative transcript levels or through alternative RNA splicing leading to changes in relative abundance of individual transcript isoforms. The objective of this research is to develop new statistical methods in detecting and analyzing both differentially expressed and spliced isoforms, which appropriately account for the dependence between isoforms and multiple testing corrections for the multi-dimensional structure of at both the gene- and isoform- level. We developed a linear mixed effects model-based approach for analyzing the complex alternative RNA splicing regulation patterns detected by whole-transcriptome RNA-sequencing technologies. This approach thoroughly characterizes and differentiates three types of genes related to alternative RNA splicing events with distinct differential expression/splicing patterns. We applied the concept of appropriately controlling for the gene-level overall false discovery rate (OFDR) in this multi-dimensional alternative RNA splicing analysis utilizing a two-step hierarchical hypothesis testing framework. In the initial screening test we identify genes that have differentially expressed or spliced isoforms; in the subsequent confirmatory testing stage we examine only the isoforms for genes that have passed the screening tests. Comparisons with other methods through application to a whole transcriptome RNA-Seq study of adenoid cystic carcinoma and extensive simulation studies have demonstrated the advantages and improved performances of our method. Our proposed method appropriately controls the gene-level OFDR, maintains statistical power, and is flexible to incorporate advanced experimental designs.

Molecular characteristics of varicocele: integration of whole-exome and transcriptome sequencing

To explore the exome and transcriptome characteristics potentially underlying the pathogenesis of varicocele (VE). Experimental study and cohort study. Academic research laboratory and university-affiliated hospital. Eleven VE patients whose fathers also had VE, plus 151 additional patients and 324 healthy men for variants genotyping; for the rat model, eight Sprague-Dawley male rats. Partial ligation of renal vein was conducted to establish VE rat models for whole-transcriptome RNA sequencing (RNA-seq). Genes with differential expression and/or harboring potential pathogenic variants detected via RNA-seq and whole-exome sequencing (WES) then subjected to population-based survey to define candidate genes of VE and analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes to identify VE-involved pathways. Whole-transcriptome RNA sequencing (RNA-seq) was performed using left spermatic veins of five rat VE models and three controls. We identified 9,688 genes and 18 pathways via RNA-seq, and via WES 160 genes harboring 279 potential deleterious variants and 16 pathways. Nine genes (AAMP, KMT2D, IRS2, SPINT1, IFT122, MKI67, DCHS1, LAMA2, and CBL) had variants in more than one patient who underwent WES, and six of these genes (AAMP, SPINT1, MKI67, IFT122, LAMA2, and DCHS1) showed differential expression. The population-based survey showed that AAMP, SPINT1, and MKI67 were strongly associated with VE risk. Together, two omic 67 data sets revealed four pathways potentially related to VE. For the first time, we have described the exome and transcriptome characteristics of VE. The bi-omics identified novel candidate genes and pathways involving the occurrence and development of VE.

Whole transcriptome RNA sequencing analysis on mesenchymal stem cells preconditioned by inflammatory stimuli

Background & Aim Mesenchymal stem cells are a promising cell type in the field of cell therapy, and the number of clinical trials utilizing different tissue sources derived MSCs continues to grow. To date, various cell preconditioning strategies have been used to boost MSC's existing immunomodulatory functions and include exposure to environmental stimuli or small molecules. However, the exact molecular mechanism by which preconditioning enhances MSCs potency, particularly with inflammatory stimuli, is still not well understood. Herein we performed whole-transcriptome RNA sequencing (RNAseq) on cytomix preconditioned MSCs (to mimic the acute inflammatory microenvironment) to specifically examine MSC molecular pathways involved by this preconditioning strategy. Methods, Results & Conclusion Human bone marrow MSCs were used between passages 3-6, and MSCs with or without cytomix preconditioning were collected for whole transcriptome RNAseq (Qiagen). Analysis of the transcriptome profile showed a robust separation of gene expression profiles for cytomix-preconditioned versus non-preconditioned MSCs. Differential expression analysis (DESEq2) revealed 1,020 upregulated genes (FDR 1) and 413 downregulated genes (FDR In summary, our preliminary analysis on the whole transcriptome of inflammatory stimuli-preconditioned MSCs showed upregulation of genes coding for secreted proteins associated with functions in immune responses. Further experiments will be performed to confirm the relationships between genes coding for secreted proteins identified in this study with MSC's immunomodulatory functions.

Identification of stable genes in the corpus luteum of lactating Holstein cows in pregnancy and luteolysis: Implications for selection of reverse-transcription quantitative PCR reference genes

In lactating dairy cattle, the corpus luteum (CL) is a dynamic endocrine tissue vital for pregnancy maintenance, fertility, and cyclicity. Understanding processes underlying luteal physiology is therefore necessary to increase reproductive efficiency in cattle. A common technique for investigating luteal physiology is reverse-transcription quantitative PCR (RT-qPCR), a valuable tool for quantifying gene expression. However, reference-gene-based RT-qPCR quantification methods require utilization of stably expressed genes to accurately assess mRNA expression. Historically, selection of reference genes in cattle has relied on subjective selection of a small pool of reference genes, many of which may have significant expression variation among different tissues or physiologic states. This is particularly concerning in dynamic tissues such as the CL, with its capacity for rapid physiologic changes during luteolysis, and likely in the less characterized period of CL maintenance during pregnancy. Thus, there is a clear need to identify reference genes well suited for the bovine CL over a wide range of physiological states. Whole-transcriptome RNA sequencing stands as an effective method to identify new reference genes by enabling the assessment of the expression profile of the entire pool of mRNA transcripts. We report the identification of 13 novel putative reference genes using RNA sequencing in the bovine CL throughout early pregnancy and luteolysis: RPL4, UQCRFS1, COX4I1, RPS4X, SSR3, CST3, ZNF266, CDC42, CD63, HIF1A, YWHAE, EIF3E, and PPIB. Independent RT-qPCR analyses were conducted confirming expression stability in another set of CL tissues from pregnancy and regression, with analyses performed for 3 groups of samples: (1) all samples, (2) samples from pregnancy alone, and (3) samples throughout the process of CL regression. Seven genes were found to be more stable in all states than 2 traditional reference genes (ACTB and GAPDH): RPS4X, COX4I1, PPIB, SSR3, RPL4, YWHAE, and CDC42. When CL tissues from pregnant animals alone were analyzed, CST3, HIF1A, and CD63 were also identified as more stable than ACTB and GAPDH. Identification of these new reference genes will aid in accurate normalization of RT-qPCR results, contributing to proper interpretation of gene expression relevant to luteal physiology. Furthermore, our analysis sheds light on the effects of luteolysis and pregnancy on the stability of gene expression in the bovine CL.

Abstract PR12: Genomic landscape of immunotherapy resistance in lung cancer

Abstract Immune checkpoint inhibitors (ICI) have emerged as an important modality in lung cancer treatment. However, studies have shown that only a fraction of patients will experience any clinical benefit when treated with ICI, and a better understanding of the mechanisms of resistance is still needed. In this study, we sought to characterize the prevalence of known mechanisms of primary and adaptive resistance in the context of the greater immunogenomic landscape in a real-world cohort of 100 non-small cell lung cancer patients treated with ICI. Targeted panel DNA sequencing and whole-transcriptome RNA sequencing were performed on a primary tumor biopsy for each patient, the clinical notes were abstracted and structured, and the digital image of the H&amp;E was processed through a tumor-infiltrating lymphocyte (TIL) analysis pipeline. We evaluated each tumor for evidence of HLA loss of heterozygosity (LOH), mutations in the antigen presentation or interferon gamma signaling pathways, expression of alternative checkpoint molecules, and immune infiltrate exclusion. We found that over 25% of patients in the cohort showed signs of at least one of these mechanisms of ICI resistance. We evaluated the real-world progression-free survival (rwPFS) for these patients and found that the majority experienced a progression event within 6 months of treatment start. This study suggests that primary resistance to ICI affects a significant population of lung cancer patients. This abstract is also being presented as Poster B22. Citation Format: Denise Lau, Michelle Stein, Jason Perera, Madhavi Kannan, Aly Khan. Genomic landscape of immunotherapy resistance in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR12.

Whole-transcriptome RNA sequencing reveals the global molecular responses and ceRNA regulatory network of mRNAs, lncRNAs, miRNAs and circRNAs in response to copper toxicity in Ziyang Xiangcheng (Citrus junos Sieb. Ex Tanaka)

BackgroundCopper (Cu) toxicity has become a potential threat for citrus production, but little is known about related mechanisms. This study aims to uncover the global landscape of mRNAs, long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) in response to Cu toxicity so as to construct a regulatory network of competing endogenous RNAs (ceRNAs) and to provide valuable knowledge pertinent to Cu response in citrus.ResultsTolerance of four commonly used rootstocks to Cu toxicity was evaluated, and ‘Ziyang Xiangcheng’ (Citrus junos) was found to be the most tolerant genotype. Then the roots and leaves sampled from ‘Ziyang Xiangcheng’ with or without Cu treatment were used for whole-transcriptome sequencing. In total, 5734 and 222 mRNAs, 164 and 5 lncRNAs, 45 and 17 circRNAs, and 147 and 130 miRNAs were identified to be differentially expressed (DE) in Cu-treated roots and leaves, respectively, in comparison with the control. Gene ontology enrichment analysis showed that most of the DEmRNAs and targets of DElncRNAs and DEmiRNAs were annotated to the categories of ‘oxidation-reduction’, ‘phosphorylation’, ‘membrane’, and ‘ion binding’. The ceRNA network was then constructed with the predicted pairs of DEmRNAs-DEmiRNAs and DElncRNAs-DEmiRNAs, which further revealed regulatory roles of these DERNAs in Cu toxicity.ConclusionsA large number of mRNAs, lncRNAs, circRNAs, and miRNAs in ‘Ziyang Xiangcheng’ were altered in response to Cu toxicity, which may play crucial roles in mitigation of Cu toxicity through the ceRNA regulatory network in this Cu-tolerant rootstock.

Molecular Alterations in Adult Histiocytic Neoplasms

Introduction Histiocytic neoplasms are rare disorders of the monocyte-macrophage-dendritic cell lineage that pose significant management challenges to the clinicians. Over the last five years, there has been emerging data on the role of MAPK/ERK pathway (RAS-RAF-MEK-ERK) in their pathogenesis. In this study, we report the molecular alterations among patients with histiocytic neoplasms seen at our institution. Methods We pursued target-capture next generation sequencing (NGS) on tissue specimen of patients with various histiocytic neoplasms- Erdheim-Chester disease (ECD), Langerhans cell histiocytosis (LCH), Rosai-Dorfman disease (RDD), histiocytic sarcoma (HS), and Langerhans cell sarcoma (LCS). We used three NGS platforms, Tempus® xO (1,714 genes), Tempus® xT (596 genes), or Foundation One® CDX (324 genes). All platforms used formalin-fixed paraffin-embedded tumor tissue specimens for detection of substitutions, insertion and deletion alterations, and copy number alterations, and gene rearrangements. The Tempus assays also utilized matched blood or saliva samples for germline DNA sequencing in addition to whole-transcriptome RNA sequencing. The tumor cellularity threshold for pursuing RNA sequencing was 30% so the test was not pursued specimens for lower cellularity. Results We included 31 cases with histiocytic neoplasms in this study. The disease distribution was as follows: ECD (n=6), LCH (n=7), RDD (n=12), ECD-RDD overlap (n=3), HS (n=2), and LCS (n=1). Among the 13 patients with ECD and LCH, BRAF-V600E mutations were found in 8 (62%) cases. We found several novel DNA alterations in this study in all disease groups: ECD (BRAFV471F), LCH (MAP2K1 in-frame deletion), and RDD (frameshift mutations of DNMT3A, SMARCA4, KMT2A, and CDC73 truncation). In one LCH patient without any DNA alterations, we found MAPK1 and MAP2K1 overexpression on RNA sequencing. We also had two cases with HS, both of which were found to have KRAS exon 3 mutations. One of these cases had concomitant JAK2V617F+ myelofibrosis and the tissue specimen for HS demonstrated TET2 and SRSF2 mutations as well. Our cohort included one patient with multifocal lymph node LCS that demonstrated NRAS and TP53 mutations, who did not respond to MEK-inhibitor therapy. Alterations in epigenetic regulators (TET2, DMNT3A, KMT2A, ASXL1) were found in 5 (16%), potentially suggesting clonal hematopoiesis in a subgroup of histiocytosis. Conclusions Histiocytic neoplasms demonstrate diverse somatic oncogenic alterations. In our study, we noted several novel alterations beyond the MAPK-ERK pathway in RDD that may point toward other potentially pathogenic mechanisms for these rare disorders. In cases where the DNA sequencing does not reveal pathogenic alterations, transcriptome analysis may aid in the diagnosis and management. OffLabel Disclosure: Trametinib use for Langerhans cell sarcoma

Genome-wide discovery and characterization of long noncoding RNAs in patients with multiple myeloma

BackgroundLong noncoding RNAs (lncRNAs) are involved in a wide range of biological processes in tumorigenesis. However, the role of lncRNA expression in the biology, prognosis, and molecular classification of human multiple myeloma (MM) remains unclear, especially the biological functions of the vast majority of lncRNAs. Recently, lncRNAs have been identified in neoplastic hematologic disorders. Evidence has accumulated on the molecular mechanisms of action of lncRNAs, providing insight into their functional roles in tumorigenesis. This study aimed to characterize potential lncRNAs in patients with MM.MethodsIn this study, the whole-transcriptome strand-specific RNA sequencing of samples from three newly diagnosed patients with MM was performed. The whole transcriptome, including lncRNAs, microRNAs, and mRNAs, was analyzed. Using these data, MM lncRNAs were systematically analyzed, and the lncRNAs involved in the occurrence of MM were identified.ResultsThe results revealed that MM lncRNAs had distinctive characteristics different from those of other malignant tumors. Further, the functions of a set of lncRNAs preferentially expressed in MM were verified, and several lncRNAs were identified as competing endogenous RNAs. More importantly, the aberrant expression of certain lncRNAs, including maternally expressed gene3, colon cancer–associated transcript1, and coiled-coil domain-containing 26, as well as some novel lncRNAs involved in the occurrence of MM was established. Further, lncRNAs were related to some microRNAs, regulated each other, and participated in MM development.ConclusionsGenome-wide screening and functional analysis enabled the identification of a set of lncRNAs involved in the occurrence of MM. The interaction exists among microRNAs and lncRNAs.

LUNG CANCER DETECTION VIA WHOLE-TRANSCRIPTOME RNA SEQUENCING OF NASAL EPITHELIUM

SESSION TITLE: Tuesday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: We have previously established the clinical utility of a molecular test (Percepta) that utilizes a brushing of normal-appearing bronchial epithelium to improve lung cancer detection in former and current smokers. This “field of injury” principle has been extended to the nose with evidence that genomic changes associated with cancer can be detected in the nasal epithelium (Perez-Rogers et al. 2017). We report the feasibility of a molecular test developed from non-invasive nasal brush samples for lung cancer detection in current and former smokers. METHODS: Brushings of the nasal epithelium were prospectively collected during the AEGIS clinical trials from current and former smokers undergoing a bronchoscopy for suspected lung cancer lesions found on chest CT. Patients were followed for up to one year until a final diagnosis of lung cancer or benign disease was made. Purified RNA was analyzed using whole-transcriptome RNA sequencing. A total of 675 patients were divided into a training set of 411 nasal samples and an independent test set of 264 nasal samples. Classifiers were developed using gene expression as well as clinical factors such as age, gender, smoking status and nodule characteristics. RESULTS: The independent test set was analyzed using the developed classifiers and results compared to the final clinical diagnosis (malignant or benign). A nasal genomic-clinical classifier demonstrated sensitivity of 97% (95% CI: 93-99) and a specificity of 46% (95% CI: 32-59). While the prevalence of lung cancer in the overall test set is 78%, reflecting the high-risk clinical setting of this bronchoscopy-based cohort, it also includes a subset of patients with low/intermediate pre-test risk of cancer (n=57). In this subset, sensitivity is 95% (95% CI: 74-100) and specificity is 45% (95% CI: 29-62), similar to that of the overall test set. CONCLUSIONS: A nasal genomic-clinical classifier developed using whole transcriptome RNA sequencing shows early feasibility in detecting lung-cancer related gene expression changes in nasal brushings from high-risk smokers. Further research is warranted to understand its impact on patient care in a real-world clinical setting. CLINICAL IMPLICATIONS: A nasal genomic-clinical classifier has the potential to serve as a non-invasive tool for lung cancer risk-stratification among patients with pulmonary nodules. DISCLOSURES: Employee relationship with Veracyte Please note: >$100000 Added 06/24/2019 by Joshua Babiarz, source=Web Response, value=Salary Employee relationship with Veracyte Please note: >$100000 Added 06/24/2019 by Joshua Babiarz, source=Web Response, value=Ownership interest Removed 06/24/2019 by Joshua Babiarz, source=Web Response Employee relationship with Veracyte, Inc Please note: >$100000 Added 06/22/2019 by Sangeeta Bhorade, source=Web Response, value=Salary Employee relationship with Veracyte, Inc. Please note: >$100000 Added 03/21/2019 by Janna Chamberlin, source=Web Response, value=Salary Employee relationship with Veracyte, Inc. Please note: $20001 - $100000 Added 03/21/2019 by Janna Chamberlin, source=Web Response, value=Stock holder Employee relationship with Veracyte, Inc. Please note: >$100000 Added 06/25/2019 by Jie Ding, source=Web Response, value=Salary Employee relationship with Veracyte Inc Please note: >$100000 Added 03/15/2019 by Jing Huang, source=Web Response, value=Salary no disclosure on file for Giulia Kennedy; Consultant relationship with Boston Scientific Please note: $1001 - $5000 Added 11/30/2018 by Carla Lamb, source=Web Response, value=Honoraria Advisory Committee Member relationship with Veracyte Please note: $1001 - $5000 Added 03/25/2019 by Carla Lamb, source=Web Response, value=Consulting fee Consultant relationship with Veracyte Please note: $5001 - $20000 Added 06/21/2019 by Marc Lenburg, source=Web Response, value=Consulting fee Consultant relationship with Johnson and Johnson Please note: $5001 - $20000 Added 06/21/2019 by Marc Lenburg, source=Web Response, value=Consulting fee Employee relationship with Veracyte Please note: >$100000 Added 03/15/2019 by Lori Lofaro, source=Web Response, value=Salary Employee relationship with Veracyte Please note: >$100000 Added 03/18/2019 by Daniel Pankratz, source=Web Response, value=Salary no disclosure on file for Saeed Saberi; Employee relationship with Johnson and Johnson Please note: >$100000 Added 06/25/2019 by Avrum Spira, source=Web Response, value=Salary Employee relationship with Johnson and Johnson Please note: >$100000 Added 07/01/2019 by Christopher Stevenson, source=Web Response, value=Salary Employee relationship with Johnson and Johnson Please note: >$100000 Added 07/01/2019 by Christopher Stevenson, source=Web Response, value=Salary Employee relationship with Veracyte Please note: >$100000 Added 03/18/2019 by Patric Walsh, source=Web Response, value=Salary Employee relationship with Johnson & Johnson Please note: >$100000 Added 06/23/2019 by Duncan Whitney, source=Web Response, value=Salary Employee relationship with Veracyte Please note: >$100000 Added 06/25/2019 by Jonathan Wilde, source=Web Response, value=Salary

IMPROVING INDETERMINANT PULMONARY NODULE MANAGEMENT WITH THE PERCEPTA GENOMIC SEQUENCING CLASSIFIER

SESSION TITLE: Late Breaking Abstracts SESSION TYPE: Original Investigations PRESENTED ON: 10/23/2019 10:45 AM - 11:45 AM PURPOSE: Guidelines recommend patients with intermediate-risk nodules undergo bronchoscopic workup, yet the diagnostic yield is only around 50%, underscoring the need for additional tools to inform patient management. We previously established the clinical accuracy of Percepta Bronchial Genomic Classifier, a molecular test that utilizes a brushing of bronchial epithelium to improve nodule management in smokers. We have since developed the second-generation Percepta Genomic Sequencing Classifier (GSC), which utilizes 1232 gene transcripts from whole-transcriptome RNA sequencing, as well as clinical factors, to achieve improved performance. Here we report the clinical validation and demonstrate improvement for nodule diagnosis. METHODS: We utilized over 1600 patient samples to train the new Percepta GSC, which is an ensemble of four machine-learning models that incorporate genomic and clinical features as well as their interactions. We established diagnostic accuracy on an independent validation set consisting of 412 patient samples from three cohorts: AEGIS I and II (246 patients) and the Percepta Registry (166 patients)—all with nondiagnostic lung nodules from bronchoscopy. We measured positive predictive value (PPV) and negative predictive value (NPV) of Percepta GSC results in physician-reported pre-test risk categories of low, intermediate or high risk. In AEGIS I/II cohorts, where patients with diagnostic bronchoscopy results are available, we calculated the sensitivities of bronchoscopy and Percepta GSC alone, as well as in combination. RESULTS: In the validation set, 80 patients (19%) had a low pre-test risk (cancer prevalence 5.0%), 188 (35%) had an intermediate pre-test risk (cancer prevalence 28.2%), and 144 (46%) had a high pre-test risk (cancer prevalence 73.6%). Percepta GSC down-classified patients with low pre-test risk with >99% NPV and intermediate pre-test risk with a 91.0% NPV. In addition, the classifier up-classified patients with intermediate pre-test risk with a 65.4% PPV and high pre-test risk with a 91.5% PPV. In total, 41.6% of intermediate pre-test risk patients were either down- or up-classified by the test. In the AEGIS cohorts, the sensitivity among low and intermediate pre-test risk patients was: 40.9% (bronchoscopy alone), 92.3% (Percepta GSC alone), and 95.5% (combined). CONCLUSIONS: Percepta GSC is clinically validated to accurately up-classify and down-classify the probability of malignancy for a substantial portion of lung nodule patients with nondiagnostic bronchoscopy. Percepta GSC significantly improves the sensitivity of bronchoscopy overall and demonstrates a combined 95%+ sensitivity for low/intermediate pre-test risk groups. CLINICAL IMPLICATIONS: Percepta GSC compliments bronchoscopy and when used together can enable improved management of pulmonary nodules. DISCLOSURES: No relevant relationships by Christina Bellinger, source=Web Response No relevant relationships by Michael Bernstein, source=Web Response Employee relationship with Veracyte, Inc Please note: >$100000 Added 06/22/2019 by Sangeeta Bhorade, source=Web Response, value=Salary Employee relationship with Veracyte Inc Please note: >$100000 Added 03/21/2019 by Yoonha Choi, source=Web Response, value=Salary Removed 03/21/2019 by Yoonha Choi, source=Web Response Employee relationship with Veracyte Inc Please note: >$100000 Added 03/21/2019 by Yoonha Choi, source=Web Response, value=Salary No relevant relationships by Travis Dotson, source=Web Response Consultant relationship with AstraZeneca Please note: $5001 - $20000 Added 11/29/2018 by David Feller-Kopman, source=Web Response, value=Consulting fee Consultant relationship with Veracyte Please note: $5001 - $20000 Added 11/29/2018 by David Feller-Kopman, source=Web Response, value=Consulting fee Consultant relationship with Veran Medical Please note: $5001 - $20000 Added 11/29/2018 by David Feller-Kopman, source=Web Response, value=Consulting fee Employee relationship with Veracyte Inc Please note: >$100000 Added 03/15/2019 by Jing Huang, source=Web Response, value=Salary no disclosure on file for Giulia Kennedy; Consultant relationship with Veracyte Please note: $5001 - $20000 Added 03/18/2019 by Hans Lee, source=Web Response, value=Consulting fee Consultant relationship with Veran medical Please note: $5001 - $20000 Added 03/18/2019 by Hans Lee, source=Web Response, value=Consulting fee Employee relationship with Veracyte Please note: >$100000 Added 03/15/2019 by Lori Lofaro, source=Web Response, value=Salary Advisory Committee Member relationship with Grail Please note: $1-$1000 Added 06/22/2019 by Peter Mazzone, source=Web Response, value=Consulting fee Consultant relationship with SEER Please note: $1-$1000 Added 06/22/2019 by Peter Mazzone, source=Web Response, value=Consulting fee Research support to my institution relationship with Veracyte Please note: $5001 - $20000 Added 06/22/2019 by Peter Mazzone, source=Web Response, value=Grant/Research Support Research support to my institution relationship with Oncocyte Please note: $5001 - $20000 Added 06/22/2019 by Peter Mazzone, source=Web Response, value=Grant/Research Support Research support to my institution relationship with Exact Sciences Please note: $1001 - $5000 Added 06/22/2019 by Peter Mazzone, source=Web Response, value=Grant/Research Support Employee relationship with Veracyte Please note: >$100000 Added 03/18/2019 by Daniel Pankratz, source=Web Response, value=Salary No relevant relationships by Momen Wahidi, source=Web Response Employee relationship with Veracyte Please note: >$100000 Added 03/18/2019 by Patric Walsh, source=Web Response, value=Salary

Analytical and Clinical Validation of Expressed Variants and Fusions From the Whole Transcriptome of Thyroid FNA Samples.

Introduction: The Afirma® Xpression Atlas (XA) detects gene variants and fusions in thyroid nodule FNA samples from a curated panel of 511 genes using whole-transcriptome RNA-sequencing. Its intended use is among cytologically indeterminate nodules that are Afirma GSC suspicious, Bethesda V/VI nodules, or known thyroid metastases. Here we report its analytical and clinical validation.Methods: DNA and RNA were purified from the same sample across 943 blinded FNAs and compared by multiple methodologies, including whole-transcriptome RNA-seq, targeted RNA-seq, and targeted DNA-seq. An additional 695 blinded FNAs were used to define performance for fusions between whole-transcriptome RNA-seq and targeted RNA-seq. We quantified the reproducibility of the whole-transcriptome RNA-seq assay across laboratories and reagent lots. Finally, variants and fusions were compared to histopathology results.Results: Of variants detected in DNA at 5 or 20% variant allele frequency, 74 and 88% were also detected by XA, respectively. XA variant detection was 89% when compared to an alternative RNA-based detection method. Low levels of expression of the DNA allele carrying the variant, compared with the wild-type allele, was found in some variants not detected by XA. 82% of gene fusions detected in a targeted RNA fusion assay were detected by XA. Conversely, nearly all variants or fusions detected by XA were confirmed by an alternative method. Analytical validation studies demonstrated high intra-plate reproducibility (89%-94%), inter-plate reproducibility (86–91%), and inter-lab accuracy (90%). Multiple variants and fusions previously described across the spectrum of thyroid cancers were identified by XA, including some with approved or investigational targeted therapies. Among 190 Bethesda III/IV nodules, the sensitivity of XA as a standalone test was 49%.Conclusion: When the Afirma Genomic Sequencing Classifier (GSC) is used first among Bethesda III/IV nodules as a rule-out test, XA supplements genomic insight among those that are GSC suspicious. Our data clinically and analytically validate XA for use among GSC suspicious, or Bethesda V/VI nodules. Genomic information provided by XA may inform clinical decision-making with precision medicine insights across a broad range of FNA sample types encountered in the care of patients with thyroid nodules and thyroid cancer.

Profiling Gene Programs in the Blood During Liver Regeneration in Living Liver Donors.

The human liver's capacity to rapidly regenerate to a full-sized functional organ after resection has allowed successful outcomes for living donor liver transplantation (LDLT) procedures. However, the ability to detect and track physiological changes occurring during liver regeneration after resection and throughout the restoration process is still lacking. We performed a comprehensive whole-transcriptome RNA sequencing analysis of liver and circulating blood tissue from 12 healthy LDLT donors to define biomarker signatures for monitoring physiological activities during liver regeneration at 14 time points for up to a 1-year procedural follow-up. LDLT donor liver tissue differentially expressed 1238 coding and noncoding genes after resection, and an additional 1260 genes were selectively regulated after LDLT. A total of 15,011 RNA transcript species were identified in the blood in response to liver resection. The transcripts most highly regulated were sequentially expressed within 3 distinct peaks that correlated with sets of functional genes involved in the induction of liver resection-specific innate immune response (peak 1), activation of the complement system (peak 2), and platelet activation and erythropoiesis (peak 3). Each peak corresponded with progressive phases of extracellular matrix degradation, remodeling, and organization during liver restoration. These processes could be tracked by distinct molecular signatures of up-regulated and down-regulated gene profiles in the blood during phases of liver repair and regeneration. In conclusion, the results establish temporal and dynamic transcriptional patterns of gene expression following surgical liver resection that can be detected in the blood and potentially used as biomarker signatures for monitoring phases of liver regeneration.