Comparison Of Gene Expression Data Research Articles

Abstract PO3-25-07: Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p< 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-07.

Fedratinib Overcomes Ruxolitinib Resistance through Inhibition of the Interferon Signaling Pathway

Background: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by hyperactive JAK-STAT signaling, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly. Ruxolitinib (RUX) and Fedratinib are both JAK inhibitors used for the treatment of patients with intermediate or higher risk MF. While RUX is the first line treatment for patients with MF, about 50% of the patients discontinue treatment due to lack of efficacy or intolerance (Harrison et al., Ann. Hematol. 2020) after 3 years. Fedratinib is used as a second line therapy in MF based on efficacy from the JAKARTA2 study. The recently concluded FREEDOM and FREEDOM2 studies further support this. In this work, using in vitro approaches, we have explored the mechanistic basis of fedratinib's ability to overcome RUX resistance. Pharmacologically, Fedratinib binds with high affinity to the substrate-binding site and with low affinity to the ATP-binding site of JAK2(Kesarwani etal., Sci. Rep. 2015) while RUX binds only to the ATP-binding site. Previous studies on RUX resistance have shown acquired mutations driving resistance to RUX but not to Fedratinib. Here, we use RUX sensitive and resistant BaF3 (expressing human JAK2 WT or V617F) cell lines to examine the mechanism of resistance to RUX and the mechanism of efficacy of Fedratinib in RUX resistance. Methods: BaF3 cell lines overexpressing JAK2 WT or JAK2 activating mutation V617F were incrementally exposed to RUX over 3 weeks (up to IC 90) to induce resistance. Cell proliferation and phosphorylation of STAT5 in Fedratinib and RUX-treated cells were measured using celltiter glo and immunoblotting, respectively. RNAseq and ATAC-seq analyses were performed in triplicates by treating the relevant cell lines with DMSO, RUX or Fedratinib at two timepoints (6 and 15 hours). Pathway analysis was performed on RNAseq data using Metacore®. Results and Discussion: Ruxolitinib resistant BaF3 JAK2V617F cells showed minimal inhibition of proliferation (<10%) when treated with RUX but maintained sensitivity towards Fedratinib (IC 50 650nM vs 1552nM in RUX sensitive and resistant cells respectively) in 24-hour proliferation assays. Biochemical analysis revealed that Fedratinib inhibited JAK-STAT signaling in the RUX resistant cell line while RUX was unable to do so as measured by p-STAT5. Similar results were observed in BaF3-JAK2-WT cells as well. To further understand the mechanism of RUX-resistance and differential activity of RUX and Fedratinib, we performed RNAseq analysis on our RUX sensitive and resistant models before and after RUX and Fedratinib treatment. Myelofibrosis is associated with aberrant cytokine production and pro-inflammatory signaling that are mediated through activation of TNF-α and NF-κB signaling (Dunbar et al., Blood 2023). Consistent with this, RNAseq analysis of BaF3 JAK2 V617F cells treated with RUX or Fedratinib showed effective inhibition of these pathways by both agents as early as 6 hours. In the RUX resistant cells, while RUX did not have any significant pathway level changes, the top enriched pathways upon treatment with Fedratinib were predominantly interferon signaling pathways which includes JAK-STAT signaling suggesting that Fedratinib's efficacy in MF is not impacted by RUX resistance. To understand the mechanism of resistance, comparison of gene expression data from RUX senstive and RUX resistant cells without any drug treatment revealed minimal gene expression/pathway level differences suggesting that the mechanism of resistance could be driven by genetic (Downes et al. NPJ Precis Oncol, 2021) or epigenetic changes(Dunbar et al., Blood 2023). Ongoing analyses includes whole genome sequencing and ATAC-seq to investigate potential mutations and chromatin accessibility changes during resistance acquisition to RUX and how Fedratinib may overcome these. Conclusion: Our stable RUX resistant preclinical model demonstrates that Fedratinib is effective in overcoming RUX resistance by continuing to target key pathways in MPN biology including interferon signaling. Resistance to RUX on the other hand seems to be driven through potential genetic or epigenetic changes based on the minimal changes observed in gene expression profiles. Our ongoing analyses will reveal these potential genetic and epigenetic modifications associated with RUX resistance and explain the differential activity of RUX versus Fedratinib.

Endothelial dysfunction at the cellular level in three dimensions: severity, acuteness, and distribution.

Dysfunction or failure of the endothelial organ is a heterogenous and often ill-described feature of both cardiovascular and noncardiovascular disorders. Although seldom recognized as a separate clinical condition, endothelial cell dysfunction (ECD) is an established catalyst of disease. However, even in recent pathophysiological studies, ECD is frequently oversimplified as a binary state without gradation, based on the assessment of a single function (e.g., synthesis or activity of nitric oxide) and without considering spatiotemporal dimensions (local vs. generalized, acute vs. chronic). In this article, we suggest a simple scale to grade the severity of ECD and a definition of ECD in three dimensions: space, time, and severity. We also adopt a broader perspective on ECD by integrating and comparing gene expression data of endothelial cells from different organs and diseases and propose a concept that links common pathophysiological mechanisms. We hope that this will enhance the understanding of the pathophysiology of ECD and stimulate discussion in this field.

Genealyzer: web application for the analysis and comparison of gene expression data

BackgroundGene expression profiling is a widely adopted method in areas like drug development or functional gene analysis. Microarray data of gene expression experiments is still commonly used and widely available for retrospective analyses. However, due to to changes of the underlying technologies data sets from different technologies are often difficult to compare and thus a multitude of already available data becomes difficult to use. We present a web application that abstracts away mathematical and programmatical details in order to enable a convenient and customizable analysis of microarray data for large-scale reproducibility studies. In addition, the web application provides a feature that allows easy access to large microarray repositories.ResultsOur web application consists of three basic steps which are necessary for a differential gene expression analysis as well as Gene Ontology (GO) enrichment analysis and the comparison of multiple analysis results. Genealyzer can handle Affymetrix data as well as one-channel and two-channel Agilent data. All steps are visualized with meaningful plots. The application offers flexible analysis while being intuitively operable.ConclusionsOur web application provides a unified platform for analysing microarray data, while allowing users to compare the results of different technologies and organisms. Beyond reproducibility, this also offers many possibilities for gaining further insights from existing study data, especially since data from different technologies or organisms can also be compared. The web application can be accessed via this URL: https://genealyzer.item.fraunhofer.de/. Login credentials can be found at the end.

A novel human tau knock-in mouse model reveals interaction of Abeta and human tau under progressing cerebral amyloidosis in 5xFAD mice

BackgroundHyperphosphorylation and intraneuronal aggregation of the microtubule-associated protein tau is a major pathological hallmark of Alzheimer’s disease (AD) brain. Of special interest is the effect of cerebral amyloid beta deposition, the second main hallmark of AD, on human tau pathology. Therefore, studying the influence of cerebral amyloidosis on human tau in a novel human tau knock-in (htau-KI) mouse model could help to reveal new details on their interplay.MethodsWe studied the effects of a novel human htau-KI under fast-progressing amyloidosis in 5xFAD mice in terms of correlation of gene expression data with human brain regions, development of Alzheimer’s-like pathology, synaptic transmission, and behavior.ResultsThe main findings are an interaction of human beta-amyloid and human tau in crossbred 5xFADxhtau-KI observed at transcriptional level and corroborated by electrophysiology and histopathology. The comparison of gene expression data of the 5xFADxhtau-KI mouse model to 5xFAD, control mice and to human AD patients revealed conspicuous changes in pathways related to mitochondria biology, extracellular matrix, and immune function. These changes were accompanied by plaque-associated MC1-positive pathological tau that required the htau-KI background. LTP deficits were noted in 5xFAD and htau-KI mice in contrast to signs of rescue in 5xFADxhtau-KI mice. Increased frequencies of miniature EPSCs and miniature IPSCs indicated an upregulated presynaptic function in 5xFADxhtau-KI.ConclusionIn summary, the multiple interactions observed between knocked-in human tau and the 5xFAD-driven progressing amyloidosis have important implications for future model development in AD.

Comparative transcriptomics analysis pipeline for the meta-analysis of phylogenetically divergent datasets (CoRMAP)

BackgroundTranscriptional regulation is a fundamental mechanism underlying biological functions. In recent years, a broad array of RNA-Seq tools have been used to measure transcription levels in biological experiments, in whole organisms, tissues, and at the single cell level. Collectively, this is a vast comparative dataset on transcriptional processes across organisms. Yet, due to technical differences between the studies (sequencing, experimental design, and analysis) extracting usable comparative information and conducting meta-analyses remains challenging.ResultsWe introduce Comparative RNA-Seq Metadata Analysis Pipeline (CoRMAP), a meta-analysis tool to retrieve comparative gene expression data from any RNA-Seq dataset using de novo assembly, standardized gene expression tools and the implementation of OrthoMCL, a gene orthology search algorithm. It employs the use of orthogroup assignments to ensure the accurate comparison of gene expression levels between experiments and species. Here we demonstrate the use of CoRMAP on two mouse brain transcriptomes with similar scope, that were collected several years from each other using different sequencing technologies and analysis methods. We also compare the performance of CoRMAP with a functional mapping tool, previously published.ConclusionCoRMAP provides a framework for the meta-analysis of RNA-Seq data from divergent taxonomic groups. This method facilitates the retrieval and comparison of gene expression levels from published data sets using standardized assembly and analysis. CoRMAP does not rely on reference genomes and consequently facilitates direct comparison between diverse studies on a range of organisms.

Identification of cancer risk assessment signature in patients with chronic obstructive pulmonary disease and exploration of the potential key genes

It is essential to assess the cancer risk for patients with chronic obstructive pulmonary disease (COPD). Comparing gene expression data from patients with lung cancer (a total of 506 samples) and those with cancer-adjacent normal lung tissues (a total of 370 samples), we generated a qualitative transcriptional signature consisting of 2046 gene pairs. The signature was verified in an evaluation dataset comprising 18 subjects with severe disease and 52 subjects with moderate disease (Wilcoxon rank-sum test; p = 7.33 × 10−5). Similar results were obtained in other independent datasets. Among the gene pairs in the signature, 326 COPD stage-related gene pairs were identified based on Spearman’s rank correlation tests and those gene pairs comprised 368 unique genes. Of these 368 genes, 16 genes were significantly dysregulated in COPD rat model data compared with control data. Some of these genes (Dhx16, Upf2, Notch3, Sec61a1, Dyrk2, and Hmmr) were altered when the COPD rat model was treated with traditional Chinese medicines (TCM), including Bufei Yishen formula, Bufei Jianpi formula, and Yiqi Zishen formula. Overall, the signature could predict the cancer incidence-risk of COPD and the identified key genes might provide guidance regarding both the treatment of COPD using TCM and the prevention of cancer in patients with COPD. KEY MESSAGES A cancer risk assessment signature was identified in patients with COPD. The signature is insensitive to batch effects and is well verified. COPD key genes identified in this study might play a crucial role in TCM treatment and cancer prevention.

A bioinformatics framework for targeted gene expression assay design: Application to in vitro developmental neurotoxicity screening in a rat model

Brain development involves a series of intricately choreographed neuronal differentiation and maturation steps that are acutely vulnerable to interferences from chemical exposures. Many genes involved in neurodevelopmental processes show evolutionarily conserved expression patterns in mammals and may constitute useful indicators/biomarkers for the evaluation of potential developmental neurotoxicity. Based on these premises, this study developed a bioinformatics framework to guide the design of a gene expression-based in vitro developmental neurotoxicity assay targeting evolutionary conserved genes associated with neuronal differentiation and maturation in rat cerebellar granule cells (CGCs). Rat, mouse and human genes involved in neurodevelopment and presenting one-to-one orthology were selected and orthologous exons within these genes were identified. PCR primer sets were designed within these orthologous exons and their specificity was evaluated in silico. The performance and specificity of rat, mouse and human PCR primer sets were then confirmed experimentally. Finally, RT-qPCR analyses in CGCs exposed in vitro to well-known neurotoxicants (Chlorpyrifos and Chlorpyrifos oxon) uncovered perturbations of expression levels for most of the selected genes. This bioinformatics framework for gene and target sequence selection may facilitate the identification of transcriptional biomarkers for developmental neurotoxicity assays and the comparison of gene expression data across experimental models from different mammalian species.

Abstract CT039: Intratumoral CD40 agonist sotigalimab with pembrolizumab induces broad innate and adaptive immune activation in local and distant tumors in metastatic melanoma

Abstract The use of immune-checkpoint inhibitors (CPI) has become an important modality in the treatment of metastatic melanoma (MM). However, most patients (pts) do not experience durable responses and new treatment options are needed to improve clinical outcomes. Our pre-clinical studies have demonstrated that intratumoral CD40 activation synergizes with anti-PD-1 based therapy and induces systemic and distant anti-tumor effects. In this ongoing phase I/II study, we assessed intratumoral sotigalimab (APX005M), a CD40 agonist antibody, in combination with systemic pembrolizumab in CPI treatment naïve, unresectable stage III or IV MM. A total of 40 participants will be enrolled. As of December 15, 2021, 30 pts were enrolled. Pts received sotigalimab every 3 weeks for a total of 4 doses. The dose escalation portion of the trial has been completed, with 14 pts enrolled in 5 dosing cohorts of sotigalimab at 0.1, 0.5, 1, 3 and 10 mg. The primary objectives include safety and tolerability, determination of the recommended phase 2 dose (RP2D), and assessment of the overall response rate (ORR) by RECIST v1.1. Biomarker analyses of blood and tumor samples were performed to measure immune activation using immunophenotyping including imaging mass cytometry, TCR sequencing, and a cross-cohort comparison of gene expression data (sotigalimab plus pembrolizumab versus anti-PD1 monotherapy). The combination therapy has been well-tolerated, and there were no study discontinuations or death due to treatment-related adverse events (TRAEs). Most common TRAEs were injection-site reactions; six pts experienced grade-3 immune-related adverse events. Efficacy analysis of 30 pts with post-baseline disease evaluations demonstrated an ORR of 50% (5 CR and 10 PR) in distant lesions and a disease control rate of 67%. The ORR at the RP2D of 10 mg is 55% (12/22). Responses were observed in PD-L1 negative pts and those with elevated LDH. Comprehensive transcriptome and immune cell profiling of peripheral blood mononuclear cells and tumor biopsies obtained from local lesions at baseline and 24 hours post sotigalimab injection demonstrate that sotigalimab effectively engaged CD40 pathway. In comparison to anti-PD1 monotherapy, the combination therapy significantly increased expression of genes associated with antigen presentation and effector T cells in local lesions accompanied by an increase in T cell activation genes at distant lesions. Additionally, T cell repertoire analysis demonstrated a significant increase in T cell clonality with expansion of new clones shared between local and distant tumors. Importantly, these immunologic changes were correlated with clinical response. Collectively, this combination therapy is well tolerated and has a notable clinical response rate, accompanied by broad innate and adaptive immune activation at both local and distant lesions. Citation Format: Salah-Eddine Bentebibel, Daniel Johnson, Rodabe Amariae, Daniel McGrail, Srisuda Lecagoonporn, Cara Haymaker, Dzifa Duose, Khalida Wani, Houssein Safa, Isabella Claudia Glitza, Sapna Pradyuman Patel, Michael K. Wong, Hussein Tawbi, Jared Burks, Xiaodong Yang, Patrick Hwu, Cassian Yee, Michael A. Davies, Ravi Murthy, Chantale Bernatchez, Suhendan Ekmekcioglu, Adi Diab, Gregory Lizée. Intratumoral CD40 agonist sotigalimab with pembrolizumab induces broad innate and adaptive immune activation in local and distant tumors in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT039.

Gapdh Shows Altered Gene Expression in Alcohol Models

Abstract Background and objectives. Though glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is one of the most commonly used housekeeping genes for comparison of gene expression data, studies have revealed that Gapdh expression is not constant at different developmental stages, and is modulated by many factors including ethanol. In view of this, in the present study, we investigated the effect of ethanol on Gapdh stability and expression levels in different model systems. Methods. Evaluation of Gapdh stability was determined by comparison with three commonly used housekeeping genes in alcohol-exposed and control mouse blastocysts, embryos, and placentas (ActB, Hsp90ab1, and Atp5b), and in alcohol-dependent and healthy humans (ACTB, ATP5B, and HSPCB), and was ranked by the software program RefFinder. To detect the Gapdh expression patterns as a target gene, qRT-PCR analysis was applied. Results. The order of expression stabilities obtained by the RefFinder ranked Gapdh as the most unstable reference gene in studied groups. The study of the expression dynamics of Gapdh showed significant upregulation in ethanol-exposed mouse blastocysts and embryos (FC = 1.56, p = 0.05 and FC = 6.0, p = 0.01) and downregulation in placentas (FC = 0.60, p = 0.01). Conclusions. Our results revealed that the expression of Gapdh can vary in different model systems under ethanol exposure and imply the need for caution when using it as an internal control. This is the first report for statistically significant change in Gapdh expression after ethanol exposure during preimplantation mouse development.

Mitochondrial-nuclear epistasis underlying phenotypic variation in breast cancer pathology

The interplay between genes harboring single nucleotide polymorphisms (SNPs) is vital to better understand underlying contributions to the etiology of breast cancer. Much attention has been paid to epistasis between nuclear genes or mutations in the mitochondrial genome. However, there is limited understanding about the epistatic effects of genetic variants in the nuclear and mitochondrial genomes jointly on breast cancer. We tested the interaction of germline SNPs in the mitochondrial (mtSNPs) and nuclear (nuSNPs) genomes of female breast cancer patients in The Cancer Genome Atlas (TCGA) for association with morphological features extracted from hematoxylin and eosin (H&E)-stained pathology images. We identified 115 significant (q-value < 0.05) mito-nuclear interactions that increased nuclei size by as much as 12%. One interaction between nuSNP rs17320521 in an intron of the WSC Domain Containing 2 (WSCD2) gene and mtSNP rs869096886, a synonymous variant mapped to the mitochondrially-encoded NADH dehydrogenase 4 (MT-ND4) gene, was confirmed in an independent breast cancer data set from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). None of the 10 mito-nuclear interactions identified from non-diseased female breast tissues from the Genotype-Expression (GTEx) project resulted in an increase in nuclei size. Comparisons of gene expression data from the TCGA breast cancer patients with the genotype homozygous for the minor alleles of the SNPs in WSCD2 and MT-ND4 versus the other genotypes revealed core transcriptional regulator interactions and an association with insulin. Finally, a Cox proportional hazards ratio = 1.7 (C.I. 0.98–2.9, p-value = 0.042) and Kaplan–Meier plot suggest that the TCGA female breast cancer patients with low gene expression of WSCD2 coupled with large nuclei have an increased risk of mortality. The intergenomic dependency between the two variants may constitute an inherent susceptibility of a more severe form of breast cancer and points to genetic targets for further investigation of additional determinants of the disease.

Drug Repositioning Ketamine as a New Treatment for Bipolar Disorder Using Text Mining

Bipolar Disorder (BD), a chronic mental illness, does not have an ideal treatment, and patients with BD have a higher chance of being diagnosed with alcohol abuse, liver disease, and diabetes. The goal of treatment is to prevent a relapse in BD episodes and find a new treatment. The research here looks at the genetics of BD and ignores environmental factors, as they are subjective. Therapy treats known environmental triggers and stressors and explores methods to reduce them. However, therapy alone cannot fully alleviate the symptoms of BD. My research employs text-mining as a primary strategy to obtain relevant genes and drugs pertaining to BD. The main gene involved is the Brain-Derived Neurotrophic Factor (BDNF). Popular drugs currently used for treatment of BD are Lithium and Carbamazepine. Using CMapPy to look at gene expression data, one sees a relationship between the two drug therapies and BDNF. Lithium fails to treat mania and Carbamazepine fails to treat depression, relatively speaking. When comparing gene expression data of Lithium and Carbamazepine with Ketamine, a newer therapy for BD, Ketamine, raises the BDNF level, keeps it elevated, and effectively controls BD episodes. Ketamine does not have the shortcomings that Lithium and Carbamazepine have. Next steps would include conducting a clinical trial with the hopeful application of Ketamine as a new treatment for BD.

Conserved Mechanisms, Novel Anatomies: The Developmental Basis of Fin Evolution and the Origin of Limbs

The transformation of paired fins into tetrapod limbs is one of the most intensively scrutinized events in animal evolution. Early anatomical and embryological datasets identified distinctive morphological regions within the appendage and posed hypotheses about how the loss, gain, and transformation of these regions could explain the observed patterns of both extant and fossil appendage diversity. These hypotheses have been put to the test by our growing understanding of patterning mechanisms that regulate formation of the appendage axes, comparisons of gene expression data from an array of phylogenetically informative taxa, and increasingly sophisticated and elegant experiments leveraging the latest molecular approaches. Together, these data demonstrate the remarkable conservation of developmental mechanisms, even across phylogenetically and morphologically disparate taxa, as well as raising new questions about the way we view homology, evolutionary novelty, and the often non-linear connection between morphology and gene expression. In this review, we present historical hypotheses regarding paired fin evolution and limb origins, summarize key aspects of central appendage patterning mechanisms in model and non-model species, address how modern comparative developmental data interface with our understanding of appendage anatomy, and highlight new approaches that promise to provide new insight into these well-traveled questions.

Estimates of gene ensemble noise highlight critical pathways and predict disease severity in H1N1, COVID-19 and mortality in sepsis patients

Finding novel biomarkers for human pathologies and predicting clinical outcomes for patients is challenging. This stems from the heterogeneous response of individuals to disease and is reflected in the inter-individual variability of gene expression responses that obscures differential gene expression analysis. Here, we developed an alternative approach that could be applied to dissect the disease-associated molecular changes. We define gene ensemble noise as a measure that represents a variance for a collection of genes encoding for either members of known biological pathways or subunits of annotated protein complexes and calculated within an individual. The gene ensemble noise allows for the holistic identification and interpretation of gene expression disbalance on the level of gene networks and systems. By comparing gene expression data from COVID-19, H1N1, and sepsis patients we identified common disturbances in a number of pathways and protein complexes relevant to the sepsis pathology. Among others, these include the mitochondrial respiratory chain complex I and peroxisomes. This suggests a Warburg effect and oxidative stress as common hallmarks of the immune host–pathogen response. Finally, we showed that gene ensemble noise could successfully be applied for the prediction of clinical outcome namely, the mortality of patients. Thus, we conclude that gene ensemble noise represents a promising approach for the investigation of molecular mechanisms of pathology through a prism of alterations in the coherent expression of gene circuits.

Differential gene expression and Ingenuity Pathway Analysis of bronchoalveolar lavage cells from horses with mild/moderate neutrophilic or mastocytic inflammation on BAL cytology

Mild to moderate equine asthma syndrome (mEAS) affects horses of all ages and breeds. To date, the etiology and pathophysiology of mEAS are active areas of research, and it remains incompletely understood whether mEAS horses with different immune cell ‘signatures’ on BAL cytology represent different phenotypes, distinct pathobiological mechanisms (endotypes), varied environmental conditions, disease severity, genetic predispositions, or all of the above. In this descriptive study, we compared gene expression data from BAL cells isolated from horses with normal BALF cytology (n = 5), to those isolated from horses with mild/moderate neutrophilic inflammation (n = 5), or mild/moderate mastocytic inflammation (n = 5). BAL cell protein lysates were analyzed for cytokine/chemokine levels using Multiplex Bead Immunoassay, and for select proteins using immunoblot. The transcriptome, determined by RNA-seq and analyzed with DEseq2, contained 20, 63, and 102 significantly differentially expressed genes in horses with normal vs. neutrophilic, normal vs. mastocytic, and neutrophilic vs. mastocytic BALF cytology, respectively. Pathway analyses revealed that BAL-isolated cells from horses with neutrophilic vs. normal cytology showed enrichment in inflammation pathways, and horses with mastocytic vs. normal cytology showed enrichment in pathways involved in fibrosis and allergic reaction. BAL cells from horses with mastocytic mEAS, compared to neutrophilic mEAS, showed enrichment in pathways involved in alteration of tissue structures. Cytokine analysis determined that IL-1β was significantly different in the lysates from horses with neutrophilic inflammation compared to those with normal or mastocytic BAL cytology. Immunoblot revealed significant difference in the relative level of MMP2 in horses with neutrophilic vs. mastocytic mEAS. Upregulation of mRNA transcripts involved in the IL-1 family cytokine signaling axis (IL1a, IL1b, and IL1R2) in neutrophilic mEAS, as well as KIT mRNA in mastocytic mEAS, are novel, potentially clinically relevant, findings of this study. These findings further inform our understanding of inflammatory cell subtypes in mEAS.

Transcriptomic profiling of long- and short-lived mutant mice implicates mitochondrial metabolism in ageing and shows signatures of normal ageing in progeroid mice

Genetically modified mouse models of ageing are the living proof that lifespan and healthspan can be lengthened or shortened, and provide a powerful context in which to unravel the molecular mechanisms at work. In this study, we analysed and compared gene expression data from 10 long-lived and 8 short-lived mouse models of ageing. Transcriptome-wide correlation analysis revealed that mutations with equivalent effects on lifespan induce more similar transcriptomic changes, especially if they target the same pathway. Using functional enrichment analysis, we identified 58 gene sets with consistent changes in long- and short-lived mice, 55 of which were up-regulated in long-lived mice and down-regulated in short-lived mice. Half of these sets represented genes involved in energy and lipid metabolism, among which Ppargc1a, Mif, Aldh5a1 and Idh1 were frequently observed. Based on the gene sets with consistent changes, and also the whole transcriptome, the gene expression changes during normal ageing resembled the transcriptome of short-lived models, suggesting that accelerated ageing models reproduce partially the molecular changes of ageing. Finally, we identified new genetic interventions that may ameliorate ageing, by comparing the transcriptomes of 51 mouse mutants not previously associated with ageing to expression signatures of long- and short-lived mice and ageing-related changes.

Differential Glial Activation in Early Epileptogenesis-Insights From Cell-Specific Analysis of DNA Methylation and Gene Expression in the Contralateral Hippocampus.

Background and Aims: Morphological changes in mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) are well-characterized. Yet, it remains elusive whether these are a consequence of seizures or originate from a hitherto unknown underlying pathology. We recently published data on changes in gene expression and DNA methylation in the ipsilateral hippocampus (ILH) using the intracortical kainate mouse model of mTLE-HS. In order to explore the effects of epileptic activity alone and also to further disentangle what triggers morphological alterations, we investigated glial and neuronal changes in gene expression and DNA methylation in the contralateral hippocampus (CLH).Methods: The intracortical kainic acid mouse model of mTLE-HS was used to elicit status epilepticus. Hippocampi contralateral to the injection site from eight kainate-injected and eight sham mice were extracted and shock frozen at 24 h post-injection. Glial and neuronal nuclei were sorted by flow cytometry. Alterations in gene expression and DNA methylation were assessed using reduced representation bisulfite sequencing and RNA sequencing. The R package edgeR was used for statistical analysis.Results: The CLH featured substantial, mostly cell-specific changes in both gene expression and DNA methylation in glia and neurons. While changes in gene expression overlapped to a great degree between CLH and ILH, alterations in DNA methylation did not. In the CLH, we found a significantly lower number of glial genes up- and downregulated compared to previous results from the ILH. Furthermore, several genes and pathways potentially involved in anti-epileptogenic effects were upregulated in the CLH. By comparing gene expression data from the CLH to previous results from the ILH (featuring hippocampal sclerosis), we derive potential upstream targets for epileptogenesis, including glial Cox2 and Cxcl10.Conclusion: Despite the absence of morphological changes, the CLH displays substantial changes in gene expression and DNA methylation. We find that gene expression changes related to potential anti-epileptogenic effects seem to dominate compared to the pro-epileptogenic effects in the CLH and speculate whether this imbalance contributes to prevent morphological alterations like neuronal death and reactive gliosis.

Renal cell tumors convert natural killer cells to a proangiogenic phenotype.

Natural killer (NK) cells are classically associated with immune surveillance and destruction of tumor cells. Inconsistent with this function, NK cells are found in advanced human tumors including renal cell carcinoma (RCC). NK cells with non-classical phenotypes (CD56+CD16dim/neg; termed decidua NK (dNK) cells) accumulate at the maternal-fetal interface during embryo implantation. These dNK cells are poorly cytotoxic, proangiogenic, and facilitate placenta development. As similarities between embryo implantation and tumor growth exist, we tested the hypothesis that an analogous shift in NK cell phenotype and function occurs in RCC tumors. Our results show that peripheral NK (pNK) cells of RCC patients were uniformly CD56+CD16bright, but lacked full cytotoxic ability. By comparison, RCC tumor-infiltrated NK (TiNK) cells were significantly enriched for CD56+CD16dim-neg cells, a phenotype of dNK cells. Gene expression analysis revealed that angiogenic and inflammatory genes were significantly increased for RCC TiNK versus RCC pNK populations, with enrichment of genes in the hypoxia inducible factor (HIF) 1α pathway. Consistent with this finding, NK cells cultured under hypoxia demonstrated limited cytotoxicity capacity, but augmented production of vascular endothelial growth factor (VEGF). Finally, comparison of gene expression data for RCC TiNK and dNK cells revealed a shared transcriptional signature of genes with known roles in angiogenesis and immunosuppression. These studies confirm conversion of pNK cells to a dNK-like phenotype in RCC tumors. These characteristics are conceivably beneficial for placentation, but likely exploited to support early tumor growth and promote metastasis.

A disintegrin and metalloproteinase domain 17-epidermal growth factor receptor signaling contributes to oral cancer pain.

Cancer cells secrete pronociceptive mediators that sensitize adjacent sensory neurons and cause pain. Identification and characterization of these mediators could pinpoint novel targets for cancer pain treatment. In this study, we identified candidate genes in cancer cell lines that encode for secreted or cell surface proteins that may drive nociception. To undertake this work, we used an acute cancer pain mouse model, transcriptomic analysis of publicly available human tumor-derived cell line data, and a literature review. Cancer cell line supernatants were assigned a phenotype based on evoked nociceptive behavior in an acute cancer pain mouse model. We compared gene expression data from nociceptive and nonnociceptive cell lines. Our analyses revealed differentially expressed genes and pathways; many of the identified genes were not previously associated with cancer pain signaling. Epidermal growth factor receptor (EGFR) and disintegrin metalloprotease domain 17 (ADAM17) were identified as potential targets among the differentially expressed genes. We found that the nociceptive cell lines contained significantly more ADAM17 protein in the cell culture supernatant compared to nonnociceptive cell lines. Cytoplasmic EGFR was present in almost all (>90%) tongue primary afferent neurons in mice. Monoclonal antibody against EGFR, cetuximab, inhibited cell line supernatant-induced nociceptive behavior in an acute oral cancer pain mouse model. We infer from these data that ADAM17-EGFR signaling is involved in cancer mediator-induced nociception. The differentially expressed genes and their secreted protein products may serve as candidate therapeutic targets for oral cancer pain and warrant further evaluation.

Abstract P4-07-06: Breast cancer subtype classification using NanoString and RNAseq technologies

Abstract Background: Immunohistochemistry (IHC) is conventionally used in clinical practice to define Breast Cancer (BC) subtypes, while PAM50 is useful in specific cases. The former is partly subjective and semi-quantitative, the latter is based on gene expression profile. We compared gene expression data obtained by NanoString BC 360™ panel with those obtained by RNAseq on 12 Formalin-Fixed Paraffin-Embedded (FFPE) BC samples. Methods: RNA was isolated from FFPE tumors and the quality was checked before performing NanoString BC 360™ assay and preparing RNAseq library (NEBNext Ultra II RNA Library Prep Kit, Illumina) following the manufacturers’ instructions. Libraries were sequenced on NextSeq500 (Illumina). Reads were aligned using Kallisto and raw read counts were normalized as Transcripts Per Million (TPM). PAM50 and Tumor Inflammation Signature (TIS) were determined on NanoString data. Two-sided t-test on TPM and on normalized NanoString counts was used to compare luminal and Triple Negative (TN) BCs (p &amp;lt;0.05 statistically significant), considering the 734 genes shared by the 2 assays. Deseq2 (1.22.1) was used to perform a differential gene expression analysis on raw RNAseq counts. Enrichment tests were performed on KEGG pathways (FDR &amp;lt; 0.05). Python and R softwares were used for statistical analyses. Results: Of the 12 BC samples, 3 were luminal A-like, 3 luminal B (HER2+)-like and 6 TN by IHC (Table 1). Pearson’s correlation coefficient between NanoString counts and RNAseq TPM was 0.72. Out of the 734 genes shared by both methods, those differentially expressed between luminal and TN BCs, defined based on IHC, were 135 according to RNAseq TPM, 155 according to NanoString counts and 88 according to both methods. These 88 genes belonged to “cell cycle”, “pathways in cancer”, “HIF-1 signaling”, “progesterone-mediated oocyte maturation”, “prostate cancer” and “basal cell carcinoma” KEGG pathways. When considering the whole transcriptome analyzed by RNAseq, the differential gene expression analysis between luminal and TN/basal BCs was performed according to both IHC and PAM50 classifications. Considering the former, there were 1143 differentially expressed genes out of a total of 20936 (5.5%) and the pathways involved were “fatty acid elongation”, “cell cycle”, “oocyte meiosis”, “p53 signaling” and “alcoholism”. The analysis on the latter classification showed 1608 differentially expressed genes out of 21048 (7.6%), and the enriched pathways were “cell cycle”, “p53 signaling”, “DNA replication”, “alcoholism” and “systemic lupus erythematosus”. Regarding the TIS, no significant differences were found between TN/basal and luminal BCs, comparing both IHC and the PAM50 subtypes. An expected heterogeneity was found among TNBCs, that showed 3 distinct signatures: basal-like immune activated, basal-like immune-suppressed and luminal androgen receptor. Conclusions: This preliminary analysis shows a good concordance between NanoString and RNAseq data. The choice of the method must take into account important factors such as the costs, the number of genes analyzed, the requirement of bioinformatic support, the working time. Further analyses on pathway enrichment will be presented. Table 1. Breast cancer samples characteristics.Sample ID Age Grade T N IHC subtype PAM50 Subtype TIS scoreTNBC signature 0172222aLumALumB7,0NA026031c2aTNBasal7,2BLIA037221cxTNLumA7,4LAR045231c0LumALumA7,5NA053531c1aTNBasal5,5BLIS0675320TNBasal6,2BLIS077931cxLumB HER2+LumB6,7NA0878220LumALumB7,7NA097431a2aTNBasal8,5BLIA106031c1LumB HER2+LumA8,2NA116621b0LumB HER2+LumA6,1NA1241321aTNBasal7,5BLIATIS: Tumor Inflammation Signature; BLIA: Basal-like Immune Activated; BLIS: Basal-like Immune-Suppressed; LAR: Luminal Androgen Receptor. Citation Format: Andrea Rocca, Sara Ravaioli, Eugenio Fonzi, Iros Barozzi, Ylenia Perone, Luca Magnani, Francesca Pirini, Giovanni Martinelli, Sara Bravaccini. Breast cancer subtype classification using NanoString and RNAseq technologies [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-06.