Detection of FGFR fusions in intrahepatic cholangiocarcinoma using targeted RNA sequencing.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e16185-e16185
Author(s):  
Maria Farooq ◽  
Havell Markus ◽  
Bradon McDonald ◽  
Jan B. Egan ◽  
Tania Contente-Cuomo ◽  
...  
Keyword(s):  
Prior Knowledge ◽  
Rna Sequencing ◽  
Intrahepatic Cholangiocarcinoma ◽  
Growth Factor Receptor ◽  
Digital Pcr ◽  
High Yield ◽  
Fusion Partner ◽  
Rna Molecules ◽  
Quantitative Performance ◽  
Highly Correlated

e16185 Background: Comprehensive sequencing efforts have identified fibroblast growth factor receptor (FGFR) translocations in numerous cancers. FGFR2 fusions were recently identified as an actionable therapeutic target in ̃17% of cholangiocarcinoma patients, predominantly found in patients with intrahepatic cholangiocarcinoma. Genomic partners for FGFR fusions are variable. In addition, obtaining high yield tissue biopsies for CCA remains challenging and tumor tissue available for molecular analyses is scarce. When combined, these two factors make detection of FGFR fusions in CCA challenging. An accurate molecular assay to detect FGFR fusions in CCA could improve utilization of FGFR-targeted therapies. Methods: The objective of our study was to develop an approach for targeted RNA analysis to improve sensitivity for FGFR fusion detection from limited tumor material. We adapted the sensitive targeted digital sequencing (TARDIS) approach recently developed in our lab for analysis of RNA. This approach utilizes a combination of open-ended targeted amplification followed by ligation, enabling detection of tyrosine kinase fusions without prior knowledge of the precise sequence of the fusion breakpoint or identity of the fusion partner. For evaluation of analytical performance, we analyzed RNA from a CCA organoid model with a known FGFR2 fusion, and Seraseq Fusion RNA Mix v3 Reference Material with two known FGFR3 fusions. Results: Without prior knowledge the partner or coordinates of the fusion breakpoint, we detected an FGFR2-KIF5C fusion in RNA from a CCA organoid model. The fusion breakpoint coordinates predicted by TARDIS-RNA were validated by comparison with RNA-Seq. To assess sensitivity and quantitative performance of the assay, we analyzed a serial dilution of the reference RNA sample from Seraseq with concentration of RNA molecules representing candidate fusions verified by digital PCR. Using TARDIS-RNA to analyze replicates with 5 ng RNA input, we were able to detect candidate fusions represented by as few as 2.7 fusion molecules on average. Concentrations of fusion molecules measured using the two methods were highly correlated (R = 0.96). Conclusions: These results demonstrate sensitivity and quantitative performance of a targeted RNA sequencing assay to detect and quantify FGFR fusions in tumor specimen from intrahepatic cholangiocarcinoma. On-going work is focused on further evaluating assay performance and characterizing FGFR fusions using additional tissue specimen.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Arne H. A. Scheu ◽  
Sheryl Y. T. Lim ◽  
Felix J. Metzner ◽  
Shabaz Mohammed ◽  
Mark Howarth
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Growth Factor Receptor ◽  
Molecular Engineering ◽  
Nucleophilic Attack ◽  
High Yield ◽  
Protein Chemistry ◽  
Transforming Growth Factor Α ◽  
Endogenous Proteins

AbstractThe Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex. Various sites on the target (α-amine or ε-amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2 min. We arm Transforming Growth Factor-α with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering.

scholarly journals Receptor-driven invasion profiles in diffuse intrinsic pontine glioma (DIPG)

2021 ◽  
Author(s):  
Anju Karki ◽  
Noah E Berlow ◽  
Jin-Ah Kim ◽  
Esther Hulleman ◽  
Qianqian Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Protein Expression ◽  
Cell Viability ◽  
Rna Sequencing ◽  
Cell Invasion ◽  
Growth Factor Receptor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Sequencing Data ◽  
Pontine Glioma

Abstract Background Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric cancer with unmet clinical need. DIPG is invasive in nature, where tumor cells interweave into the fiber nerve tracts of the pons making the tumor unresectable. Accordingly, novel approaches in combating the disease is of utmost importance and receptor-driven cell invasion in the context of DIPG is under-researched area. Here we investigated the impact on cell invasion mediated by PLEXINB1, PLEXINB2, platelet growth factor receptor (PDGFR)α, PDGFRβ, epithelial growth factor receptor (EGFR), activin receptor 1 (ACVR1), chemokine receptor 4 (CXCR4) and NOTCH1. Methods We used previously published RNA-sequencing data to measure gene expression of selected receptors in DIPG tumor tissue versus matched normal tissue controls (n=18). We assessed protein expression of the corresponding genes using DIPG cell culture models. Then, we performed cell viability and cell invasion assays of DIPG cells stimulated with chemoattractants/ligands. Results RNA-sequencing data showed increased gene expression of receptor genes such as PLEXINB2, PDGFRα, EGFR, ACVR1, CXCR4 and NOTCH1 in DIPG tumors compared to the control tissues. Representative DIPG cell lines demonstrated correspondingly increased protein expression levels of these genes. Cell viability assays showed minimal effects of growth factors/chemokines on tumor cell growth in most instances. Recombinant SEMA4C, SEM4D, PDGF-AA, PDGF-BB, ACVA, CXCL12 and DLL4 ligand stimulation altered invasion in DIPG cells. Conclusions We show that no single growth factor-ligand pair universally induces DIPG cell invasion. However, our results reveal a potential to create a composite of cytokines or anti-cytokines to modulate DIPG cell invasion.

scholarly journals Global increase in circRNA levels in myotonic dystrophy

2018 ◽  
Author(s):  
Karol Czubak ◽  
Katarzyna Taylor ◽  
Agnieszka Piasecka ◽  
Krzysztof Sobczak ◽  
Katarzyna Kozlowska ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Digital Pcr ◽  
Circular Rnas ◽  
Splicing Factors ◽  
Rna Molecules ◽  
Experimental Systems ◽  
Muscle Tissues ◽  
Protein Interactome ◽  
Global Increase ◽  
And Function

AbstractSplicing aberrations induced as a consequence of the sequestration of MBNL splicing factors on the DMPK transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As MBNLs may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected twenty well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1 and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity.Author SummaryRecently, a great deal of interest has been focused on a new class of RNA molecules called circular RNAs (circRNAs). To date, thousands of circRNAs have been found in different human cells/tissues. Although the function of circRNAs remains mostly unknown, circRNAs have emerged as an important component of the RNA-RNA and RNA-protein interactome. Thus, intensive efforts are being made to fully understand the biology and function of circRNAs, especially their role in human diseases. As an important role in the biogenesis of circRNA may be played by MBNL splicing factors, in this study we used DM1 (to a lesser extent, DM2) as a natural model in which the level of MBNLs is decreased. In contrast to the expected effect, our results consistently showed a global increase in circRNA levels in DM1. As a consequence, whole genome transcriptome analysis revealed dozens of circRNAs with significantly altered (mostly increased) levels in DM1. Furthermore, we observed that the circRNA levels were in many cases strongly associated with DM1 severity.

scholarly journals Rice cold tolerance at the reproductive stage in a controlled environment

2006 ◽  
Vol 63 (3) ◽  
pp. 255-261 ◽  
Author(s):  
Renata Pereira da Cruz ◽  
Sandra Cristina Kothe Milach ◽  
Luiz Carlos Federizzi
Keyword(s):  
Cold Tolerance ◽  
Oryza Sativa L ◽  
Variable Temperature ◽  
Reproductive Stage ◽  
Spikelet Fertility ◽  
High Yield ◽  
Cold Temperatures ◽  
Cold Tolerant ◽  
Rice Genotypes ◽  
Highly Correlated

Cold tolerance of rice (Oryza sativa L.) during the reproductive stage is important to guarantee high yield under low temperature environments. Field selection, however, does not allow identification of adequate tolerance sources and limits selection of segregating lines due to variable temperature. The objective of this study was to devise methods for distinguishing rice genotypes as to their cold tolerance at the reproductive stage when evaluated under controlled temperature. The effect of cold temperatures was investigated in six rice genotypes at 17°C for varying length of time (three, five, seven and ten days) at two reproductive stages (microsporogenesis and anthesis). Cold tolerance was measured as the percentage of reduction in panicle exsertion and in spikelet fertility. Evaluating cold tolerance through the reduction in panicle exsertion did not allow for the distinction between cold tolerant from cold sensitive genotypes and, when the reduction in spikelet fertility was considered, a minimum of seven days was required to differentiate the genotypes for cold tolerance. Genotypes were more sensitive to cold at anthesis than at microsporogenesis and, as these stages were highly correlated, cold screening could be performed at anthesis only, since it is easier to determine. Rice cold tolerance at the reproductive stage may be characterized by the reduction in spikelet fertility due to cold temperature (17°C) applied for seven days at anthesis.

Apatinib Inhibits Angiogenesis in Intrahepatic Cholangiocarcinoma by Regulating the Vascular Endothelial Growth Factor Receptor-2/Signal Transducer and Activator of Transcription Factor 3/Hypoxia Inducible Factor 1 Subunit Alpha Signaling Axis

Pharmacology ◽  
2021 ◽  
pp. 1-11
Author(s):  
Man-Ping Huang ◽  
Shan-Zhi Gu ◽  
Bin Huang ◽  
Guo-Wen Li ◽  
Zheng-Ping Xiong ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Transcription Factor ◽  
Growth Factor ◽  
Intrahepatic Cholangiocarcinoma ◽  
Hypoxia Inducible Factor ◽  
Growth Factor Receptor ◽  
Vascular Endothelial ◽  
Signal Transducer ◽  
Hypoxia Inducible Factor 1 ◽  
Endothelial Growth Factor

<b><i>Introduction:</i></b> Intrahepatic cholangiocarcinoma (ICC), which is difficult to diagnose and is usually fatal due to its late clinical presentation and a lack of effective treatment, has risen over the past decades but without much improvement in prognosis. <b><i>Objective:</i></b> The study aimed to investigate the role of apatinib that targets vascular endothelial growth factor receptor-2 (VEGFR2) in ICC. <b><i>Methods:</i></b> MTT assays, cell scratch assays, and tube formation assays were used to assess the effect of apatinib on human ICC cell line (HuCCT-1) and RBE cells proliferation, migration, and angiogenic capacity, respectively. Expression of vascular endothelial growth factor (VEGF), VEGFR2, signal transducer and activator of transcription factor 3 (STAT3), pSTAT3, and hypoxia inducible factor 1 subunit alpha (HIF-1α) pathway proteins was assessed using Western blotting and mRNA expression analysis in HuCCT-1 was performed using RT-qPCR assays. The pcDNA 3.1(-)-VEGFR2 and pcDNA 3.1(-)-HIF-1α were transfected into HuCCT-1 and RBE cells using Lipofectamine 2,000 to obtain overexpressed HuCCT-1 and RBE cells. <b><i>Results:</i></b> We found that apatinib-inhibited proliferation, migration, and angiogenesis of HuCCT-1 and RBE cells in vitro in a dose-dependent manner. We also proved that apatinib effectively inhibits angiogenesis in tumor cells by blocking the expression of VEGF and VEGFR2 in these cells. In addition, we demonstrated that apatinib regulates the expression of STAT3 phosphorylation by inhibiting VEGFR2. Finally, we showed that apatinib regulates ICC angiogenesis and HIF-1α/VEGF expression via STAT3. <b><i>Conclusions:</i></b> Based on the above findings, we conclude that apatinib inhibits HuCCT-1 and RBE cell proliferation, migration, and tumor angiogenesis by inhibiting the VEGFR2/STAT3/HIF-1α axis signaling pathway. Apatinib can be a promising drug for ICC-targeted molecular therapy.

Expanding the molecular taxonomy of NUT midline carcinomas with multiomic analyses.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e21008-e21008
Author(s):  
Henry G. Kaplan ◽  
Alex Barrett ◽  
Jiaxin Niu ◽  
Somasundaram Subramaniam ◽  
Maria Matsangou
Keyword(s):  
Dna Sequencing ◽  
Rna Sequencing ◽  
Molecular Taxonomy ◽  
Fusion Partner ◽  
Targeted Proteomics ◽  
Single Nucleotide Variants ◽  
Proteomic Data ◽  
Spatial Profiling ◽  
Tumor Mutational Burden ◽  
Custom Panel

e21008 Background: NUT midline carcinoma (NMC) is an aggressive squamous cell carcinoma molecularly defined by a chromosomal rearrangement of nuclear protein in testis (NUTM1) with bromodomain-containing protein 3 or 4 (BRD3/4). While NMCs are characterized by this rare canonical gene rearrangement little is known about the transcriptome and proteosome of this rare disease. As such, we set out to comprehensively characterize five NMC cases in which we attained targeted DNA sequencing, full-transcriptome RNA sequencing, and targeted proteomics. We further examine and integrate these results in order to better understand the relationship between gene expression and protein abundance within the context of NMC. Methods: All cases were analyzed for genomic and transcriptomic alterations against a custom panel via the Tempus xT tissue biopsy assay (DNA sequencing of 648 genes in tumor and matched normal samples at 500x depth and full-transcriptome RNA sequencing) for germline and/or somatic mutations. The xT assay detects single nucleotide variants, specific insertion/deletions, amplifications and gene fusions, as well as tumor mutational burden (TMB) and microsatellite instability (MSI) status. Proteomic data were obtained utilizing digital spatial profiling through Nanostring immune, MAPK and PI3/AKT, and pan tumor nCounter GeoMix panels. Results: Clinical characteristics, histology, and genomic/proteomic alterations for 5 NMC cases are presented. Cases were defined by pathological assessment and the identification of the canonical NUTM1 fusion, further broken down by fusion partner with three patients having NUTM1-BRD4 fusions, one NUT-BRD3, and one NUT-ZMYND8. TMBs ranged for 0.8-.6 mutations/megabases (n=5). All patients were MSI stable (5/5). Of three patients with available PD-L1 IHC result, one had elevated PD-L1 tumor staining at 70%. Results will be presented from full-transcriptome RNA expression analysis indicating overexpression of BRAF, MYC, mTOR, and EGFR, among others. Targeted proteomics were performed to assess relative abundance at the protein level (results to be presented). Clinical follow up for the five patients revealed that two have survived beyond 7 months. A lung primary patient treated with surgical resection and post op radiation (XRT) is NED at 63 months. A sinus primary patient is NED at 16 months after a partial response (PR) to taxotere/5FU/Cisplatin followed by resection and XRT/cis platin. One patient had a brief PR from ifosphamide/etoposide/vorinostat. One patient's tumor grew through XRT/cisplatin. Conclusions: Multi-omic analysis has the potential to further elucidate the mechanisms of tumor growth in NMC and identify new targets for the treatment of this aggressive and poor prognosis disease.

RNA sequencing of intrahepatic cholangiocarcinoma reveals two prognostic subclasses

2017 ◽  
Vol 66 (1) ◽  
pp. S460
Author(s):  
K.S. Ahn ◽  
D. O’Brien ◽  
Y.N. Kang ◽  
Y.H. Kim ◽  
T.S. Kim ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Intrahepatic Cholangiocarcinoma

The murine transcriptome reveals global aging nodes with organ-specific phase and amplitude

2019 ◽  
Author(s):  
Nicholas Schaum ◽  
Benoit Lehallier ◽  
Oliver Hahn ◽  
Shayan Hosseinzadeh ◽  
Song E. Lee ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Mitochondrial Function ◽  
Cell Activation ◽  
Plasma Cells ◽  
Immune Cell ◽  
Age Of Onset ◽  
Systemic Circulation ◽  
Protein Levels ◽  
Organ Specific ◽  
Highly Correlated

Aging is the single greatest cause of disease and death worldwide, and so understanding the associated processes could vastly improve quality of life. While the field has identified major categories of aging damage such as altered intercellular communication, loss of proteostasis, and eroded mitochondrial function1, these deleterious processes interact with extraordinary complexity within and between organs. Yet, a comprehensive analysis of aging dynamics organism-wide is lacking. Here we performed RNA-sequencing of 17 organs and plasma proteomics at 10 ages across the mouse lifespan. We uncover previously unknown linear and non-linear expression shifts during aging, which cluster in strikingly consistent trajectory groups with coherent biological functions, including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Remarkably, these gene sets are expressed similarly across tissues, differing merely in age of onset and amplitude. Especially pronounced is widespread immune cell activation, detectable first in white adipose depots in middle age. Single-cell RNA-sequencing confirms the accumulation of adipose T and B cells, including immunoglobulin J-expressing plasma cells, which also accrue concurrently across diverse organs. Finally, we show how expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to aging of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of aging, thereby providing a foundation to track systemic sources of declining health at old age.

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