RNA sequencing effectively identifies gene fusions undetected by DNA sequencing in lung adenocarcinomas.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3052-3052
Author(s):  
Ruiying Zhao ◽  
Yuchen Han ◽  
Chan Xiang ◽  
Shengnan Chen ◽  
Jikai Zhao ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Rna Sequencing ◽  
Genomic Rearrangements ◽  
Driver Gene ◽  
Functional Domain ◽  
Gene Fusions ◽  
Rna Seq ◽  
Probe Coverage ◽  
Therapeutic Decision Making ◽  
Lung Adenocarcinomas

3052 Background: Next-generation sequencing of DNA, which can provide valid information for clinical therapeutic decision-making, has been widely used in the management of lung cancer especially adenocarcinoma. However, due to its technical limitations for detecting certain alterations such as gene rearrangement, the DNA-based sequencing (DNA-seq) may miss the actionable alteration in some cases, who would have benefited from targeted therapy. The study aimed to evaluate the capability of RNA sequencing (RNA-seq) in identifying DNA-seq undetectable gene alterations in lung adenocarcinomas. Methods: A total of 219 lung adenocarcinomas, which had no driver alteration detected by DNA-seq (OncoScreen Plus, Burning Rock Biotech) and had a max AF ≥5%, underwent capture-based RNA-seq using a custom panel (OncoRNA, Burning Rock Biotech) spanning full transcripts of 115 genes commonly involved in cancer genomic rearrangements. Furthermore, an independent cohort of 100 DNA-seq driver–negative lung adenocarcinomas were also subjected to RNA-seq with the same panel. Results: In the discovery cohort, 166/219 samples (75.8%) generated qualified RNA-seq data for subsequent analyses. RNA-seq identified 44 previously undetected alterations (26.5%), including 40 gene fusions (24.1%), 1 MET exon14 skipping variant ( METex14, 0.6%) and 3 other alternative splicing variants (1.8%). Among them, 14 (8.4%) were potential actionable alterations, consisting of METex14 and in-frame fusions containing functional domain of the driver gene (4 ROS1 fusions, 3 BRAF fusions, 2 NRG1 fusions, 2 EGFR fusions, 1 ALK fusion and 1 MET fusion). In the validation cohort, 69/100 samples (69.0%) generated qualified data. RNA-seq identified 22 DNA-seq undetected alterations (31.9%), with 7 of them being potential actionable fusions (10.1%). ROS1 fusion remained as the most common actionable alteration (n = 3), followed by ALK fusion (n = 2), EGFR fusion (n = 1) and MET fusion (n = 1). Further analyses of the two datasets revealed that lacking sufficient coverage spanning the rearrangement breakpoint in the DNA-seq panel mainly accounted for the failure of DNA-seq on detecting these fusions. This can be improved by increasing the corresponding probe coverage in the DNA-seq panel. In addition, complex genomic rearrangement at DNA level and the presence of repetitive sequence in the intronic region spanning or adjacent to the breakpoint might lead to missed calling of canonical fusions by DNA-seq. Conclusions: Targeted RNA-seq can effectively identify genomic rearrangements that are undetectable by DNA-seq and provide lung adenocarcinoma patients with more opportunities for targeted therapy. Therefore, it should be recommended for all patients, in whom DNA-seq fails to detect driver alteration.

scholarly journals Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors

2018 ◽  
Vol 20 (4) ◽  
pp. 495-511 ◽  
Author(s):  
Jennifer L. Winters ◽  
Jaime I. Davila ◽  
Amber M. McDonald ◽  
Asha A. Nair ◽  
Numrah Fadra ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Gene Fusions ◽  
Rna Seq

scholarly journals High Frequency of PDGFRA and MUC Family Gene Mutations in Diffuse Hemispheric Glioma, H3 G34-mutant: A Glimmer of Hope?

2021 ◽  
Author(s):  
Wanming Hu ◽  
Hao Duan ◽  
Sheng Zhong ◽  
Jing Zeng ◽  
Yonggao Mou
Keyword(s):  
Targeted Therapy ◽  
Rna Sequencing ◽  
High Frequency ◽  
High Grade Glioma ◽  
Chinese Patients ◽  
Molecular Characteristics ◽  
High Grade ◽  
Rna Seq ◽  
Immune Infiltration ◽  
Genome Atlas

Abstract BackgroundDiffuse hemispheric glioma H3 G34-mutant (G34-DHG) is a new type of pediatric-type diffuse high-grade glioma in the fifth edition of the WHO Classification of Tumors of the Central Nervous System. The current treatment for G34-DHG involves a combination of surgery and conventional radiotherapy or chemotherapy; however, the therapeutic efficacy of this approach is not satisfactory. In recent years, molecular targeted therapy and immunotherapy have achieved significant benefits in a variety of tumors. In-depth understanding of molecular changes and immune infiltration in G34-DHGs will help to establish personalized tumor treatment strategies. Here, we report the clinicopathological, molecular and immune infiltration characteristics of G34-DHG cases from our center along with cases from the HERBY Trial and the Chinese Glioma Genome Atlas database (CGGA). MethodsHematoxylin-eosin (HE) and immunohistochemistry (IHC) staining were used to present the clinicopathological characteristics of 10 Chinese G34-DHG patients treated at our institution. To address the molecular characteristics of G34-DHG, we performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) analyses of 5 patients from our center and 3 Chinese patients from the Chinese Glioma Genome Atlas (CGGA) database. Additionally, 7 European G34-DHG patients from the HERBY Trail were also subjected to analyses, with 7 cases of WES data and 2 cases of RNA-seq data.ResultsWES showed a high frequency of PDGFRA mutation in G34-DHGs (12/15). We further identified frequent mutations in MUC family genes in G34-DHGs, including MUC16 (8/15) and MUC17 (8/15). Although no statistical difference was found, PDGFRA mutation tended to be an indicator for worse prognosis whereas MUC16/MUC17 mutation indicated a favorable prognosis in G34-DHGs. RNA sequencing results revealed that most G34-DHG are considered to be immune cold tumors. However, one patient in our cohort with MUC16 mutation showed significant immune infiltration, and the total overall survival of this patient reached 75 months. ConclusionsOur results demonstrate that G34-DHG is a new high-grade glioma with high frequency of PDGFRA and MUC gene family mutations. PDGFRA may serve as an indicator of poor prognosis and an effective therapeutic target. Moreover, MUC16 tends to be a favorable prognostic factor and indicates high immune infiltration in certain patients, and these findings may provide a new direction for targeted therapy and immunotherapy of patients with G34-DHGs.

scholarly journals MA 06.12 Genomic Rearrangements of Lung Adenocarcinomas with Fusion Driver Gene

2017 ◽  
Vol 12 (11) ◽  
pp. S1824
Author(s):  
S. Park ◽  
J. Lee ◽  
J. Lee ◽  
K. Yi ◽  
K.H. Kang ◽  
...  
Keyword(s):  
Genomic Rearrangements ◽  
Driver Gene ◽  
Lung Adenocarcinomas

scholarly journals The application of RNA sequencing for the diagnosis and genomic classification of pediatric acute lymphoblastic leukemia

2020 ◽  
Vol 4 (5) ◽  
pp. 930-942 ◽  
Author(s):  
Lauren M. Brown ◽  
Andrew Lonsdale ◽  
Andrea Zhu ◽  
Nadia M. Davidson ◽  
Breon Schmidt ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Rna Sequencing ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Standard Of Care ◽  
Clinical Service ◽  
Molecular Diagnostic ◽  
Gene Fusions ◽  
Rna Seq

Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, and implementation of risk-adapted therapy has been instrumental in the dramatic improvements in clinical outcomes. A key to risk-adapted therapies includes the identification of genomic features of individual tumors, including chromosome number (for hyper- and hypodiploidy) and gene fusions, notably ETV6-RUNX1, TCF3-PBX1, and BCR-ABL1 in B-cell ALL (B-ALL). RNA-sequencing (RNA-seq) of large ALL cohorts has expanded the number of recurrent gene fusions recognized as drivers in ALL, and identification of these new entities will contribute to refining ALL risk stratification. We used RNA-seq on 126 ALL patients from our clinical service to test the utility of including RNA-seq in standard-of-care diagnostic pipelines to detect gene rearrangements and IKZF1 deletions. RNA-seq identified 86% of rearrangements detected by standard-of-care diagnostics. KMT2A (MLL) rearrangements, although usually identified, were the most commonly missed by RNA-seq as a result of low expression. RNA-seq identified rearrangements that were not detected by standard-of-care testing in 9 patients. These were found in patients who were not classifiable using standard molecular assessment. We developed an approach to detect the most common IKZF1 deletion from RNA-seq data and validated this using an RQ-PCR assay. We applied an expression classifier to identify Philadelphia chromosome–like B-ALL patients. T-ALL proved a rich source of novel gene fusions, which have clinical implications or provide insights into disease biology. Our experience shows that RNA-seq can be implemented within an individual clinical service to enhance the current molecular diagnostic risk classification of ALL.

scholarly journals Platform study of genotyping-guided precision medicine for rare solid tumours: a study protocol for a phase II, non-randomised, 18-month, open-label, multiarm, single-centre clinical trial testing the safety and efficacy of multiple Chinese-approved targeted drugs and PD-1 inhibitors in the treatment of metastatic rare tumours

BMJ Open ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. e044543
Author(s):  
Shuhang Wang ◽  
Hui-Yao Huang ◽  
Dawei Wu ◽  
Hong Fang ◽  
Jianming Ying ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Targeted Therapy ◽  
Single Agent ◽  
Solid Tumours ◽  
Targeted Drugs ◽  
Gene Fusions ◽  
Single Centre ◽  
Open Label ◽  
Safety And Efficacy ◽  
Rare Tumours

IntroductionLimited clinical studies have been conducted on rare solid tumours, and there are few guidelines on the diagnosis and treatment, including experiences with targeted therapy and immunotherapy, of rare solid tumours in China, resulting in limited treatment options and poor outcomes. This study first proposes a definition of rare tumours and is designed to test the preliminary efficacy of targeted and immunotherapy drugs for the treatment of rare tumours.Methods and analysisThis is a phase II, open-label, non-randomised, multiarm, single-centre clinical trial in patients with advanced rare solid tumours who failed standard treatment; the study aims to evaluate the safety and efficacy of targeted drugs in patients with advanced rare solid tumours with corresponding actionable alterations, as well as the safety and efficacy of immune checkpoint (programmed death receptor inhibitor 1, PD-1) inhibitors in patients with advanced rare solid tumours without actionable alterations. Patients with advanced rare tumours who fail standardised treatment and carry actionable alterations (Epidermal growth factor receptor (EGFR) mutations, ALK gene fusions, ROS-1 gene fusions, C-MET gene amplifications/mutations, BRAF mutations, CDKN2A mutations, BRCA1/2 mutations, HER-2 mutations/overexpressions/amplifications or C-KIT mutations) will be enrolled in the targeted therapy arm and be given the corresponding targeted drugs. Patients without actionable alterations will be enrolled in the PD-1 inhibitor arm and be treated with sintilimab. After the patients treated with vemurafenib, niraparib and palbociclib acquire resistance, they will receive combination treatment with sintilimab or atezolizumab. With the use of Simon’s two-stage Minimax design, and the sample size was estimated to be 770. The primary endpoint of this study is the objective response rate. The secondary endpoints are progression-free survival in the targeted treatment group and single-agent immunotherapy group; the duration of response in the targeted therapy and single-agent immunotherapy groups; durable clinical benefit in the single-agent immunotherapy group; and the incidence of adverse events.Ethics and disseminationEthics approval was obtained from the Chinese Academy of Medical Sciences (ID: 20/132-2328). The results from this study will be actively disseminated through manuscript publications and conference presentations.Trial registration numbersNCT04423185; ChiCTR2000039310.

scholarly journals Identification of NTRK gene fusions in lung adenocarcinomas in the Chinese population

2021 ◽  
Author(s):  
Ruiying Zhao ◽  
Feng Yao ◽  
Chan Xiang ◽  
Jikai Zhao ◽  
Zhanxian Shang ◽  
...  
Keyword(s):  
Chinese Population ◽  
Gene Fusions ◽  
Lung Adenocarcinomas

scholarly journals Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming

2021 ◽  
Author(s):  
Ping Huang ◽  
Jieying Zhu ◽  
Yu Liu ◽  
Guihuan Liu ◽  
Ran Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Rna Sequencing ◽  
Human Urine ◽  
Embryonic Stem ◽  
Rna Seq ◽  
Reprogramming Efficiency ◽  
Yamanaka Factors ◽  
Urine Cells

Abstract Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. Methods The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. Results ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. Conclusions In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease. Graphical abstract

scholarly journals Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Milda Mickutė ◽  
Kotryna Kvederavičiūtė ◽  
Aleksandr Osipenko ◽  
Raminta Mineikaitė ◽  
Saulius Klimašauskas ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Regulatory Networks ◽  
Library Preparation ◽  
Rna Seq ◽  
Basic Principles ◽  
Cofactor Binding ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

scholarly journals Mechanism of protective effect of xuan-bai-cheng-qi decoction on LPS-induced acute lung injury based on an integrated network pharmacology and RNA-sequencing approach

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Huahe Zhu ◽  
Shun Wang ◽  
Cong Shan ◽  
Xiaoqian Li ◽  
Bo Tan ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Rna Sequencing ◽  
Target Genes ◽  
Mammalian Target Of Rapamycin ◽  
Network Pharmacology ◽  
Protective Mechanism ◽  
Rna Seq ◽  
Active Components ◽  
Integrated Network

AbstractXuan-bai-cheng-qi decoction (XCD), a traditional Chinese medicine (TCM) prescription, has been widely used to treat a variety of respiratory diseases in China, especially to seriously infectious diseases such as acute lung injury (ALI). Due to the complexity of the chemical constituent, however, the underlying pharmacological mechanism of action of XCD is still unclear. To explore its protective mechanism on ALI, firstly, a network pharmacology experiment was conducted to construct a component-target network of XCD, which identified 46 active components and 280 predicted target genes. Then, RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) between ALI model rats treated with and without XCD and 753 DEGs were found. By overlapping the target genes identified using network pharmacology and DEGs using RNA-seq, and subsequent protein–protein interaction (PPI) network analysis, 6 kernel targets such as vascular epidermal growth factor (VEGF), mammalian target of rapamycin (mTOR), AKT1, hypoxia-inducible factor-1α (HIF-1α), and phosphoinositide 3-kinase (PI3K) and gene of phosphate and tension homology deleted on chromsome ten (PTEN) were screened out to be closely relevant to ALI treatment. Verification experiments in the LPS-induced ALI model rats showed that XCD could alleviate lung tissue pathological injury through attenuating proinflammatory cytokines release such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Meanwhile, both the mRNA and protein expression levels of PI3K, mTOR, HIF-1α, and VEGF in the lung tissues were down-regulated with XCD treatment. Therefore, the regulations of XCD on PI3K/mTOR/HIF-1α/VEGF signaling pathway was probably a crucial mechanism involved in the protective mechanism of XCD on ALI treatment.

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