Detection of Multiple Types of Cancer Driver Mutations Us-ing Targeted RNA Sequencing in NSCLC

Currently, DNA and RNA are used separately to capture different types of gene mutations. DNA is commonly used for the detection of SNVs, indels and CNVs; RNA is used for analysis of gene fusion and gene expression. To perform both DNA sequencing (DNA-seq) and RNA-seq, material is divided into two copies, and two different proce-dures are required for sequencing. Due to overconsumption of samples and experimental process complexity, it is necessary to create an experimental method capable of analyzing SNVs, indels, fusions and expression. We developed an RNA-based hybridization capture panel targeting actionable driver oncogenes in solid tumors and corresponding sample preparation and bioinformatics workflows. Analytical validation with an RNA standard reference containing 16 known fusion mutations and 6 SNV mutations demonstrated a detection specificity of 100.0% [95% CI 88.7%~100.0%] for SNVs and 100.0% [95% CI 95.4%~100.0%] for fusions. The targeted RNA panel achieved a 0.73-2.63 copies/ng RNA lower limit of detection (LOD) for SNVs and 0.21-6.48 copies/ng RNA for fusions. Gene expression analysis revealed a correlation greater than 0.9 across all 15 cancer-related genes between the RNA-seq re-sults and targeted RNA panel. Among 1253 NSCLC FFPE tumor samples, multiple mutation types were called from DNA- and RNA-seq data and compared between the two assays. The DNA panel detected 103 fusions and 21 METex14 skipping events; 124 fusions and 26 METex14 skipping events were detected by the target RNA panel; 21 fusions and 4 METex14 skipping events were only detected by the target RNA panel. Among the 173 NSCLC samples negative for targetable mutations by DNA-seq, 15 (15/173, 8.67%) showed targetable gene fusions that may change clinical decisions with RNA-seq. In total, 226 tier I and tier II missense variants for NSCLC were analyzed at ge-nomic (DNA-seq) and transcriptomic (RNA-seq) levels. The positive percent agreement (PPA) was 97.8%, and the positive predictive value (PPV) was 98.6%. Interestingly, var-iant allele frequencies were generally higher at the RNA level than at the DNA level, suggesting relatively dominant expression of mutant alleles. PPA was 97.6% and PPV 99.38% for EGFR 19del and 20ins variants. We also explored the relationship of RNA expression with gene copy number and protein expression. The RPKM of EGFR transcripts assessed by the RNA panel showed a linear relationship with copy number quantified by the DNA panel, with an R of 0.8 in 1253 samples. In contrast, MET gene expression is regulated in a more complex manner. In IHC analysis, all 3+ samples exhibited higher RPKM levels; IHC level of 2+ and below showed lower RNA expression. Parallel DNA- and RNA-seq and systematic analysis demonstrated the accuracy and robustness of the RNA sequencing panel in identifying multiple types of variants for cancer therapy.

Download Full-text

Targeted DNA and RNA sequencing of paired urothelial and squamous bladder cancers to reveal discordant genomic and transcriptomic events and unique therapeutic opportunities.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.296 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. 296-296 ◽

Cited By ~ 2

Author(s):

Daniel H. Hovelson ◽

Lorena Lazo De La Vega ◽

Andrew McDaniel ◽

Aaron Udager ◽

Rohit Mehra ◽

...

Keyword(s):

Bladder Cancer ◽

Rna Sequencing ◽

Copy Number ◽

Clinical Treatment ◽

Gene Copy ◽

Molecular Heterogeneity ◽

Copy Number Alterations ◽

Dna And Rna ◽

Divergent Differentiation ◽

High Level

296 Background: Expression-based molecular subtypes thought to be intrinsic in bladder cancer have been widely reported, carrying important potential clinical treatment implications. Histologically, bladder cancers are also heterogeneous diseases, with a large portion of urothelial carcinomas exhibiting divergent differentiation. Previous subtyping efforts have been carried out using predominantly fresh frozen tissue samples, potentially obscuring this known differentiation heterogeneity. Methods: Here we performed targeted multiplexed, amplicon-based DNA and RNA sequencing on 100 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples (including 12 paired urothelial / squamous lesions). High-confidence somatic point mutations, short insertions/deletions (indels), and copy number alterations were detected using the DNA component of the Oncomine Comprehensive Assay (OCP). Targeted RNA sequencing was carried out using a custom Ampliseq panel comprised of 8 housekeeping genes and 103 target genes assessing major transcriptional programs as identified from publically available data. Results: By DNA analysis, we observe frequent TP53 (35%) and activating hotspot PIK3CA (23%) somatic mutations across the cohort, as well as targetable high-level (log-2 copy number ratio > = 1.5) focal amplifications of ERBB2 (3%) or EGFR (3%) in a subset of samples. We report a novel approach for detecting sub-gene copy-number alterations, and confirm several detectable multi-exon losses using whole transcriptome RNA sequencing. Pairing targeted RNA expression analysis with DNA-based alterations, we show high level expression of EGFR and ERBB2 in focally-amplified samples. Most importantly, we show that despite identical prioritized somatic genomic alterations, we observe divergent expression-based profiles in 3 of 12 (25%) paired urothelial and squamous samples. Conclusions: Taken together, these results highlight the importance of molecular heterogeneity in bladder cancer and suggest important considerations for using existing expression-based clustering approaches to guide clinical treatment decisions.

Download Full-text

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

BMC Biology ◽

10.1186/s12915-021-01053-w ◽

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.

Download Full-text

Insulin-like Growth Factor Receptor 1 (IGF1R) Gene Copy Number Is Associated With Survival in Operable Non–Small-Cell Lung Cancer: A Comparison Between IGF1R Fluorescent In Situ Hybridization, Protein Expression, and mRNA Expression

Journal of Clinical Oncology ◽

10.1200/jco.2009.24.6611 ◽

2010 ◽

Vol 28 (13) ◽

pp. 2174-2180 ◽

Cited By ~ 89

Author(s):

Rafal Dziadziuszko ◽

Daniel T. Merrick ◽

Samir E. Witta ◽

Adelita D. Mendoza ◽

Barbara Szostakiewicz ◽

...

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Protein Expression ◽

Squamous Cell ◽

Copy Number ◽

Gene Copy Number ◽

Growth Factor Receptor ◽

Gene Copy ◽

Cell Versus

PurposeThe purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non–small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis.Patients and MethodsOne hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181).ResultsIGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis.ConclusionIGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

Download Full-text

Drug Repositioning Based on the Reversal of Gene Expression Signatures Identifies TOP2A as a Therapeutic Target for Rectal Cancer

Cancers ◽

10.3390/cancers13215492 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5492

Author(s):

Robson Francisco Carvalho ◽

Luisa Matos do Canto ◽

Sarah Santiloni Cury ◽

Torben Frøstrup Hansen ◽

Lars Henrik Jensen ◽

...

Keyword(s):

Gene Expression ◽

Rectal Cancer ◽

Cancer Patients ◽

Copy Number ◽

Topoisomerase Ii ◽

Drug Repositioning ◽

Gene Copy ◽

Common Disease ◽

Topoisomerase Ii Inhibitors ◽

Gene Expression Signatures

Rectal cancer is a common disease with high mortality rates and limited therapeutic options. Here we combined the gene expression signatures of rectal cancer patients with the reverse drug-induced gene-expression profiles to identify drug repositioning candidates for cancer therapy. Among the predicted repurposable drugs, topoisomerase II inhibitors (doxorubicin, teniposide, idarubicin, mitoxantrone, and epirubicin) presented a high potential to reverse rectal cancer gene expression signatures. We showed that these drugs effectively reduced the growth of colorectal cancer cell lines closely representing rectal cancer signatures. We also found a clear correlation between topoisomerase 2A (TOP2A) gene copy number or expression levels with the sensitivity to topoisomerase II inhibitors. Furthermore, CRISPR-Cas9 and shRNA screenings confirmed that loss-of-function of the TOP2A has the highest efficacy in reducing cellular proliferation. Finally, we observed significant TOP2A copy number gains and increased expression in independent cohorts of rectal cancer patients. These findings can be translated into clinical practice to evaluate TOP2A status for targeted and personalized therapies based on topoisomerase II inhibitors in rectal cancer patients.

Download Full-text

Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing

PeerJ ◽

10.7717/peerj.11875 ◽

2021 ◽

Vol 9 ◽

pp. e11875

Author(s):

Tomoko Matsuda

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Time Course ◽

Sequence Data ◽

Specific Gene ◽

Time Interval ◽

Short Time Interval ◽

Rna Seq ◽

Lysis Buffer ◽

Rna Protection

Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database.

Download Full-text

Frequent EPAS1/HIF2α exons 9 and 12 mutations in non-familial pheochromocytoma

Endocrine Related Cancer ◽

10.1530/erc-13-0384 ◽

2014 ◽

Vol 21 (3) ◽

pp. 495-504 ◽

Cited By ~ 39

Author(s):

Jenny Welander ◽

Adam Andreasson ◽

Michael Brauckhoff ◽

Martin Bäckdahl ◽

Catharina Larsson ◽

...

Keyword(s):

Gene Expression ◽

Copy Number ◽

Gene Expression Pattern ◽

Exon 2 ◽

Dna And Rna ◽

Familial Pheochromocytoma ◽

Predisposing Genes ◽

Adrenal Pheochromocytoma ◽

New Gene ◽

Sporadic Pheochromocytoma

Pheochromocytomas are neuroendocrine tumors arising from the adrenal medulla. While heritable mutations are frequently described, less is known about the genetics of sporadic pheochromocytoma. Mutations in genes involved in the cellular hypoxia response have been identified in tumors, and recentlyEPAS1, encoding HIF2α, has been revealed to be a new gene involved in the pathogenesis of pheochromocytoma and abdominal paraganglioma. The aim of this study was to further characterizeEPAS1alterations in non-familial pheochromocytomas. Tumor DNA from 42 adrenal pheochromocytoma cases with apparently sporadic presentation, without known hereditary mutations in predisposing genes, were analyzed for mutations inEPAS1by sequencing of exons 9 and 12, which contain the two hydroxylation sites involved in HIF2α degradation, and also exon 2. In addition, the copy number at theEPAS1locus as well as transcriptome-wide gene expression were studied by DNA and RNA microarray analyses, respectively. We identified six missenseEPAS1mutations, three in exon 9 and three in exon 12, in five of 42 pheochromocytomas (12%). The mutations were both somatic and constitutional, and had no overlap in 11 cases (26%) with somatic mutations inNF1orRET. One sample had two differentEPAS1mutations, shown by cloning to occur incis, possibly indicating a novel mechanism of HIF2α stabilization through inactivation of both hydroxylation sites. One of the tumors with anEPAS1mutation also had a gain in DNA copy number at theEPAS1locus. AllEPAS1-mutated tumors displayed a pseudo-hypoxic gene expression pattern, indicating an oncogenic role of the identified mutations.

Download Full-text

Shared DNA-based copy number with divided methylation changes differentiate and clinically associate early stage pancreatic cancer tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.3_suppl.434 ◽

2021 ◽

Vol 39 (3_suppl) ◽

pp. 434-434

Author(s):

Eva Chao ◽

Kyaw Lwin Aung ◽

Qi Xu ◽

William H. Matsui ◽

Jeanne Kowalski

Keyword(s):

Gene Expression ◽

Pancreatic Cancer ◽

Overall Survival ◽

Clinical Outcomes ◽

Copy Number ◽

Cancer Biology ◽

Early Stage ◽

Molecular Taxonomy ◽

Unmet Need ◽

Gene Copy

434 Background: There is no known molecular taxonomy of pancreatic cancer that can guide therapeutic strategies. Understanding the fundamental molecular mechanism underlying pancreatic cancer biology remains an unmet need. We explore the extent to which combinations of DNA-based molecular changes in copy number (CN) and methylation separate early stage PAAD tumors and associated with survival outcomes. Methods: We performed genome-wide combined cluster analyses on DNA-based CN and methylation changes using TCGA data. We examined cluster associations with clinical outcomes by comparing in months (mos), Kaplan--Meier estimated overall survival (OS) and disease-free interval (DFI) using a log-rank test. We performed additional comparisons among CN-Methylation derived clusters with respect to PAAD phenotypes. Results: Using 78 early stage pancreatic cancer tumors from TCGA with CN, methylation and clinical outcomes data, we identified two patient clusters with distinct gene copy number signatures that when combined with three methylation signatures, resulted in three additional clusters. Thus, the same gene CN signature, when combined with different methylation signatures, further differentiated tumors into sub-clusters with varying levels of associations with clinical outcome. Among them, analogous to current gene-expression based subtypes, we also identified an immune-rich subtype that was associated with improved overall survival (n=21, median OS=16mos; DFI=16mos), and an extracellular matrix (ECM)-rich with worse survival (n=19, median OS=12mos; DFI=8mos). Unlike previous expression subtypes, we identified another metabolic-enriched subtype with the same worse median OS and DFI, differentiated by methylation with the ECM-rich subtype. The improved OS cluster had a signature of CN neutral and increased methylation, while the poor cluster had a signature of CN gains and increased methylation among a set of genes distinct from the improved cluster. No significant differences in age, site, microsatellite instability and KRAS status among clusters were noted. Notably, in a multivariable model that included gene expression-based subtypes, only our DNA-level subtypes remained significantly associated with overall survival. Conclusions: While RNA-level changes often display large variations, DNA-level changes are more robust. Copy number changes appear to separate tumors into poor and improved prognosis clusters, while methylation appears to inform on the further separation of poor prognosis into various levels. A DNA-based molecular taxonomy for early stage pancreatic cancer could prove invaluable when used in combination with methylation-based circulating tumor DNA assays for clinical trial monitoring of tumor responses.

Download Full-text

Integrated genomic and transcriptomic analysis revealed mutation patterns of de-differentiated liposarcoma and leiomyosarcoma

BMC Cancer ◽

10.1186/s12885-020-07456-2 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Wenshuai Liu ◽

Hanxing Tong ◽

Chenlu Zhang ◽

Rongyuan Zhuang ◽

He Guo ◽

...

Keyword(s):

Gene Expression ◽

Sanger Sequencing ◽

Immune Cell ◽

Gene Copy Number ◽

Gene Copy ◽

Rna Seq ◽

Loss Of Function ◽

Rt Pcr ◽

The Difference ◽

Histologic Types

Abstract Background Treating patients with advanced sarcomas is challenging due to great histologic diversity among its subtypes. Leiomyosarcoma (LMS) and de-differentiated liposarcoma (DDLPS) are two common and aggressive subtypes of soft tissue sarcoma (STS). They differ significantly in histology and clinical behaviors. However, the molecular driving force behind the difference is unclear. Methods We collected 20 LMS and 12 DDLPS samples and performed whole exome sequencing (WES) to obtain their somatic mutation profiles. We also performed RNA-Seq to analyze the transcriptomes of 8 each of the LMS and DDLPS samples and obtained information about differential gene expression, pathway enrichment, immune cell infiltration in tumor microenvironment, and chromosomal rearrangement including gene fusions. Selected gene fusion events from the RNA-seq prediction were checked by RT-PCR in tandem with Sanger sequencing. Results We detected loss of function mutation and deletion of tumor suppressors mostly in LMS, and oncogene amplification mostly in DDLPS. A focal amplification affecting chromosome 12q13–15 region which encodes MDM2, CDK4 and HMGA2 is notable in DDLPS. Mutations in TP53, ATRX, PTEN, and RB1 are identified in LMS but not DDLPS, while mutation of HERC2 is only identified in DDLPS but not LMS. RNA-seq revealed overexpression of MDM2, CDK4 and HMGA2 in DDLPS and down-regulation of TP53 and RB1 in LMS. It also detected more fusion events in DDLPS than LMS (4.5 vs. 1, p = 0.0195), and the ones involving chromosome 12 in DDLPS stand out. RT-PCR and Sanger sequencing verified the majority of the fusion events in DDLPS but only one event in LMS selected to be tested. The tumor microenvironmental signatures are highly correlated with histologic types. DDLPS has more endothelial cells and fibroblasts content than LMS. Conclusions Our analysis revealed different recurrent genetic variations in LMS and DDLPS including simultaneous upregulation of gene expression and gene copy number amplification of MDM2 and CDK4. Up-regulation of tumor related genes is favored in DDLPS, while loss of suppressor function is favored in LMS. DDLPS harbors more frequent fusion events which can generate neoepitopes and potentially targeted by personalized immune treatment.

Download Full-text

141 A nutrigenomics approach using RNA sequencing technology to study nutrient-gene interactions in agricultural animals

Journal of Animal Science ◽

10.1093/jas/skz258.275 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 135-135

Author(s):

Shengfa F Liao ◽

Shamimul Hasan ◽

Jean M Feugang

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Gene Interactions ◽

Animal Nutrition ◽

Rna Seq ◽

Bottom Line ◽

Holistic View ◽

Nutrition Research ◽

Dietary Nutrients ◽

A Genome

Abstract Animal life essentially is a set of gene expression processes. Thorough understanding of these processes driven by dietary nutrients and other environmental factors can be regarded as a bottom line of modern advanced animal nutrition research for improving animal growth, development, health, production, and reproduction performance. Nutrigenomics, a genome-wide approach using the knowledge and techniques obtained from the disciplines of genomics (including transcriptomics) and molecular biology, is to study the effects of dietary nutrients on cellular gene expression, cellular metabolic responses and, ultimately, the phenotypic changes of a living organism. Transcriptomics can be applied to investigate animal tissue transcriptome at a defined physiological or nutritional state, which provides a holistic view of the intracellular expression of RNA, especially mRNA. As a novel, promising transcriptomics approach, RNA sequencing (RNA-Seq) technology can monitor all-gene expressions simultaneously in response to dietary intervention. The principle and history of RNA-Seq technology will be briefly reviewed, and the three principal steps of this methodology, including the laboratory analysis of tissue samples, the bioinformatics analysis of the generated sequence data, and the subsequent biological interpretation of the data, will be described. The application of RNA-Seq technology in different areas of animal nutrition research, which include maternal nutrition, feeding strategy and gut microbiota, will be summarized. Lastly, the application of RNA-Seq technology in swine science and nutrition research will also be discussed. In short, to further improve animal feeding or production efficiency, RNA-Seq technology holds a great potential to be employed to explore the new insights into better understanding of nutrient-gene interactions in agricultural animals, and it is expected that the application of this cutting-edge technology in animal nutrition research will continue to grow in the foreseeable future. This research was supported in part by a USDA-NIFA Multistate Project (No. 1007691).

Download Full-text