scholarly journals Development of an RNA sequencing panel to detect gene fusions in thyroid cancer

2021 ◽  
Vol 19 (4) ◽  
pp. e41
Author(s):  
Dongmoung Kim ◽  
Seung-Hyun Jung ◽  
Yeun-Jun Chung
Keyword(s):  
Quality Control ◽  
Thyroid Cancer ◽  
Rna Sequencing ◽  
Copy Number ◽  
Gene Fusion ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Housekeeping Genes ◽  
Gene Fusions ◽  
Copy Number Alterations

In addition to mutations and copy number alterations, gene fusions are commonly identified in cancers. In thyroid cancer, fusions of important cancer-related genes have been commonly reported; however, extant panels do not cover all clinically important gene fusions. In this study, we aimed to develop a custom RNA-based sequencing panel to identify the key fusions in thyroid cancer. Our ThyChase panel was designed to detect 87 types of gene fusion. As quality control of RNA sequencing, five housekeeping genes were included in this panel. When we applied this panel for the analysis of fusions containing reference RNA (HD796), three expected fusions (EML4-ALK, CCDC6-RET, and TPM3-NTRK1) were successfully identified. We confirmed the fusion breakpoint sequences of the three fusions from HD796 by Sanger sequencing. Regarding the limit of detection, this panel could detect the target fusions from a tumor sample containing a 1% fusion-positive tumor cellular fraction. Taken together, our ThyChase panel would be useful to identify gene fusions in the clinical field.

Development of a breast and lung cancer research panel to target therapeutically relevant copy number and gene fusion variants from blood.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e12518-e12518
Author(s):  
Varun Bagai ◽  
Jeoffrey Schageman ◽  
Dumitru Brinza ◽  
Yanchun Li ◽  
Jian Gu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Somatic Mutation ◽  
Copy Number ◽  
Gene Fusion ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Negative Impact ◽  
Detection Sensitivity ◽  
Gene Fusions ◽  
Blood Samples

e12518 Background: With recent advances in next-generation sequencing (NGS) technologies, it is now possible to detect somatic mutations with allele frequencies in blood samples as low as 0.1% from circulating tumor DNA. A natural extension to this achievement is adding the ability to simultaneously detect copy number variants and gene fusions. A panel such as this addresses a full repertoire of variant classes found to be linked with certain tumors and would enable researchers additional tools to profile cancer samples more dynamically thus enriching current diagnostic tool sets. Here, we present progress on such an approach and apply current NGS technology to achieve our goals. Methods: Samples were sequecned using the Ion S5™ system. Results: Using control samples, we can reproducibly demonstrate detection of ERBB2 (HER2/neu) gene amplifications with high statistical significance and as low as a 2 fold difference versus non-amplified loci in titration experiments. In addition, this ERBB2 gene amplification was detected in the context of a validated breast cancer somatic mutation panel in which no negative impact was exhibited and mutation detection specificity and sensitivity were both greater than 90%. Lastly, we developed an additional panel to detect gene fusions relevant to lung cancer. Using the titration approach above, the EML4-ALK fusion variant was shown to have a limit of detection near 1% with no negative impact on detection sensitivity and specificity when combined with the validated lung cfDNA somatic mutation panel. Conclusions: From the outcomes of these experiments, we have shown the ability to reproducibly and simultaneously detect copy number and gene fusion variants as well as somatic mutations at very low limits of detection in a cell free DNA background derived from blood samples.

scholarly journals A pediatric case of pigmented epithelioid melanocytoma with chromosomal copy number alterations in 15q and 17q and a novel NTRK3‐SCAPER gene fusion

2019 ◽  
Vol 47 (1) ◽  
pp. 70-75 ◽  
Author(s):  
Ben J. Friedman ◽  
Simon Hernandez ◽  
Chelsea Fidai ◽  
Angela Jiang ◽  
Tor A. Shwayder ◽  
...  
Keyword(s):  
Copy Number ◽  
Gene Fusion ◽  
Copy Number Alterations ◽  
Pediatric Case ◽  
Chromosomal Copy Number ◽  
Chromosomal Copy

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

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. 296-296 ◽  
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.

Targeted deep sequencing as a diagnostic and research method for detecting EGFR and ALK pathway driver genes in FFPE tissue of lung and colorectal carcinomas.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 39-39
Author(s):  
Sakari Knuutila ◽  
Katja Merkkiniemi ◽  
Mikko Rönty ◽  
Aino Wirtanen ◽  
Satu Maria Remes ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Deep Sequencing ◽  
Lung Carcinoma ◽  
Copy Number ◽  
Mutation Screening ◽  
Gene Fusions ◽  
Copy Number Alterations ◽  
Hospital District ◽  
Targeted Deep Sequencing ◽  
Diagnostic Validation

39 Background: Molecular targeted tyrosine kinase inhibitor (TKI) treatments have made it crucial to perform diagnostic tests of multiple molecular targets. In lung carcinoma there are close to ten clinically relevant gene mutations, copy number alterations and/or gene fusions, such as ALK, EGFR, ERBB2, KRAS, BRAF, MET, PTEN, PI3KCA, ROS1 and RET. Presently, several different tests are utilized, requiring a high amount of tumor material and long turnaround time. Next generation sequencing or targeted deep sequencing (TDS) has opened a new era for rapid genome-wide analyses of mutations, copy number alterations and gene fusions. Our aimwas to 1) prove feasibility for applying TDS to FFPE samples, 2) compare mutations detected by prevalent methods & TDS, and 3) mine novel clinically and biologically relevant genes in lung and colorectal carcinoma. Methods: For TDS, we selected 192 lung carcinoma and colorectal carcinoma related genes and microRNA genes, focusing on the EGFR and ALK pathways. In total, 98 FFPE specimens were studied. Agilent SureSelect system and Illumina sequencing was adopted for the analysis. For diagnostic validation the following genes were selected: EGFR, KRAS, BRAF, PTEN, PI3K, RET and ALK. TDS results were confirmed by PCR, FISH and IHC. Results: We focused on the genes selected for diagnostic validation. Successful results were obtained from all specimens. The results from TDS correlated significantly with those obtained from PCR, FISH, and IHC. Importantly, TDS revealed novel mutations not detected by targeted PCR. Conclusions: An enormous advantage of TDS is that multiple mutation screening can be achieved in one analysis (saving time and material), and most importantly, provides enormous amounts of novel information, for example understanding mechanisms for drug resistance. This study was supported by Finnish Academy, Sigrid Jusélius Foundation, Finnish Cancer Organizations, the special governmental subsidy research funds appropriated to the Helsinki and Uusimaa Hospital District (HUS EVO), Pfizer Oy, AstraZeneca AS, Lab21 Ltd, Abbott Molecular Inc.

scholarly journals Analytical and Clinical Validation of an Amplicon-based Next Generation Sequencing Assay for Ultrasensitive Detection of Circulating Tumor DNA

2021 ◽  
Author(s):  
Jonathan Poh ◽  
Kao Chin Ngeow ◽  
Michelle Pek ◽  
Kian-Hin Tan ◽  
Jing Shan Lim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Point Mutations ◽  
Circulating Tumor Dna ◽  
Clinical Validation ◽  
Gene Fusions ◽  
Copy Number Alterations ◽  
Next Generation ◽  
Tumor Dna ◽  
Generation Sequencing

Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA. The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (EBV and HBV) and microsatellite instability. Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF, with high specificity even at 0.1% VAF (99.11% per-base). The limit of detection for copy number alterations, EBV, HBV, and microsatellite instability were also empirically determined. Orthogonal comparison of EGFR variant calls made by LiquidHALLMARK and a reference allele-specific PCR method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% alteration-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.

scholarly journals A Highly Sensitive and Specific Gene Fusion Algorithm Based on Multiple Fusion Callers and an Ensemble Machine Learning Approach

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Brad B Thomas ◽  
Yanglong Mou ◽  
Lauryn Keeler ◽  
Christophe Magnan ◽  
Vincent Funari ◽  
...  
Keyword(s):  
Random Forest ◽  
Rna Sequencing ◽  
False Positive ◽  
Gene Fusion ◽  
Training Data ◽  
Specific Gene ◽  
Learning Approach ◽  
Gene Fusions ◽  
Forest Models ◽  
Random Forest Models

Background: Gene Fusion events are common occurrences in malignancies, and are frequently drivers of malignancy. FISH and qPCR are two methods often used for identifying highly prevalent gene fusions/translocations. However, these are single target assays, requiring a lot of effort and sample if multiple assays are needed for multiple targets like sarcoma. High-throughput parallel (NextGen) DNA and RNA sequencing are also in current use to detect and characterize gene fusions. RNA sequencing (RNAseq) has the advantage that multiple markers can be targeted at one time and RNA fusions are readily identified from their product transcripts. While many fusion calling algorithms exist for use on RNAseq data, sensitive fusion callers, needed for samples of low tumor content, often present high false positive rates. Further, there currently is no single variable or element in NGS data that can be used to filter out false positive calls by extant callers. Individual sensitive fusion callers may be considered weak predictors of gene fusions. Combining their results into a single fusion call involves evaluating many elements, which can be a time consuming and difficult manual task. In order to achieve higher accuracy in fusion calls than can be achieved using individual fusion callers, we have combined the results of multiple fusion callers by use of an ensemble learning approach based on random forest models. Our method selects the best group of callers from among several callers, and provides an algorithmic means of combining their results, presenting a metric that can be immediately interpreted as the probability that a called fusion is a true fusion call. Methods: Random forest models were generated with the randomForest package in R, and then tuned using the R caret package. Training data sets consisted of fusion calls deemed true by review and by orthogonal methods including PCR/Sanger sequencing and the commercial Archer™ fusion calling system. We present the results of training on calls made by five fusion callers Arriba, STAR-Fusion, FusionCatcher, deFuse, and Kallisto/pizzly. Logistic training variables (seen vs not seen by the fusion caller) were used for the five callers. Variables also included metrics for the magnitude and balance of coverage on either side of candidate fusion breakpoints reported by Arriba and STAR Fusion ("coverage balance") and a single metric consisting of the number of sequencing reads that cross the candidate breakpoint. The model was validated by 10-fold cross-validation on 598 fusion calls by the five callers. Results: The resulting model is superior to the simple strategy of requiring agreement by n of five callers, particularly with regard to specificity (Table 1). Also, "importance of variables," reported by randomForest, gauges the relative contribution of variables in the model. Here it shows that one caller, Kallisto\pizzly, does not contribute to the model (Table 2). Conclusion: Random Forest modeling provides a viable means of combining gene fusion call data from multiple callers into a single fusion calling tool with improved performance over simple combinations of fusion calls. An additional benefit is seen in that building and evaluating such models can guide the selection of fusion callers, thereby eliminating non-contributory calling methods and ensuring optimal utilization of computational resources. Disclosures Thomas: NeoGenomics,Inc.: Current Employment. Mou:NeoGenomics: Current Employment. Keeler:NeoGenomics: Current Employment. Magnan:NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment. Weiss:Merck: Other: Speaker; Bayer: Other: speaker; Genentech: Other: Speaker; NeoGenomics: Current Employment. Brown:NeoGenomics,Inc.: Current Employment. Agersborg:NeoGenomics: Current Employment.

scholarly journals The genetic landscape of choroid plexus tumors in children and adults

2020 ◽  
Author(s):  
Christian Thomas ◽  
Patrick Soschinski ◽  
Melissa Zwaig ◽  
Spyridon Oikonomopoulos ◽  
Konstantin Okonechnikov ◽  
...  
Keyword(s):  
Choroid Plexus ◽  
Rna Sequencing ◽  
Copy Number ◽  
Clinical Course ◽  
Copy Number Alterations ◽  
Choroid Plexus Tumors ◽  
Tp53 Mutations ◽  
Tert Promoter Mutations ◽  
Promoter Mutations ◽  
Choroid Plexus Papillomas

Abstract Background Choroid plexus tumors (CPTs) are intraventricular brain tumors predominantly arising in children but also affecting adults. In most cases, driver mutations have not been identified, although there are reports of frequent chromosome-wide copy-number alterations and TP53 mutations, especially in choroid plexus carcinomas (CPCs). Methods DNA methylation profiling and RNA-sequencing was performed in a series of 47 CPTs. Samples comprised 35 choroid plexus papillomas (CPPs), 6 atypical choroid plexus papillomas (aCPPs) and 6 CPCs plus three recurrences thereof. Targeted TP53 and TERT promotor sequencing was performed in all samples. Whole exome sequencing (WES) and linked-read whole genome sequencing (WGS) was performed in 25 and 4 samples, respectively. Results Tumors comprised the molecular subgroups “pediatric A” (N=11), “pediatric B” (N=12) and “adult” (N=27). Copy-number alterations mainly represented whole-chromosomal alterations with subgroup-specific enrichments (gains of Chr1, 2 and 21q in “pediatric B” and gains of Chr5 and 9 and loss of Chr21q in “adult”). RNA sequencing yielded a novel CCDC47-PRKCA fusion transcript in one adult choroid plexus papilloma patient with aggressive clinical course; an underlying Chr17 inversion was demonstrated by linked-read WGS. WES and targeted sequencing showed TP53 mutations in 7/47 CPTs (15%), five of which were children. On the contrary, TERT promoter mutations were encountered in 7/28 adult patients (25%) and associated with shorter progression-free survival (log-rank test, p=0.015). Conclusion Pediatric CPTs lack recurrent driver alterations except for TP53, whereas CPTs in adults show TERT promoter mutations or a novel CCDC47-PRKCA gene fusion, being associated with a more unfavorable clinical course.

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