Artículos de revistas: "Targeted gene panels"

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Kanygina, A. V., E. I. Sharova, R. I. Sultanov, Y. A. Schelygin, Y. V. Doludin, E. S. Kostryukova y E. V. Generozov. "Targeted gene sequencing panels: applicability for neoantigen profiling of colon and rectal adenocarcinoma". Biomeditsinskaya Khimiya 64, n.º 6 (2018): 517–24. http://dx.doi.org/10.18097/pbmc20186406517.

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Cancer immunotherapy represents a promising and rapidly developing approach for the treatment of oncological diseases. Among the methods of personalized adjuvant immunotherapy, neoantigenic peptide-based drugs have demonstrated substantial efficiency. These drugs are designed to target mutant proteins arising from somatic alterations in the genome of tumor cells and thus stimulate immune response against tumor tissues. The methods of individual screening for potentially immunogenic mutations are mostly based on next-generation exome sequencing of tumor samples, which is a complex and costly procedure for clinical application. Targeted gene sequencing panels limited to a certain set of genes represent a reasonable alternative to WES. Targeted sequencing is also more efficient when there is a low amount of the sample DNA available. We have estimated the potential efficiency of targeted oncological panels in terms of somatic neoantigen profiling in colorectal cancer (colon and rectal adenocarcinoma). The clinical practice of identification of frequent somatic variants does not provide enough data for designing an efficient personalized drug when applied to low and medium mutated cancers such as colorectal cancer. Our analysis of 11 commercially available panels containing different number of genes has shown that neither the larger size of a panel nor its initial customization for colorectal cancer provides a significantly better estimation of an individual somatic mutation profile. The optimal approach is to use the general-purpose medium-sized cancer panels (2300-11200 amplicons and/or 150-600 genes). These panels allow to detect a sufficient number of immunogenic epitopes (>3) per patient for over 30-50% of patients.

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Romanov, Dmitriy y Nikolai Skoblikow. "Linkage Disequilibrium in Targeted Sequencing". Mathematical Biology and Bioinformatics 17, n.º 2 (22 de noviembre de 2022): 325–37. http://dx.doi.org/10.17537/2022.17.325.

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We propose an approach for optimizing the development of gene diagnostic panels, which is based on the construction of non-equilibrium linkage maps. In the process of gene selection we essentially use genome-wide association analysis (GWAS). Whole-genome analysis of associations makes it possible to reveal the relationship of genomic variants with the studied phenotype. However, the nucleotide variants that showed the highest degree of association can only be statistically associated with the phenotype, not being the true cause of the phenotype. In this case, they may be in the block of linked inheritance with nucleotide variants that really affect the manifestation of the phenotype. The construction of maps of non-equilibrium linkage of nucleotides makes it possible to optimally determine the boundaries of linkage blocks, in which the desired variants fall. The aim of this study was to optimize the demarcation of genomic loci to create targeted panels aimed at predicting susceptibility to SARS-CoV-2 and the severity of COVID-19. The proposed method for selecting loci for a target panel, taking into account nonequilibrium linkage, makes it possible to use the phenomenon of nonequilibrium linkage in order to maximally cover the regions involved in the development of the phenotype, while simultaneously minimizing the length of these regions, and, at the same time, the cost of sequencing.

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Santani, Avni, Jill Murrell, Birgit Funke, Zhenming Yu, Madhuri Hegde, Rong Mao, Andrea Ferreira-Gonzalez, Karl V. Voelkerding y Karen E. Weck. "Development and Validation of Targeted Next-Generation Sequencing Panels for Detection of Germline Variants in Inherited Diseases". Archives of Pathology & Laboratory Medicine 141, n.º 6 (21 de marzo de 2017): 787–97. http://dx.doi.org/10.5858/arpa.2016-0517-ra.

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Context.— The number of targeted next-generation sequencing (NGS) panels for genetic diseases offered by clinical laboratories is rapidly increasing. Before an NGS-based test is implemented in a clinical laboratory, appropriate validation studies are needed to determine the performance characteristics of the test. Objective.— To provide examples of assay design and validation of targeted NGS gene panels for the detection of germline variants associated with inherited disorders. Data Sources.— The approaches used by 2 clinical laboratories for the development and validation of targeted NGS gene panels are described. Important design and validation considerations are examined. Conclusions.— Clinical laboratories must validate performance specifications of each test prior to implementation. Test design specifications and validation data are provided, outlining important steps in validation of targeted NGS panels by clinical diagnostic laboratories.

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Bhattacharya, Arjun, Alina M. Hamilton, Melissa A. Troester y Michael I. Love. "DeCompress: tissue compartment deconvolution of targeted mRNA expression panels using compressed sensing". Nucleic Acids Research 49, n.º 8 (1 de febrero de 2021): e48-e48. http://dx.doi.org/10.1093/nar/gkab031.

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Abstract Targeted mRNA expression panels, measuring up to 800 genes, are used in academic and clinical settings due to low cost and high sensitivity for archived samples. Most samples assayed on targeted panels originate from bulk tissue comprised of many cell types, and cell-type heterogeneity confounds biological signals. Reference-free methods are used when cell-type-specific expression references are unavailable, but limited feature spaces render implementation challenging in targeted panels. Here, we present DeCompress, a semi-reference-free deconvolution method for targeted panels. DeCompress leverages a reference RNA-seq or microarray dataset from similar tissue to expand the feature space of targeted panels using compressed sensing. Ensemble reference-free deconvolution is performed on this artificially expanded dataset to estimate cell-type proportions and gene signatures. In simulated mixtures, four public cell line mixtures, and a targeted panel (1199 samples; 406 genes) from the Carolina Breast Cancer Study, DeCompress recapitulates cell-type proportions with less error than reference-free methods and finds biologically relevant compartments. We integrate compartment estimates into cis-eQTL mapping in breast cancer, identifying a tumor-specific cis-eQTL for CCR3 (C–C Motif Chemokine Receptor 3) at a risk locus. DeCompress improves upon reference-free methods without requiring expression profiles from pure cell populations, with applications in genomic analyses and clinical settings.

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Adeboyeje, Gboyega, Eleanor O. Caplan, Yihua Xu, Monica Chase, Sheetal Sheth, Brandon T. Suehs y Nicole Myer. "Abstract 4111: Trends in the use of broad genomic sequencing-directed therapy among Medicare patients with newly diagnosed advanced cancer in the United States from 2018-2020: A retrospective analysis from the SEQUENCE study". Cancer Research 82, n.º 12_Supplement (15 de junio de 2022): 4111. http://dx.doi.org/10.1158/1538-7445.am2022-4111.

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Abstract Introduction/Purpose: Recent advances have led to approval of multiple new targeted drugs across a variety of indications based on genomic markers. In part due to the increasing number of therapeutically actionable genomic markers in many cancers, whether a strategy of upfront broad next-generation sequencing (NGS) could potentially improve selection of patients for targeted drugs compared to narrow/single gene panels remains uncertain. We examined recent trends in the proportions of patients receiving sequencing-directed therapy following the use of broad NGS panel testing among newly diagnosed patients with advanced cancer in the United States (US). Methods: Using a retrospective cohort study design to analyze administrative claims data on patients (65-89 years) enrolled in a national US payer Medicare Advantage plans from 2018 through 2020, we identified patients with select incident advanced/metastatic cancer diagnoses (lung, colorectal [CRC], breast, ovarian) based on previously validated algorithms. We defined 2 cohorts by the occurrence of broad NGS (51+ genes) versus narrow (≤50 genes) sequencing within 182 days of diagnosis using a previously validated algorithm based on laboratory tax identification numbers, current procedural terminology codes, and diagnosis codes. We described the rates of sequencing-directed therapy (defined as receipt of an FDA-approved genomically targeted drug) within 90 days of testing by broad NGS versus narrow panels across tumor types and subgroups defined by race (White versus Black) and annual income (<$50,000 versus ≥$50,000) data. Results: We identified 32,130 patients with incident advanced cancer during the study period. Overall (broad plus narrow panels) testing rates varied by cancer type (lung, 40.1%; CRC 28.6%; breast 53.9%; ovarian 41.7%). Compared to narrow gene panels, a higher proportion of patients with lung and ovarian cancer sequenced with broad NGS panels initiated an FDA-approved genomically targeted drug within 90 days of testing (lung: 8.6% versus 7.5%; ovarian: 9.2% versus 5.5%). For CRC and breast cancer, narrow gene panels matched a higher proportion of patients with targeted drug within 90 days of testing versus broad NGS panel testing (CRC: 5.1 versus 4.5%; breast: 41.7% versus 35.7%). Conclusions: A higher proportion of patients initiated a genomically-targeted drug after broad NGS panel testing compared with narrow gene panels in lung and ovarian cancer. As the number of actionable genomic markers in advanced cancers increase, it will be important to ensure that this technology is adopted to improve upon the existing standard of care and that aligns with values important to patients. Citation Format: Gboyega Adeboyeje, Eleanor O. Caplan, Yihua Xu, Monica Chase, Sheetal Sheth, Brandon T. Suehs, Nicole Myer. Trends in the use of broad genomic sequencing-directed therapy among Medicare patients with newly diagnosed advanced cancer in the United States from 2018-2020: A retrospective analysis from the SEQUENCE study [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 4111.

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Bevins, Nicholas, Shulei Sun, Zied Gaieb, John A. Thorson y Sarah S. Murray. "Comparison of commonly used solid tumor targeted gene sequencing panels for estimating tumor mutation burden shows analytical and prognostic concordance within the cancer genome atlas cohort". Journal for ImmunoTherapy of Cancer 8, n.º 1 (marzo de 2020): e000613. http://dx.doi.org/10.1136/jitc-2020-000613.

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BackgroundTumor mutation burden (TMB) is a biomarker frequently reported by clinical laboratories, which is derived by quantifying of the number of single nucleotide or indel variants (mutations) identified by next-generation sequencing of tumors. TMB values can inform prognosis or predict the response of a patient’s tumor to immune checkpoint inhibitor therapy. Methods for the calculation of TMB are not standardized between laboratories, with significant variables being the gene content of the panels sequenced and the inclusion or exclusion of synonymous variants in the calculations. The impact of these methodological differences has not been investigated and the concordance of reported TMB values between laboratories is unknown.MethodsSequence variant lists from more than 9000 tumors of various types were downloaded from The Cancer Genome Atlas. Variant lists were filtered to include only appropriate variant types (ie, non-synonymous only or synonymous and non-synonymous variants) within the genes found in five commonly used targeted solid tumor gene panels as well as an in-house gene panel. Calculated TMB was paired with corresponding overall survival (OS) data of each patient.ResultsRegression analysis indicates high concordance of TMB as derived from the examined panels. TMB derived from panels was consistently and significantly lower than that derived from a whole exome. TMB, as derived from whole exome or the examined panels, showed a significant correlation with OS in the examined data.ConclusionsTMB derived from the examined gene panels was analytically equivalent between panels, but not between panels and whole-exome sequencing. Correlation between TMB and OS is significant if TMB method-specific cut-offs are used. These results suggest that TMB values, as derived from the gene panels examined, are analytically and prognostically equivalent.

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Gierman, Hinco J., Nikhil Pai, Casey Catasus, Alvin Tam, Monica Labrador, Joseph Donaldson, Mallika Singaraju et al. "A retrospective three-year analysis using real-world data on uptake of broad-based NextGen sequencing panels in community oncology practices." Journal of Clinical Oncology 38, n.º 15_suppl (20 de mayo de 2020): e13668-e13668. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e13668.

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e13668 Background: There are over 100 FDA approved targeted therapies across 15 cancer types, offering improved outcomes over existing therapies. However, many of these require genetic testing, for example, advanced non-small cell lung cancer (aNSCLC) patients have over 15 targeted therapies requiring a DNA-based test. Doing multiple tests can exhaust sample, while increasing cost and turn-around time. NGS panels, often with hundreds of genes, can address some of these issues. Here we asked across aNSCLC patients if the use of NGS panels has increased over the last 3 years in community oncology practices. Methods: The Integra Connect database, which includes electronic medical record (EMR) and claims data on over 1,000,000 US cancer patients, was queried across five community oncology practices to identify aNSCLC patients (stage IIIB or IV) treated between January 2017 and January 2020. Manual chart review abstracted tumor type, stage, treatment, and testing. Patients tested for all 7 NCCN recommended genes (EGFR, ALK, ROS1, BRAF, MET, RET, ERBB2) were grouped as “NGS Panel”, patients with less genes as “Single Gene/Small Panel”, and patients with no evidence of testing as “No Test”. A Chi-Square test was used to compare actionable results between patients with NGS panels versus small panels. Results: 1,007 aNSCLC patients were analyzed and showed a doubling of the use of broad-based NGS testing from 13% in 2017 to 26% in 2019 across over 100 oncologists (table). 23% of patients had actionable results when tested on broad-based panels versus 17% using single gene or small panels (p = .048). Targeted therapies were used in 17% of broad-based tested patients, versus 15% in patients tested for single genes or small panels. Conclusions: We see an uptake of broad-based NGS testing in community oncology, which can lead to more actionable results and better utilization of targeted therapies for those patients. However, this seems to be caused by providers shifting from small panels to large panels, rather than an overall increase in testing, as we do not see the percentage of untested patients decrease. [Table: see text]

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Bansal, Nidhanjali, Hye-Won Song, Silin Sa, Woodrow E. Lomas, Gisele V. Baracho, Ian Taylor, Stephanie Widmann y Stefanie Mortimer. "Single cell whole transcriptome analysis of disease cells to generate a targeted RNA-sequencing gene panel for the simultaneous analysis of targeted mRNA and protein". Journal of Immunology 202, n.º 1_Supplement (1 de mayo de 2019): 131.35. http://dx.doi.org/10.4049/jimmunol.202.supp.131.35.

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Abstract Single cell RNA-sequencing (scRNA-seq) is a powerful tool for understanding the sample heterogeneity of individual cells. Many methods rely on whole transcriptome analysis (WTA) to get a snapshot of the entire cellular landscape. Although WTA analysis can be used to discover novel biomarkers, this technique can be expensive. To enable scaling of experiments, WTA data can be mined to design targeted gene panels. To showcase this, we used single cell sequencing to examine thousands of B cells isolated from the bone marrow and peripheral blood of chronic lymphocytic leukemia (CLL) and healthy donors. B cells that had been sorted using the BD FACSMelody™ cell sorter were multiplexed using the BD™ Human Single-Cell Multiplexing Kit and pooled before being processed on the BD Rhapsody™ system, thereby minimizing batch effects. The resulting data was mined to design a panel of differentially expressed genes between CLL and healthy B cells that could be used for subsequent CLL phenotyping. By combining this panel with the BD Rhapsody™ Immune Response Panel (together comprising ~500 mRNAs), along with 36 DNA-barcoded BD™ AbSeq antibodies, we were able to simultaneously analyze mRNA and protein targets from a new subset of CLL and healthy B cells for additional high-resolution analysis. This study showcases the power of using WTA data to design specific gene panels that can be used alone or in combination with existing targeted panels for routine and cost-effective transcriptional profiling at a single cell level. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, FACSMelody, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

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Wilson, Parker C., Latisha Love-Gregory, Meagan Corliss, Samantha McNulty, Jonathan W. Heusel y Joseph P. Gaut. "Beyond Panel-Based Testing: Exome Analysis Increases Sensitivity for Diagnosis of Genetic Kidney Disease". Kidney360 1, n.º 8 (13 de mayo de 2020): 772–80. http://dx.doi.org/10.34067/kid.0001342020.

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BackgroundNext-generation sequencing (NGS) is a useful tool for evaluating patients with suspected genetic kidney disease. Clinical practice relies on the use of targeted gene panels that are ordered based on patient presentation. We compare the diagnostic yield of clinical panel-based testing to exome analysis.MethodsIn total, 324 consecutive patients underwent physician-ordered, panel-based NGS testing between December 2014 and October 2018. Gene panels were available for four clinical phenotypes, including atypical hemolytic uremic syndrome (n=224), nephrotic syndrome (n=56), cystic kidney disease (n=26), and Alport syndrome (n=13). Variants were analyzed and clinical reports were signed out by a pathologist or clinical geneticist at the time of testing. Subsequently, all patients underwent retrospective exome analysis to detect additional clinically significant variants in kidney disease genes that were not analyzed as part of the initial clinical gene panel. Resulting variants were classified according to the American College of Medical Genetics and Genomics 2015 guidelines.ResultsIn the initial physician-ordered gene panels, we identified clinically significant pathogenic or likely pathogenic variants in 13% of patients (n=42/324). CFHR3-CFHR1 homozygous deletion was detected in an additional 13 patients with aHUS without a pathogenic or likely pathogenic variant. Diagnostic yield of the initial physician-ordered gene panel was 20% and varied between groups. Retrospective exome analysis identified 18 patients with a previously unknown pathogenic or likely pathogenic variant in a kidney disease gene and eight patients with a high-risk APOL1 genotype. Overall, retrospective exome analysis increased the diagnostic yield of panel-based testing from 20% to 30%.ConclusionsThese results highlight the importance of a broad and collaborative approach between the clinical laboratory and their physician clients that employs additional analysis when a targeted panel of kidney disease–causing genes does not return a clinically meaningful result.

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Barbosa-Gouveia, Sofia, María E. Vázquez-Mosquera, Emiliano González-Vioque, José V. Álvarez, Roi Chans, Francisco Laranjeira, Esmeralda Martins, Ana Cristina Ferreira, Alejandro Avila-Alvarez y María L. Couce. "Utility of Gene Panels for the Diagnosis of Inborn Errors of Metabolism in a Metabolic Reference Center". Genes 12, n.º 8 (19 de agosto de 2021): 1262. http://dx.doi.org/10.3390/genes12081262.

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Next-generation sequencing (NGS) technologies have been proposed as a first-line test for the diagnosis of inborn errors of metabolism (IEM), a group of genetically heterogeneous disorders with overlapping or nonspecific phenotypes. Over a 3-year period, we prospectively analyzed 311 pediatric patients with a suspected IEM using four targeted gene panels. The rate of positive diagnosis was 61.86% for intermediary metabolism defects, 32.84% for complex molecular defects, 19% for hypoglycemic/hyperglycemic events, and 17% for mitochondrial diseases, and a conclusive molecular diagnosis was established in 2–4 weeks. Forty-one patients for whom negative results were obtained with the mitochondrial diseases panel underwent subsequent analyses using the NeuroSeq panel, which groups all genes from the individual panels together with genes associated with neurological disorders (1870 genes in total). This achieved a diagnostic rate of 32%. We next evaluated the utility of a tool, Phenomizer, for differential diagnosis, and established a correlation between phenotype and molecular findings in 39.3% of patients. Finally, we evaluated the mutational architecture of the genes analyzed by determining z-scores, loss-of-function observed/expected upper bound fraction (LOEUF), and haploinsufficiency (HI) scores. In summary, targeted gene panels for specific groups of IEMs enabled rapid and effective diagnosis, which is critical for the therapeutic management of IEM patients.

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Vashistha, Vishal, Jenna Armstrong, David Winski, Pradeep J. Poonnen, Bradley Hintze, Meghan Price, Jane L. Snowdon et al. "Barriers to Prescribing Targeted Therapies for Patients With NSCLC With Highly Actionable Gene Variants in the Veterans Affairs National Precision Oncology Program". JCO Oncology Practice 17, n.º 7 (julio de 2021): e1012-e1020. http://dx.doi.org/10.1200/op.20.00703.

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PURPOSE: Next-generation sequencing (NGS) gene panels are frequently completed for patients with advanced non–small-cell lung cancer (NSCLC). Patients with highly actionable gene variants have improved outcomes and reduced toxicities with the use of corresponding targeted agents. We sought to identify barriers to targeted agent use within the Veterans Health Affairs' National Precision Oncology Program (NPOP). METHODS: A retrospective evaluation of patients with NSCLC who underwent NGS multigene panels through NPOP between July 2015 and February 2019 was conducted. Patients who were assigned level 1 or 2A evidence for oncogenic gene variants by an artificial intelligence offering (IBM Watson for Genomics [WfG]) and NPOP staff were selected. Antineoplastic drug prescriptions and provider notes were reviewed. Reasons for withholding targeted treatments were categorized. RESULTS: Of 1,749 patients with NSCLC who successfully underwent NGS gene panel testing, 112 (6.4%) patients were assigned level 1 and/or 2A evidence for available targeted treatments by WfG and NPOP staff. All highly actionable gene variants were within ALK, BRAF, EGFR, ERBB2, MET, RET, and ROS1. Of these, 36 (32.1%) patients were not prescribed targeted agents. The three most common reasons were (1) patient did not carry a diagnosis of metastatic disease (33.3%), (2) treating provider did not comment on the NGS results (25.0%), and (3) provider felt that patient could not tolerate therapy (19.4%). No patients were denied access to level 1 or 2A targeted drugs because of rejection of a nonformulary drug request. CONCLUSION: A substantial minority of patients with NSCLC bearing highly actionable gene variants are not prescribed targeted agents. Further provider- and pathologist-directed educational efforts and implementation of health informatics systems to provide real-time decision support for test ordering and interpretation are needed.

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Barnell, Erica K., Kenneth F. Newcomer, Zachary L. Skidmore, Kilannin Krysiak, Sydney R. Anderson, Lukas D. Wartman, Stephen T. Oh et al. "Impact of a 40-Gene Targeted Panel Test on Physician Decision Making for Patients With Acute Myeloid Leukemia". JCO Precision Oncology, n.º 5 (enero de 2021): 191–203. http://dx.doi.org/10.1200/po.20.00182.

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PURPOSE Physicians treating hematologic malignancies increasingly order targeted sequencing panels to interrogate recurrently mutated genes. The precise impact of these panels on clinical decision making is not well understood. METHODS Here, we report our institutional experience with a targeted 40-gene panel (MyeloSeq) that is used to generate a report for both genetic variants and variant allele frequencies for the treating physician (the limit of mutation detection is approximately one AML cell in 50). RESULTS In total, 346 sequencing reports were generated for 325 patients with suspected hematologic malignancies over an 8-month period (August 2018 to April 2019). To determine the influence of genomic data on clinical care for patients with acute myeloid leukemia (AML), we analyzed 122 consecutive reports from 109 patients diagnosed with AML and surveyed the treating physicians with a standardized questionnaire. The panel was ordered most commonly at diagnosis (61.5%), but was also used to assess response to therapy (22.9%) and to detect suspected relapse (15.6%). The panel was ordered at multiple timepoints during the disease course for 11% of patients. Physicians self-reported that 50 of 114 sequencing reports (44%) influenced clinical care decisions in 44 individual patients. Influences were often nuanced and extended beyond identifying actionable genetic variants with US Food and Drug Administration–approved drugs. CONCLUSION This study provides insights into how physicians are currently using multigene panels capable of detecting relatively rare AML cells. The most influential way to integrate these tools into clinical practice will be to perform prospective clinical trials that assess patient outcomes in response to genomically driven interventions.

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Nakashima, Takuma, Yusuke Funakoshi, Atsuhito Uneda, Shohei Nambu, Mai Kitahara, Shunsuke Yanagisawa, Makoto Ohno et al. "COT-8 DEVELOPMENT OF TARGETED GENE PANEL FOR RAPID MOLECULAR DIAGNOSIS OF BRAIN TUMORS". Neuro-Oncology Advances 4, Supplement_3 (1 de diciembre de 2022): iii25. http://dx.doi.org/10.1093/noajnl/vdac167.098.

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Abstract Background Brain tumors are diagnosed based on pathological and genetic features defined by WHO classification. Although targeted gene panels are clinically available, most of them do not cover all the necessary genes for the diagnosis of brain tumors. Moreover, broad copy number analysis, which the current WHO classification requires, usually lacks in the gene panel. Another problem is that those panels demand a high burden of time and cost, which disturbs rapid diagnosis and broad application. To overcome those problems, we developed a rapid and cost-effective workflow of molecular diagnosis for brain tumors. Methods Our panel contains 109 genes of which 68 are necessary for fundamental molecular diagnosis and 41 are other common driver genes. To detect copy number changes and structural variants, which generate a fused gene, additional probes are placed on common SNPs and introns containing common breakpoints. MGMT methylation status is examined at the same time using bisulfite-converted DNA amplification. Sequencing data is analyzed using a supercomputer. Results The analysis time is within 4 days: 2 days for library preparation, 1 day for sequencing, and 12 hours for analysis. Detected driver alterations were validated by whole genome sequencing data. MGMT methylation status was correlated between the results of our workflow and pyrosequencing. Conclusions We have developed a rapid comprehensive molecular analysis workflow that detects genetic alterations and MGMT methylation. Our method allows a cost-effective molecular diagnosis with high accuracy, which would improve molecular diagnosis for brain tumors.

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Vashistha, Vishal, Jenna Armstrong, David Winski, Meghan Price, Bradley J. Hintze, Pradeep Poonnen, Jane Snowdon et al. "Barriers to prescribing targeted therapies for NSCLC patients with highly actionable gene variants in the VA National Precision Oncology Program." Journal of Clinical Oncology 38, n.º 15_suppl (20 de mayo de 2020): 2005. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.2005.

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2005 Background: Next-Generation Sequencing (NGS) gene panels are often completed to guide therapeutic decisions for patients with advanced stage non-small cell lung cancer (NSCLC). Patients with highly-actionable gene variants may experience improved therapeutic treatments and reduced toxicities with use of targeted agents. Ensuring appropriate prescription of targeted therapies is therefore of high importance. We sought to identify barriers to targeted agent use within the Veterans Health Affairs’ (VHA) National Precision Oncology Program (NPOP). Methods: A retrospective evaluation examined the cohort of NSCLC patients who underwent NGS multi-gene panels through NPOP between July 2015 and February 2019. A level of evidence for drug actionability was assigned to each observed oncogenic gene variant using an artificial intelligence offering (IBM Watson for Genomics: WfG). WfG level 1 and 2A evidence was reviewed by NPOP staff to exclude gene variants that did not conform to NPOP level 1 and 2A definitions. Anti-neoplastic drug prescriptions and oncology provider notes were obtained for all included patients from the VHA Corporate Data Warehouse. Review of clinical notes of patients who did not receive targeted agents was performed to categorize the reason(s). Results: Of 1764 NSCLC patients who successfully underwent NGS gene panel testing, 156 (8.9%) received therapeutic level 1 (7.3%) or 2A (1.6%) options for targeted agents based on WfG evidence analysis. In total, 117 (6.6%) patients had NPOP level 1 and 2A gene variants, all within ALK, BRAF, EGFR, ERBB2, MET, and RET. Of these, 49 (41.2%) patients were not prescribed available targeted agents. The three most common reasons were: (1) treating provider did not comment on NGS results (30.7%), (2) patient did not carry a diagnosis of advanced stage disease (18.4%), and (3) patient had begun an alternative systemic therapy prior to completion of sequencing (16.3%). No patient was denied access to a level 1 or 2A targeted drug due to utilization-management review. Conclusions: A substantial minority of patients with advanced NSCLC bearing highly-actionable gene variants are not prescribed available targeted agents. Further provider- and pathologist-directed educational effort are needed, as well as implementation of health informatics systems to provide near real-time decision support for test ordering and interpretation.

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Ahram, Dina F., Vimla S. Aggarwal y Simone Sanna-Cherchi. "Phenocopies, Phenotypic Expansion, and Coincidental Diagnoses: Time to Abandon Targeted Gene Panels?" American Journal of Kidney Diseases 76, n.º 4 (octubre de 2020): 451–53. http://dx.doi.org/10.1053/j.ajkd.2020.07.003.

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Tredan, Olivier, Damien Pouessel, Nicolas Penel, Sylvie Chabaud, Carlos A. Gomez-Roca, Diane Pannier, Mehdi Brahmi et al. "Increasing targeted therapy options for patients with relapsed cancer with broader somatic gene panel analysis from the primary tumor: The Profiler02 randomized phase II trial." Journal of Clinical Oncology 40, n.º 16_suppl (1 de junio de 2022): 3130. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.3130.

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3130 Background: PROFILER-02 is a multicenter randomized prospective study comparing the proportion of metastatic cancer patients (pts) with Targeted Agent (TA) recommendation provided by large NGS panel (FOne panel, 324 genes) vs home 87-gene NGS panel (CTRL) (PMID 30865223). Methods: Adult pts with advanced/metastatic cancer during 1st or 2nd line of therapy without known targetable gene alteration were eligible and randomized (1:1) to FOne vs CTRL panel. Both panels were performed for each patient. The randomization arm defined the first panel reviewed by dedicated Molecular Tumor Board (MTB) at disease progression while the 2nd panel remained blinded. The primary objective was the pts rate with at least one TA recommendation by the MTB using either FOne or CTRL panel. The study was designed in order to detect difference in proportions of 10% between the two panels. A sample size of 289 pts with both panels were requested to show this difference with an expected proportion of discordant pairs of 20% using a McNemar's test with 98% power and 5% two-sided significance level. Secondary endpoints included number of pts receiving at least one TA, progression free survival (PFS) and overall survival (OS). Results: From June 2017 to June 2019, among the 339 included pts 171 and 168 pts were randomized in FOne or CTRL panels’ first use, respectively. Median age was 57 years [19.0 - 85.0]; 54.9% were female. The median time from randomization to first MTB was 7.62 months [range 0.80 - 48.1]. Among the 339 pts, 147 pts (43.4%) had no TA recommendation, 108 pts (31.9%) had at least one TA recommendation according to both panels, 67 pts (19.8%) had one or more TA recommendation according to FOne panel only and 17 pts (5%) according to CTRL panel only (McNemar p < 0.001). At the time of the analysis, 51/339 (15%) pts started recommended treatment: 27 pts (8%) with TA recommendation from both panels, 21 pts (6.2%) from FOne only and 3 pts (0.3%) from CTRL only. Main initiated TA were PARP inh. (FOne n = 12; CTRL n = 9), PI3K/AKT/mTOR inh. (FOne n = 10; CTRL n = 9) and immunotherapy (ICI) (FOne n = 7; CTRL n = 0). Median PFS following first MTB were 3.2 months (95% CI 2.5-3.8) and 2.6 months (95% CI 2.0-3.8), median OS were 8.7 months (95% CI 6.6-10.8) and 8.4 months (95% CI 6.4-9.7), in the FOne and CTRL arm, respectively. Conclusions: Larger NGS panel including Tumor Mutational Burden increased the number of recommended options (TA and ICI), as well as the number of treatment initiation. Clinical trial information: NCT03163732.

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Merino, Diana M., Lisa McShane, Matt Butler, Vincent Anthony Funari, Matthew David Hellmann, Ruchi Chaudhary, Shu-Jen Chen et al. "TMB standardization by alignment to reference standards: Phase II of the Friends of Cancer Research TMB Harmonization Project." Journal of Clinical Oncology 37, n.º 15_suppl (20 de mayo de 2019): 2624. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.2624.

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2624 Background: Tumor mutational burden (TMB) is a predictive biomarker of response to immune checkpoint inhibitors across multiple cancers. In Phase 1 of the Friends of Cancer Research Harmonization Project, we demonstrated a robust correlation between TMB estimated using targeted next-generation sequencing (NGS) gene panels and whole exome sequencing (WES) applied to MC3-TCGA data. These findings demonstrated variability in TMB estimates across different panels. Phase 2 evaluates sustainable TMB reference standard materials for TMB alignment to assess this variability. The goal of this effort is to establish best practices for estimating TMB in order to improve consistency across panels, for the sake of optimizing clinical application and facilitating integration of datasets generated from multiple assays. Methods: Fifteen laboratories with targeted panels at different stages of development participated. We identified a set of reference standards consisting of 10 well-characterized human-derived lung and breast tumor-normal matched cell lines. WES was performed using a uniform bioinformatics pipeline agreed upon by all team members (WES-TMB). Each laboratory used their own sequencing and bioinformatics pipelines (tumor-only and tumor-normal) to estimate TMB according to genes represented in their respective panels (panel-TMB). The association between WES-TMB and each panel-TMB was investigated using regression analyses. Bias (relative to WES-TMB) and variability in TMB estimates across panels were rigorously assessed. All analyses were blinded. Results: The set of reference standards spanned a clinically meaningful TMB range (4.3 to 31.4 mut/Mb). Preliminary data from 12 laboratories shows a good correlation between panel-TMB and WES-TMB in this empirical analysis. Across panels, regression R2 values range 0.77-0.96 with slopes ranging 0.60-1.26. Calibration analyses that seek to minimize variability of TMB estimates across panels using the established set of reference standards are ongoing, as well as investigating cancer type dependence on the relationship between panel-TMB vs. WES-TMB, which will be available at the time of presentation. Conclusions: Preliminary findings demonstrate feasibility of using sustainable reference control cell lines to standardize and align estimation of TMB across different targeted NGS assays. Future studies aim to validate reference standard material as a reliable alignment tool by using formalin-fixed paraffin-embedded human tumor samples.

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Kopetz, Scott, Kenna R. Mills Shaw, J. Jack Lee, Jiexin Zhang, Beate Litzenburger, Vijaykumar Holla, Walter Kinyua et al. "Use of a Targeted Exome Next-Generation Sequencing Panel Offers Therapeutic Opportunity and Clinical Benefit in a Subset of Patients With Advanced Cancers". JCO Precision Oncology, n.º 3 (diciembre de 2019): 1–14. http://dx.doi.org/10.1200/po.18.00213.

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PURPOSE Smaller hotspot-based next-generation sequencing (NGS) panels have emerged to support standard of care therapy for patients with cancer. When standard treatments fail, it is unknown whether additional testing using an expanded panel of genes provides any benefit. The purpose of this study was to determine if larger sequencing panels that capture additional actionable genes, coupled with decision support, translates into treatment with matched therapy after frontline therapy has failed. PATIENTS AND METHODS A prospective protocol accrued 521 patients with a wide variety of refractory cancers. NGS testing using a 46- or 50-gene hotspot assay, then a 409-gene whole-exome assay, was sequentially performed in a Clinical Laboratory Improvement Amendments–certified clinical laboratory. A decision-support team annotated somatic alterations in clinically actionable genes for function and facilitated therapeutic matching. Survival and the impact of matched therapy use were determined by Kaplan-Meier estimate, log-rank test, and Cox proportional hazards regression. RESULTS The larger NGS panel identified at least one alteration in an actionable gene not previously identified in the smaller sequencing panel in 214 (41%) of 521 of enrolled patients. After the application of decision support, 41% of the alterations in actionable genes were considered to affect the function of the gene and were deemed actionable. Forty patients (40 of 214 [19%]) were subsequently treated with matched therapy. Treatment with matched therapy was associated with significantly improved overall survival compared with treatment with nonmatched therapy ( P = .017). CONCLUSION Combining decision support with larger NGS panels that incorporate genes beyond those recommended in current treatment guidelines helped to identify patients who were eligible for matched therapy while improving overall treatment selection and survival. This survival benefit was restricted to a small subset of patients.

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Min, Byung-Joo, Woo Seung Lee, Myung-Eui Seo, Kye-Hwa Lee, Seung-Yong Jeong, Ja-Lok Ku, Yeul Hong Kim, Sang-Won Shin y Ju Han Kim. "Development and Validation of Targeted Gene Sequencing Panel Based Companion Diagnostic for Korean Patients with Solid Tumors". Cancers 13, n.º 20 (12 de octubre de 2021): 5112. http://dx.doi.org/10.3390/cancers13205112.

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Recently, several panels using two representative targeting methods have been developed but they do not reflect racial specificity, especially for Asians. We have developed and analytically validated the Korean Pan-cancer Companion Diagnostic (CDX) Panel to apply targeted anticancer drugs to Korean patients based on the molecular characteristics of tumors using tumor samples without matched patient normal samples. The panel included 31 genes with reported single nucleotide variants, 9 genes with reported copy number variations, and 15 genes with predictive responses to targeted drugs under clinical testing, enabling the panel to be analyzed for the targets of 30 targeted anticancer drugs. It is cost-effective and optimized for cancer type-specific therapy in Korean cancer patients across solid cancer types while minimizing the limitations of existing approaches. In addition, the optimized filtering protocol for somatic variants from tumor-only samples enables researchers to use this panel without matched normal samples. To verify the panel, 241 frozen tumor tissues and 71 formalin-fixed paraffin-embedded (FFPE) samples from several institutes were registered. This gene screening method is expected to reduce test turnaround time and cost, making it a balanced approach to investigate solid cancer-related gene regions.

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Maxwell, Perry, Seán O. Hynes, Marc Fuchs, Stephanie Craig, Claire McGready, Fiona McLean, Stephen McQuaid, Jacqueline James y Manuel Salto-Tellez. "Practical guide for the comparison of two next-generation sequencing systems for solid tumour analysis in a universal healthcare system". Journal of Clinical Pathology 72, n.º 3 (31 de enero de 2018): 225–31. http://dx.doi.org/10.1136/jclinpath-2017-204917.

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AimsAlthough there have been excellent reports in the literature of validating next-generation sequencing, comparisons between two systems are not often published due to cost and time. We set out to establish that targetable mutations could be reliably detected with different gene panels and different chemistries using a common bioinformatics pipeline for meaningful comparisons to be made.MethodsAfter running selected formalin-fixed, paraffin-embedded samples through QPCR, Sanger sequencing and the 50 gene hotspot v2 panel from Life Technologies to determine standard-of-care variants, we compared the Oncomine panel from Life Technologies performed on a Personal Genome Machine (PGM) and the eight-gene actionable panel from Qiagen performed on a MiSeq platform. We used a common bioinformatics program following the creation of respective VCF files.ResultsBoth panels were accurate to above 90%, the actionable panel workflow was easier to perform but the lowest effective starting DNA load was obtained on the Oncomine workflow at 4 ng. Such minimal DNA can help with samples where there is limited material such as those for lung cancer molecular studies. We also discuss gene panel content and propose that increasing the gene profile of a panel will not benefit clinical laboratories where standard-of-care testing is all that is required.ConclusionsOnce recognised, it may be cost-effective for such laboratories to begin validation with an appropriate bioinformatics pipeline for targeted multigene hotspot molecular testing.

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Kanygina, A. V., E. I. Sharova, R. I. Sultanov, Y. A. Shelygin, Y. V. Doludin, E. S. Kostryukova y E. V. Generozov. "Targeted Gene Sequencing Panels: Applicability for Neoantigen Profiling of Colon and Rectal Adenocarcinoma". Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry 13, n.º 2 (abril de 2019): 146–53. http://dx.doi.org/10.1134/s1990750819020045.

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Tsoulos, Nikolaos, Eirini Papadopoulou, Vasiliki Metaxa-Mariatou, Georgios Tsaousis, Chrisoula Efstathiadou, Georgia Tounta, Katerina Skapeti et al. "Molecular profiling of 502 patient cohort with NSCLC using a 27 somatic gene panel." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): e23193-e23193. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e23193.

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e23193 Background: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using the Next Generation Sequencing (NGS) technology, has become a key tool for NSCLC patients’ treatment decision and clinical management. Methods: The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq Technology, was evaluated for the analysis of tumor DNA extracted from FFPE (Formalin Fixed Parraffin Embedded) tissue. Furthermore the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. Tumors’ mutation spectrum was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. Results: The panel used for tumor DNA analysis in this study exhibit high rates (100%) of sensitivity, specificity and reproducibility at a mutation frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK ROS1) and 9.4% had an alteration in a gene related to emerging targeted therapies according the NCCN guidelines. These alterations include ERBB2, BRAF and MET mutations, MET amplification and RET rearrangements. The remaining 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or give them access to a clinical trial. Conclusions: Thus the NGS panel validated is a reliable approach of clinical applicability for tumor molecular profile detection in NSCLC patients.

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Burghel, George J., Carolyn D. Hurst, Christopher M. Watson, Phillip A. Chambers, Helen Dickinson, Paul Roberts y Margaret A. Knowles. "Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms". BioMed Research International 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/478017.

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Detection of clinically actionable mutations in diagnostic tumour specimens aids in the selection of targeted therapeutics. With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis. As it is still not feasible for routine diagnostic laboratories to perform sequencing of the entire cancer genome, our approach was to undertake targeted mutation detection. To optimise our diagnostic workflow, we evaluated three target enrichment strategies using two next-generation sequencing (NGS) platforms (Illumina MiSeq and Ion PGM). The target enrichment strategies were Fluidigm Access Array custom amplicon panel including 13 genes (MiSeq sequencing), the Oxford Gene Technologies (OGT) SureSeq Solid Tumour hybridisation panel including 60 genes (MiSeq sequencing), and an Ion AmpliSeq Cancer Hotspot Panel including 50 genes (Ion PGM sequencing). DNA extracted from formalin-fixed paraffin-embedded (FFPE) blocks of eight previously characterised cancer cell lines was tested using the three panels. Matching genomic DNA from fresh cultures of these cell lines was also tested using the custom Fluidigm panel and the OGT SureSeq Solid Tumour panel. Each panel allowed mutation detection of core cancer genes includingKRAS,BRAF, andEGFR. Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.

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Fields, Jessica, Dimitrios Nasioudis, Zhen Ni Zhou, Ann Carlson, Melissa Kristen Frey, Kevin Holcomb y Eloise Chapman-Davis. "Underutilization of multigene panels among Ashkenazi Jewish patients." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): 1533. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.1533.

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1533 Background: Approximately one in forty Ashkenazi Jewish (AJ) individuals carry a BRCA1/2 mutation and genetic screening in this population has largely focused on these two genes. With the recent rapid uptake of multigene panel testing for cancer genetic assessment, we sought to explore multigene panels in our cohort which is comprised of AJ and non-AJ patients. Methods: The results of all patients with known ancestry who underwent genetic testing and counseling at the hereditary breast and ovarian cancer center at a single institution between 7/1/2013-12/31/2016 were reviewed. Results: One thousand six hundred and fifty patients with known ancestry underwent genetic testing over the study period, including 681 AJ patients. The median age was 49 (range 20-86). AJ patients were more likely to undergo targeted testing than non-AJ patients (74% vs. 61 %, P<0.001). The use of multigene panels in AJ patients increased over time (2013 – 3.2%, 2014 – 18.7%, 2015 – 27.4%, 2016 – 48.4%, P<0.001). Mutations were more common in AJ patients (75, 11% vs. 66, 7%, P=0.003). Variants of uncertain significance (VUS) were less common in AJ patients (40, 6% vs. 124, 13%, P<0.001), even when excluding patients with single gene testing (32, 19% vs. 98, 27%, P=0.05). Among all patients, mutations in BRCA1/2 were most common (75%). The majority (69%) of non- BRCA1/2 mutations were identified on multigene panels. Rates of mutations in non- BRCA1/2 genes were the same among AJ and non-AJ patients (16, 21% vs. 20, 30%, P=0.3, Table 1). Conclusions: AJ patients have equivalent rates of non- BRCA1/2 mutations and on multigene panels have lower rates of VUS compared to non-AJ patients. However, the majority of AJ patients underwent targeted gene testing. These findings suggest consideration of a change in paradigm for genetic assessment of AJ patients with a focus on BRCA and non- BRCAassociated cancer genes through multigene panel testing. [Table: see text]

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Bhadada, Sanjay K., Subbiah Sridhar, Vandana Dhiman, Karen Wong, Bruce Bennetts, Dorit Naot, Sangumani Jayaraman y Tim Cundy. "HYPOPHOSPHATEMIC RICKETS WITH HYPERCALCIURIA: A NOVEL HOMOZYGOUS MUTATION IN SLC34A3 AND LITERATURE REVIEW". AACE Clinical Case Reports 6, n.º 3 (mayo de 2020): e105-e112. http://dx.doi.org/10.4158/accr-2019-0456.

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Objective: Hypophosphatemic rickets with hypercalciuria (HHRH) is a rare, recessively-inherited form of rickets caused by homozygous or compound heterozygous mutations in the SLC34A3 gene that encodes the renal tubular phosphate transporter protein NaPi2c. The bone phenotype varies from severe rickets to no disease. Accurate diagnosis is important as the treatment differs from other forms of rickets. Methods: The patient was a 12-year-old boy from the Indian subcontinent with florid hypophosphatemic rickets. A targeted gene panel to search for mutations in genes associated with inherited forms of rickets was performed. We also completed a literature search of published cases of HHRH. Results: The targeted gene panel demonstrated a novel homozygous SLC34A3 mutation: c.1339 G>A (p.Ala447Thr). His parents were heterozygous for the mutation. In our literature review we found that people with homozygous SLC34A3 mutations were more likely to have rickets than those with compound heterozygous mutations (85% versus 45%, p<0.002) and that serum phosphate z scores were lower in those with rickets than those without (−3.3 with a standard deviation of 1.5 versus −2.1 with a standard deviation of 1.5, p<0.005). Conclusion: The bone phenotype of HHRH is related to the nature of the mutation and serum phosphate levels. Targeted gene panels can aid in the accurate diagnosis of inherited forms of rickets, and facilitate correct treatment.

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Hussain, Hafiz Muhammad Jafar, Meng Wang, Austin Huang, Ryan Schmidt, Xinye Qian, Paul Yang, Molly Marra, Yumei Li, Mark E. Pennesi y Rui Chen. "Novel Pathogenic Mutations Identified from Whole-Genome Sequencing in Unsolved Cases of Patients Affected with Inherited Retinal Diseases". Genes 14, n.º 2 (9 de febrero de 2023): 447. http://dx.doi.org/10.3390/genes14020447.

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Inherited retinal diseases (IRDs) are a diverse set of visual disorders that collectively represent a major cause of early-onset blindness. With the reduction in sequencing costs in recent years, whole-genome sequencing (WGS) is being used more frequently, particularly when targeted gene panels and whole-exome sequencing (WES) fail to detect pathogenic mutations in patients. In this study, we performed mutation screens using WGS for a cohort of 311 IRD patients whose mutations were undetermined. A total of nine putative pathogenic mutations in six IRD patients were identified, including six novel mutations. Among them, four were deep intronic mutations that affected mRNA splicing, while the other five affected protein-coding sequences. Our results suggested that the rate of resolution of unsolved cases via targeted gene panels and WES can be further enhanced with WGS; however, the overall improvement may be limited.

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Zhou, Zhen Ni, Melissa K. Frey, Dimitrios Nasioudis, Ann Carlson, Jessica Fields, Kevin Holcomb y Eloise Chapman-Davis. "Patterns of genetic screening for hereditary cancer syndromes: Effect of Supreme Court’s ruling invalidating single gene patent rights." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): 1580. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.1580.

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1580 Background: In 6/2013 the Supreme Court ruled that isolated DNA sequences found in nature could not be patented, resulting in rapid uptake of multigene panels. We sought to explore trends in genetic testing since this ruling. Methods: Results of all patients undergoing genetic testing and counseling at a single institution between 7/1/13 and 12/31/16 were reviewed. Associations between categorical variables were evaluated by chi-square tests or Fisher's exact tests as appropriate for category size. Results: 1663 patients underwent genetic testing over the study period. The median age was 49 years (range 18-86). Use of multigene panels versus targeted gene testing increased significantly in the years following the Supreme Court ruling (Table 1, P<0.001). While the percentage of patients found to have pathogenic mutations remained stable over the study period (9%), detection of variants of uncertain significance (VUS) increased significantly (Table 1, P<0.001). In 2013 BRCA1/2 mutations accounted for 91% of identified mutations; however this number decreased over time (2014-83%, 2015-70%, 2016-58%, P=0.01). Use of multigene panels detected 71% of mutations in non- BRCA1/2 genes such as CHEK(19), APC(44), MSH6(1), P53(1), and PTEN(1). Patients with a personal history of breast and/or ovarian cancer were more likely to have targeted testing than patients with other cancer types (590, 66% vs. 9, 33%, P=0.001). Conclusions: The uptake of multigene panels has increased since the 2013 Supreme Court ruling. While this technology allowed for the identification of many cancer-related genes that would be missed on targeted BRCA1/2 testing, it also resulted in a significantly increased detection of VUS, a finding with unknown clinical implications. [Table: see text]

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Bruno, Rossella y Gabriella Fontanini. "Next Generation Sequencing for Gene Fusion Analysis in Lung Cancer: A Literature Review". Diagnostics 10, n.º 8 (27 de julio de 2020): 521. http://dx.doi.org/10.3390/diagnostics10080521.

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Gene fusions have a pivotal role in non-small cell lung cancer (NSCLC) precision medicine. Several techniques can be used, from fluorescence in situ hybridization and immunohistochemistry to next generation sequencing (NGS). Although several NGS panels are available, gene fusion testing presents more technical challenges than other variants. This is a PubMed-based narrative review aiming to summarize NGS approaches for gene fusion analysis and their performance on NSCLC clinical samples. The analysis can be performed at DNA or RNA levels, using different target enrichment (hybrid-capture or amplicon-based) and sequencing chemistries, with both custom and commercially available panels. DNA sequencing evaluates different alteration types simultaneously, but large introns and repetitive sequences can impact on the performance and it does not discriminate between expressed and unexpressed gene fusions. RNA-based targeted approach analyses and quantifies directly fusion transcripts and is more accurate than DNA panels on tumor tissue, but it can be limited by RNA quality and quantity. On liquid biopsy, satisfying data have been published on circulating tumor DNA hybrid-capture panels. There is not a perfect method for gene fusion analysis, but NGS approaches, though still needing a complete standardization and optimization, present several advantages for the clinical practice.

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Benz, Stephen Charles, Shahrooz Rabizadeh, John Z. Sanborn, Charles Joseph Vaske, Gary A. Palmer y Patrick Soon-Shiong. "Protein expression by genetic mutations identified in gene panels (hotspots) and efficacy of targeted treatments." Journal of Clinical Oncology 33, n.º 15_suppl (20 de mayo de 2015): 11005. http://dx.doi.org/10.1200/jco.2015.33.15_suppl.11005.

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Schlicker, Andreas, Garry Beran, Christine M. Chresta, Gael McWalter, Alison Pritchard, Susie Weston, Sarah Runswick et al. "Use of colorectal cancer subtypes identified through iterative clustering to predict response to therapy." Journal of Clinical Oncology 30, n.º 4_suppl (1 de febrero de 2012): 482. http://dx.doi.org/10.1200/jco.2012.30.4_suppl.482.

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482 Background: Colorectal cancer (CRC) is generally stratified based on genetic and epigenetic features, such as KRAS mutation and microsatellite instability status. In order to facilitate the development of new targeted drugs and treatment regimens, it is important to redefine CRC at the molecular level by identifying subtypes that are relevant for response to targeted therapy. Methods: We applied a new unsupervised approach for iteratively stratifying tumor samples using genome-wide mRNA expression data. The resulting gene expression signatures were used to subtype CRC cell line panels and publicly available CRC tumor datasets. We employed pharmacological data on the cell line panels to link the subtypes to therapy response. Results: Starting from a gene expression dataset of 63 CRC tumor samples, we employed non-negative matrix factorization (NMF) and identified two dominant CRC subtypes. In agreement with previously published results, one of the types showed a mesenchymal and the other an epithelial-like gene expression pattern. In a second step, we applied NMF on these two dominant subtypes and further stratified them into two and three subtypes, respectively. The resulting five CRC subtypes show many differences, most notably activation of specific signaling pathways. Importantly, we recovered these five subtypes in several independent, publicly available CRC datasets. This strongly suggests that the signatures capture disease-relevant features of CRC. Furthermore, we found that the different subtypes corresponded to different cell lines in a panel of CRC cell lines. The clustered CRC cell lines displayed differential responses to a number of targeted compounds, indicating that the new CRC clusters may represent disease subtypes that of differential drug sensitivity. Conclusions: The CRC subtypes discovered using our new method offer new insights into the functional and molecular processes driving CRC. Furthermore, the evidence suggests that these subtypes may differ in activated pathway status and the response to some targeted inhibitors, indicating that targeting pathways conserved in these subtypes may provide new drug discovery opportunities.

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Chen, Penghui, Longxia He, Xiuhong Pang, Xiaowen Wang, Tao Yang y Hao Wu. "NLRP3 Is Expressed in the Spiral Ganglion Neurons and Associated with Both Syndromic and Nonsyndromic Sensorineural Deafness". Neural Plasticity 2016 (2016): 1–6. http://dx.doi.org/10.1155/2016/3018132.

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Nonsyndromic deafness is genetically heterogeneous but phenotypically similar among many cases. Though a variety of targeted next-generation sequencing (NGS) panels has been recently developed to facilitate genetic screening of nonsyndromic deafness, some syndromic deafness genes outside the panels may lead to clinical phenotypes similar to nonsyndromic deafness. In this study, we performed comprehensive genetic screening in a dominant family in which the proband was initially diagnosed with nonsyndromic deafness. No pathogenic mutation was identified by targeted NGS in 72 nonsyndromic and another 72 syndromic deafness genes. Whole exome sequencing, however, identified a p.E313K mutation in NLRP3, a gene reported to cause syndromic deafness Muckle-Wells Syndrome (MWS) but not included in any targeted NGS panels for deafness in previous reports. Follow-up clinical evaluation revealed only minor inflammatory symptoms in addition to deafness in six of the nine affected members, while the rest, three affected members, including the proband had no obvious MWS-related inflammatory symptoms. Immunostaining of the mouse cochlea showed a strong expression of NLRP3 in the spiral ganglion neurons. Our results suggested that NLRP3 may have specific function in the spiral ganglion neurons and can be associated with both syndromic and nonsyndromic sensorineural deafness.

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Kuang, Shelley, Andrea S. Fung, Kirstin A. Perdrizet, Kaitlin Chen, Janice J. N. Li, Lisa W. Le, Michael Cabanero et al. "Upfront Next Generation Sequencing in Non-Small Cell Lung Cancer". Current Oncology 29, n.º 7 (22 de junio de 2022): 4428–37. http://dx.doi.org/10.3390/curroncol29070352.

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In advanced non-small cell lung cancer (NSCLC), patients with actionable genomic alterations may derive additional clinical benefit from targeted treatment compared to cytotoxic chemotherapy. Current guidelines recommend extensive testing with next generation sequencing (NGS) panels. We investigated the impact of using a targeted NGS panel (TruSight Tumor 15, Illumina) as reflex testing for NSCLC samples at a single institution. Molecular analysis examined 15 genes for hotspot mutation variants, including AKT1, BRAF, EGFR, ERBB2, FOXL2, GNA11, GNAQ, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, RET and TP53 genes. Between February 2017 and October 2020, 1460 samples from 1395 patients were analyzed. 1201 patients (86.1%) had at least one variant identified, most frequently TP53 (47.5%), KRAS (32.2%) or EGFR (24.2%). Among these, 994 patients (71.3%) had clinically relevant variants eligible for treatment with approved therapies or clinical trial enrollment. The incremental cost of NGS beyond single gene testing (EGFR, ALK) was CAD $233 per case. Reflex upfront NGS identified at least one actionable variant in more than 70% of patients with NSCLC, with minimal increase in testing cost. Implementation of NGS panels remains essential as treatment paradigms continue to evolve.

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Han, Ji Yoon y In Goo Lee. "Genetic tests by next-generation sequencing in children with developmental delay and/or intellectual disability". Clinical and Experimental Pediatrics 63, n.º 6 (15 de junio de 2020): 195–202. http://dx.doi.org/10.3345/kjp.2019.00808.

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Developments in next-generation sequencing (NGS) techogies have assisted in clarifying the diagnosis and treatment of developmental delay/intellectual disability (DD/ID) via molecular genetic testing. Advances in DNA sequencing technology have not only allowed the evolution of targeted panels but also, and more currently enabled genome-wide analyses to progress from research era to clinical practice. Broad acceptance of accuracy- guided targeted gene panel, whole-exome sequencing (WES), and whole-genome sequencing (WGS) for DD/ID need prospective analyses of the increasing cost-effectiveness versus conventional genetic testing. Choosing the appropriate sequencing method requires individual planning. Data are required to guide best-practice recommendations for genomic testing, regarding various clinical phenotypes in an etiologic approach. Targeted panel testing may be recommended as a first-tier testing approach for children with DD/ID. Family-based trio testing by WES/WGS can be used as a second test for DD/ ID in undiagnosed children who previously tested negative on a targeted panel. The role of NGS in molecular diagnostics, treatment, prediction of prognosis will continue to increase further in the coming years. Given the rapid pace of changes in the past 10 years, all medical providers should be aware of the changes in the transformative genetics field.

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Maksemous, Neven, Robert A. Smith, Heidi G. Sutherland, Bridget H. Maher, Omar Ibrahim, Garth A. Nicholson, Elisabeth P. Carpenter, Rod A. Lea, M. Zameel Cader y Lyn R. Griffiths. "Targeted next generation sequencing identifies a genetic spectrum of DNA variants in patients with hemiplegic migraine". Cephalalgia Reports 2 (1 de enero de 2019): 251581631988163. http://dx.doi.org/10.1177/2515816319881630.

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Objective: Hemiplegic migraine in both familial (FHM) and sporadic (SHM) forms is a rare subtype of migraine with aura that can be traced to mutations in the CACNA1A, ATP1A2 and SCN1A genes. It is characterised by severe attacks of typical migraine accompanied by hemiparesis, as well as episodes of complex aura that vary significantly between individuals. Methods: Using a targeted next generation sequencing (NGS) multigene panel, we have sequenced the genomic DNA of 172 suspected hemiplegic migraine cases, in whom no mutation had previously been found by Sanger sequencing (SS) of a limited number of exons with high mutation frequency in FHM genes. Results: Genetic screening identified 29 variants, 10 of which were novel, in 35 cases in the three FHM genes ( CACNA1A, ATP1A2 and SCN1A). Interestingly, in this suspected HM cohort, the ATP1A2 gene harboured the highest number of variants with 24/35 cases (68.6%), while CACNA1A ranked the second gene, with 5 variants identified in 7/35 cases (20%). All detected variants were confirmed by SS and were absent in 100 non-migraine healthy control individuals. Assessment of variants with the American College of Medical Genetics and Genomics guidelines classified 8 variants as pathogenic, 3 as likely pathogenic and 18 as variants of unknown significance. Targeted NGS gene panel increased the diagnostic yield by fourfold over iterative SS in our diagnostics facility. Conclusion: We have identified 29 potentially causative variants in an Australian and New Zealand cohort of suspected HM cases and found that the ATP1A2 gene was the most commonly mutated gene. Our results suggest that screening using NGS multigene panels to investigate ATP1A2 alongside CACNA1A and SCN1A is a clinically useful and efficient method.

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Grossi, Alice, Maurizio Miano, Marina Lanciotti, Francesca Fioredda, Daniela Guardo, Elena Palmisani, Paola Terranova et al. "Targeted NGS Yields Plentiful Ultra-Rare Variants in Inborn Errors of Immunity Patients". Genes 12, n.º 9 (24 de agosto de 2021): 1299. http://dx.doi.org/10.3390/genes12091299.

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Inborn errors of immunity (IEI) include a large group of inherited diseases sharing either poor, dysregulated, or absent and/or acquired function in one or more components of the immune system. Next-generation sequencing (NGS) has driven a rapid increase in the recognition of such defects, though the wide heterogeneity of genetically diverse but phenotypically overlapping diseases has often prevented the molecular characterization of the most complex patients. Two hundred and seventy-two patients were submitted to three successive NGS-based gene panels composed of 58, 146, and 312 genes. Along with pathogenic and likely pathogenic causative gene variants, accounting for the corresponding disorders (37/272 patients, 13.6%), a number of either rare (probably) damaging variants in genes unrelated to patients’ phenotype, variants of unknown significance (VUS) in genes consistent with their clinics, or apparently inconsistent benign, likely benign, or VUS variants were also detected. Finally, a remarkable amount of yet unreported variants of unknown significance were also found, often recurring in our dataset. The NGS approach demonstrated an expected IEI diagnostic rate. However, defining the appropriate list of genes for these panels may not be straightforward, and the application of unbiased approaches should be taken into consideration, especially when patients show atypical clinical pictures.

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Prieto-Potin, Iván, Nerea Carvajal, Jenifer Plaza-Sánchez, Rebeca Manso, Carmen Laura Aúz-Alexandre, Cristina Chamizo, Sandra Zazo et al. "Validation and clinical application of a targeted next-generation sequencing gene panel for solid and hematologic malignancies". PeerJ 8 (6 de octubre de 2020): e10069. http://dx.doi.org/10.7717/peerj.10069.

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Background Next-generation sequencing (NGS) is a high-throughput technology that has become widely integrated in molecular diagnostics laboratories. Among the large diversity of NGS-based panels, the Trusight Tumor 26 (TsT26) enables the detection of low-frequency variants across 26 genes using the MiSeq platform. Methods We describe the inter-laboratory validation and subsequent clinical application of the panel in 399 patients presenting a range of tumor types, including gastrointestinal (GI, 29%), hematologic (18%), lung (13%), gynecological and breast (8% each), among others. Results The panel is highly accurate with a test sensitivity of 92%, and demonstrated high specificity and positive predictive values (95% and 96%, respectively). Sequencing testing was successful in two-thirds of patients, while the remaining third failed due to unsuccessful quality-control filtering. Most detected variants were observed in the TP53 (28%), KRAS (16%), APC (10%) and PIK3CA (8%) genes. Overall, 372 variants were identified, primarily distributed as missense (81%), stop gain (9%) and frameshift (7%) altered sequences and mostly reported as pathogenic (78%) and variants of uncertain significance (19%). Only 14% of patients received targeted treatment based on the variant determined by the panel. The variants most frequently observed in GI and lung tumors were: KRAS c.35G > A (p.G12D), c.35G > T (p.G12V) and c.34G > T (p.G12C). Conclusions Prior panel validation allowed its use in the laboratory daily practice by providing several relevant and potentially targetable variants across multiple tumors. However, this study is limited by high sample inadequacy rate, raising doubts as to continuity in the clinical setting.

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Kolokotronis, Konstantinos, Natalie Pluta, Eva Klopocki, Erdmute Kunstmann, Daniel Messroghli, Christoph Maack, Shai Tejman-Yarden, Michael Arad, Simone Rost y Brenda Gerull. "New Insights on Genetic Diagnostics in Cardiomyopathy and Arrhythmia Patients Gained by Stepwise Exome Data Analysis". Journal of Clinical Medicine 9, n.º 7 (9 de julio de 2020): 2168. http://dx.doi.org/10.3390/jcm9072168.

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Inherited cardiomyopathies are characterized by clinical and genetic heterogeneity that challenge genetic diagnostics. In this study, we examined the diagnostic benefit of exome data compared to targeted gene panel analyses, and we propose new candidate genes. We performed exome sequencing in a cohort of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia, and we analyzed the data following a stepwise approach. Overall, in 64% of patients, a variant of interest (VOI) was detected. The detection rate in the main sub-cohort consisting of patients with dilated cardiomyopathy (DCM) was much higher than previously reported (25/36; 69%). The majority of VOIs were found in disease-specific panels, while a further analysis of an extended panel and exome data led to an additional diagnostic yield of 13% and 5%, respectively. Exome data analysis also detected variants in candidate genes whose functional profile suggested a probable pathogenetic role, the strongest candidate being a truncating variant in STK38. In conclusion, although the diagnostic yield of gene panels is acceptable for routine diagnostics, the genetic heterogeneity of cardiomyopathies and the presence of still-unknown causes favor exome sequencing, which enables the detection of interesting phenotype–genotype correlations, as well as the identification of novel candidate genes.

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Fowler, Anna, Shazia Mahamdallie, Elise Ruark, Sheila Seal, Emma Ramsay, Matthew Clarke, Imran Uddin et al. "Accurate clinical detection of exon copy number variants in a targeted NGS panel using DECoN". Wellcome Open Research 1 (25 de noviembre de 2016): 20. http://dx.doi.org/10.12688/wellcomeopenres.10069.1.

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Background: Targeted next generation sequencing (NGS) panels are increasingly being used in clinical genomics to increase capacity, throughput and affordability of gene testing. Identifying whole exon deletions or duplications (termed exon copy number variants, ‘exon CNVs’) in exon-targeted NGS panels has proved challenging, particularly for single exon CNVs. Methods: We developed a tool for the Detection of Exon Copy Number variants (DECoN), which is optimised for analysis of exon-targeted NGS panels in clinical settings. We evaluated DECoN performance using 96 samples with independently validated exon CNV data. We performed simulations to evaluate DECoN detection performance of single exon CNVs and evaluate performance using different coverage levels and sample numbers. Finally, we implemented DECoN in a clinical laboratory that tests BRCA1 and BRCA2 with the TruSight Cancer Panel (TSCP). We used DECoN to analyse 1,919 samples, validating exon CNV detections by multiplex ligation-dependent probe amplification (MLPA). Results: In the evaluation set, DECoN achieved 100% sensitivity and 99% specificity for BRCA exon CNVs, including identification of 8 single exon CNVs. DECoN also identified 14/15 exon CNVs in 8 other genes. Simulations of all possible BRCA single exon CNVs gave a mean sensitivity of 98% for deletions and 95% for duplications. DECoN performance remained excellent with different levels of coverage and sample numbers; sensitivity and specificity was >98% with the typical NGS run parameters. In the clinical pipeline, DECoN automatically analyses pools of 48 samples at a time, taking 24 minutes per pool, on average. DECoN detected 24 BRCA exon CNVs, of which 23 were confirmed by MLPA, giving a false discovery rate of 4%. Specificity was 99.7%. Conclusions: DECoN is a fast, accurate, exon CNV detection tool readily implementable in research and clinical NGS pipelines. It has high sensitivity and specificity and acceptable false discovery rate. DECoN is freely available at www.icr.ac.uk/decon.

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Bayle, Arnaud, Debora Basile, Simon Garinet, Bastien Rance, Pierre Laurent-Puig, Hélène Blons, Julien Taieb y Geraldine Perkins. "Next-Generation Sequencing Targeted Panel in Routine Care for Metastatic Colon Cancers". Cancers 13, n.º 22 (17 de noviembre de 2021): 5750. http://dx.doi.org/10.3390/cancers13225750.

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In digestive oncology, the clinical impact of targeted next-generation sequencing (NGS) in routine practice should be addressed. In this work, we studied the impact of a 22-gene NGS amplicon-based panel with Ion Torrent Proton Sequencing, prospectively performed in routine practice. We analyzed the results of extended molecular testing, beyond RAS and BRAF, in metastatic colorectal cancer (mCRC) patients in a single-center, retrospective, observational study of consecutive mCRC patients followed up at the Georges Pompidou European Hospital between January 2016 and December 2018. Overall, 210 patients with mCRC were included. Median follow-up was 25.4 months (IQR: 14.9–39.5). The three most frequently mutated genes were: TP53 (63%), KRAS (41%) and PIK3CA (19%). A positive association was found between overall survival and performance status (PS) ≥ 2 (HR: 4.91 (1.84–13.1); p = 0.001) and differentiation (HR: 4.70 (1.51–14.6); p = 0.007) in multivariate analysis. The NGS panel enabled five patients to access a targeted therapy not currently registered for CRC. In conclusion, targeted NGS panels in mCRC are feasible in routine practice, but need to be regularly updated and in-depth studies are needed to better analyze the prognostic factors.

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Dasdemir, Selcuk, Mehmet Yildiz, Damla Celebi, Sezgin Sahin, Numune Aliyeva, Fatih Haslak, Aybuke Gunalp et al. "Genetic screening of early-onset patients with systemic lupus erythematosus by a targeted next-generation sequencing gene panel". Lupus 31, n.º 3 (27 de enero de 2022): 330–37. http://dx.doi.org/10.1177/09612033221076733.

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Objective In this study, we aimed to screen 31 genes (C1QA, C1QB, C1QC, C1R, C1S, C2, C3, TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, DNASE1, DNASE1L3, PRKCD, ACP5, SLC7A7, IFIH1, TMEM173, ISG15, CYBB, FAS, FASLG, KRAS, NRAS, MAN2B1, PEPD, PTPN11, RAG2, and SHOC2), that we have categorized under the umbrella term “monogenic lupus” using a targeted next-generation sequencing (NGS) panel in 24 individuals with early-onset (≤10 years of age) systemic lupus erythematosus (SLE) and in 24 patients with late-onset (>10 years of age) disease. Methods A total of 48 SLE patients (24 with disease onset ≤10 years of age and 24 with disease onset >10 years of age) were included. Patients with late-onset disease have been used as patient controls. Sequencing was carried out using 400 bp kit on the Ion S5 system. Results Among the 48 patients, three had one pathogenic variant and 45 patients had at least one rare variant classified as benign, likely benign or variant of unknown significance (VUS). In all three patients with a pathogenic variant, the onset of disease was before 10 years of age. Two patients (they were siblings) carried C1QA homozygote pathogenic allele (p.Gln208Ter, rs121909581), and one patient carried PEPD heterozygote pathogenic allele (p.Arg184Gln, rs121917722). Conclusion We demonstrated a pathogenic variant in our target gene panel with a frequency of 9.52% in patients with a disease onset ≤10 years of age. All patients with early-onset SLE phenotype, irrespective of a positive family history for SLE or parental consanguinity, should be scanned for a single-gene defect by a targeted gene panel sequencing. With the discovery of many single-gene defects and ongoing efforts to identify novel genes in SLE, similar gene panels including even more genes will possibly become more necessary and practical in the future.

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Blanch, Salvador, Antonio Fernandez-Serra, Ignacio Romero, Zaida Garcia-Casado, Carmen Illueca, Pedro Mallol, Jose Antonio Lopez-Guerrero y Andres Poveda. "Genomic characterization of high-grade serous ovarian Cancer by using targeted RNA and DNAseq gene panels." Journal of Clinical Oncology 34, n.º 15_suppl (20 de mayo de 2016): e17060-e17060. http://dx.doi.org/10.1200/jco.2016.34.15_suppl.e17060.

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Manca, P., I. Mallona, D. Santini, G. Tonini, C. D. Rolfo, M. D. Robinson y F. Pantano. "A new bioinformatic pipeline allows the design of small, targeted gene panels for efficient TMB estimation". Annals of Oncology 30 (abril de 2019): ii8. http://dx.doi.org/10.1093/annonc/mdz073.003.

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Gil, José Vicente, Esperanza Such, Claudia Sargas, Javier Simarro, Alberto Miralles, Gema Pérez, Inmaculada de de Juan et al. "Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia". International Journal of Molecular Sciences 24, n.º 5 (23 de febrero de 2023): 4440. http://dx.doi.org/10.3390/ijms24054440.

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The molecular landscape of acute lymphoblastic leukemia (ALL) is highly heterogeneous, and genetic lesions are clinically relevant for diagnosis, risk stratification, and treatment guidance. Next-generation sequencing (NGS) has become an essential tool for clinical laboratories, where disease-targeted panels are able to capture the most relevant alterations in a cost-effective and fast way. However, comprehensive ALL panels assessing all relevant alterations are scarce. Here, we design and validate an NGS panel including single-nucleotide variants (SNVs), insertion–deletions (indels), copy number variations (CNVs), fusions, and gene expression (ALLseq). ALLseq sequencing metrics were acceptable for clinical use and showed 100% sensitivity and specificity for virtually all types of alterations. The limit of detection was established at a 2% variant allele frequency for SNVs and indels, and at a 0.5 copy number ratio for CNVs. Overall, ALLseq is able to provide clinically relevant information to more than 83% of pediatric patients, making it an attractive tool for the molecular characterization of ALL in clinical settings.

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Bayegan, Amir, Julien Tessier, Emma Wang, Adalis Maisonet, Shu Yan, Shannon McGrath, Donald G. Jackson y Jack Pollard. "Abstract 762: Practical guidelines for the design of single cell sequencing studies". Cancer Research 82, n.º 12_Supplement (15 de junio de 2022): 762. http://dx.doi.org/10.1158/1538-7445.am2022-762.

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Abstract Performance of Single Cell RNA sequencing (scRNA-seq) experiments depends on multiple factors including the number of cells loaded, cell recovery rates, and sequencing coverage. We optimized such factors in 10x Genomics gene expression profiling and compared targeted panels to whole transcriptome sequencing using Human donor PBMC samples. We expect that our findings will apply to scRNA-seq studies of other sample types as well. Optimizing cell recovery in scRNA-seq experiments is essential in order to ensure that changes in low-abundance cell populations (e.g. Treg) can be accurately quantified. Statistical analysis indicated that at least 100 cells are needed to assess cell-type specific state changes. We expect to recover 40-65% of cells loaded into the 10x chip. We found that loading 20k-30k cells/lane combined with cell hashtags for improved doublet detection improved cell recovery over the recommended loading of 10K-16K cells. We also surveyed internal single cell experiments and observed variable cell recovery across different sample types. The amount of ambient RNA in the library was correlated with lower performance in single cell studies, suggesting that cell death or damage was causing lower recovery. We found that using PBS for cell resuspension (which is less likely to cause cell rupture) performs as well as the nuclease-free water suggested by 10x. We also assessed the impact of sequencing depth and protocol on scRNA-seq data quality. Sequencing depth impacts both experiment cost and data quality due to dropouts. We performed computational simulations of lower depth sequencing by subsampling various number of reads from PBMC experiments to obtain coverages of 10K, 20K, 40K and 80K reads per cell. We observed that 40K reads per cell, yielding about 70% sequencing saturation, provided a good balance between cost and sequencing depth. Finally, we compared dropout rates and cell type annotation for two targeted panels, the Human Gene Signature and Human Immunology panels to whole transcriptome scRNAseq. On average we detected only 200 genes out of over 1000 represented in each panel. Dropout rates for targeted panels were only reduced at low coverage; at read depths higher than 40K reads per cell the whole transcriptome and targeted panels had similar dropout rates. Both methods detected major immune cell types, but targeted sequencing could not accurately identify some subtypes due to low number of detected genes. We conclude that for immune cell profiling whole-transcriptome analysis at coverage of 40K reads per cell or higher with inputs of 20k-30k cells and use of sample hashtag antibodies provides the best balance of experiment cost, cell recovery and transcriptome coverage. Although these guidelines were established for Human PBMCs we expect similar outcomes with other complex cell mixtures such as dissociated tissues. Citation Format: Amir Bayegan, Julien Tessier, Emma Wang, Adalis Maisonet, Shu Yan, Shannon McGrath, Donald G. Jackson, Jack Pollard. Practical guidelines for the design of single cell sequencing studies [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 762.

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Yan, Benedict, Yongli Hu, Christopher Ng, Kenneth H. K. Ban, Tin Wee Tan, Pei Tee Huan, Peak-Ling Lee et al. "Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms". Journal of Clinical Pathology 69, n.º 9 (19 de febrero de 2016): 801–4. http://dx.doi.org/10.1136/jclinpath-2015-203580.

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AimsPCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in an off-the-shelf NGS panel (TruSight Myeloid Sequencing Panel) for myeloid neoplasms and attempted to patch the low read depth for one of the affected genes, CEBPA.MethodsWe performed targeted NGS of 158 acute myeloid leukaemia samples and analysed the amplicon read depths across 568 amplicons to identify lower-performance amplicons. We also correlated the amplicon read depths with the template GC content. Finally, we attempted to patch the low read depth for CEBPA using a parallel library preparation (Nextera XT) workflow.ResultsWe identified 16 lower-performance amplicons affecting nine genes, including CEBPA. There was a slight negative correlation between the amplicon read depths and template GC content. Addition of the separate CEBPA library generated a minimum read depth per base across the CEBPA gene ranging from 268x to 758x across eight samples.ConclusionsThe identification of lower-performance amplicons will be informative to laboratories intending to use this panel. We have also demonstrated proof-of-concept that different libraries (TruSight Myeloid and Nextera XT) can be combined and sequenced on the same flow cell to generate additional reads for CEBPA.

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Vansant, Gordon, Mark Landers, Lien Vo, Kahuku Oades, Hyunsoo Kim, Jerry Lee, Rhonda Meredith, Byung-In Lee y Joseph Monforte. "NGS-based targeted RNA sequencing for expression profiling and relative quantitation of specific gene isoforms and fusions in tumor-specific panels." Journal of Clinical Oncology 31, n.º 15_suppl (20 de mayo de 2013): 11108. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.11108.

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11108 Background: Gene expression signatures have become a useful tool for the identification of tumor subtypes and response to specific therapies. Expression of tumor, metastatic and macrophage specific transcripts utilizing alternative promoters and transcriptional start sites can further characterize these tumors . NGS is a powerful tool for gene expression analysis, however larger sample input requirements (>100ng) and excessive sequencing depth requirements (30-40M tags/sample) to detect the expression of rare isoforms or fusions in tumor samples are prohibitive for clinical assay development. We describe the development of a targeted RNA sequencing assay for the relative quantitation of specific gene expression signatures, known splice variants and gene fusions from less than 100 ng of starting material in a single tube universal amplification format. Methods: Primers for 52 genes, isoforms and gene fusion products were designed using the universal amplification strategy. 10 ng of RNA from 5 matched tumor/adjacent normal breast cancer tumor pairs were assayed. Libraries were prepared for sequencing by emPCR and sequenced on Ion Torrent PGM. Data were aligned via TMAP. Relative expression was determined vs. housekeeping genes or wild type transcripts. Results: All gene targets were detected at significant levels in at least one tumor sample. Robust expression profiling (5 log dynamic range) was obtained from FFPE macrodissected tumor and normal samples with as little as 200K reads/sample. Immune specific transcripts demonstrated differential expression (CCL3, AIF, FCGR3A and CSF1) across patients and matched pairs as well as an upregulation of CXCL12, indicative of tumor associated macrophages. Conclusions: Targeted RNAseq demonstrates detection and quantitation of relative expression levels of not only tumor subclass specific gene expression signatures, but immune cell specific transcripts from 10ng of FFPE derived total RNA derived from macrodissected tumor samples. The lower input requirements, quicker turnaround time and incredible sensitivity of targeted RNAseq make this assay a useful tool for clinical assay development.

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Seeboeck, Rita, Victoria Sarne y Johannes Haybaeck. "Current Coverage of the mTOR Pathway by Next-Generation Sequencing Oncology Panels". International Journal of Molecular Sciences 20, n.º 3 (5 de febrero de 2019): 690. http://dx.doi.org/10.3390/ijms20030690.

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The mTOR pathway is in the process of establishing itself as a key access-point of novel oncological drugs and targeted therapies. This is also reflected by the growing number of mTOR pathway genes included in commercially available next-generation sequencing (NGS) oncology panels. This review summarizes the portfolio of medium sized diagnostic, as well as research destined NGS panels and their coverage of the mTOR pathway, including 16 DNA-based panels and the current gene list of Foundation One as a major reference entity. In addition, we give an overview of interesting, mTOR-associated somatic mutations that are not yet incorporated. Especially eukaryotic translation initiation factors (eIFs), a group of mTOR downstream proteins, are on the rise as far as diagnostics and drug targeting in precision medicine are concerned. This review aims to raise awareness for the true coverage of NGS panels, which should be valuable in selecting the ideal platform for diagnostics and research.

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Avram, Carmen M., Aaron B. Caughey, Mary E. Norton y Teresa N. Sparks. "Cost-Effectiveness of Exome Sequencing versus Targeted Gene Panels for Prenatal Diagnosis of Non-Immune Hydrops Fetalis". American Journal of Obstetrics and Gynecology 226, n.º 1 (enero de 2022): S176—S177. http://dx.doi.org/10.1016/j.ajog.2021.11.307.

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Lynce, Filipa y Claudine Isaacs. "How Far Do We Go With Genetic Evaluation? Gene, Panel, and Tumor Testing". American Society of Clinical Oncology Educational Book, n.º 36 (mayo de 2016): e72-e78. http://dx.doi.org/10.1200/edbk_160391.

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The traditional model by which an individual was identified as harboring a hereditary susceptibility to cancer was to test for a mutation in a single gene or a finite number of genes associated with a particular syndrome (e.g., BRCA1 and BRCA2 for hereditary breast and ovarian cancer or mismatch repair genes for Lynch syndrome). The decision regarding which gene or genes to test for was based on a review of the patient’s personal medical history and their family history. With advances in next-generation DNA sequencing technology, offering simultaneous testing for multiple genes associated with a hereditary susceptibility to cancer is now possible. These panels typically include high-penetrance genes, but they also often include moderate- and low-penetrance genes. A number of the genes included in these panels have not been fully characterized either in terms of their cancer risks or their management options. Another way some patients are unexpectedly identified as carrying a germline mutation in a cancer susceptibility gene is at the time they undergo molecular profiling of their tumor, which typically has been carried out to guide treatment choices for their cancer. This article first focuses on the issues that need to be considered when deciding between recommending more targeted testing of a single or a small number of genes associated with a particular syndrome (single/limited gene testing) versus performing a multigene panel. This article also reviews the issues regarding germline risk that occur within the setting of ordering molecular profiling of tumors.

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Atli, Emine, Hakan Gurkan, Engin Atli, Hakki Onur Kirkizlar, Sinem Yalcintepe, Selma Demir, Ufuk Demirci et al. "THE IMPORTANCE OF TARGETED NEXT-GENERATION SEQUENCING USAGE IN CYTOGENETICALLY NORMAL MYELOID MALIGNANCIES". Mediterranean Journal of Hematology and Infectious Diseases 13, n.º 1 (31 de diciembre de 2020): e2021013. http://dx.doi.org/10.4084/mjhid.2021.013.

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Advanced diagnostic methods give an advantage for the identification of the abnormalities in myeloid malignancies. Various researchers have shown the potential importance of genetic tests both before the onset of the disease and during the remission. Large testing panels prevents false negative results in myeloid malignancies. But the important question is how the results of conventional cytogenetic and molecular cytogenetic techniques can be merged together with NGS technologies. In this paper, we drew an algorithm for evaluation of the myeloid malignancies. In order to evaluate genetic abnormalities, we performed cytogenetics, molecular cytogenetics and NGS testing in hematologic malignancies. In this study, we analyzed 100 patients who admitted to Medical Genetics Laboratory within different type of myeloid malignancies. We highlighted the possible diagnostic algorithm for cytogenetically normal cases. We applied NGS 141 gene panel for cytogenetically normal patients and we detected two or more pathogenic variations in 61 out of 100 patients (61%). The pathogenic variation detection rate of NGS varies in disease groups: AML were 85% and MDS were 23%. Here, we identified 24 novel variation out of total pathogenic variations in myeloid malignancies. A total 18 novel variation were identified in AML and 6 novel variation were identified in MDS. Despite of long turnaround time, conventional techniques are still golden standard for myeloid malignancies but sometimes cryptic gene fusions or complex abnormalities cannot be identified easily by conventional techniques. In these conditions, advanced technologies like NGS are highly recommended.

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