The large outbreak of diarrhea and hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli O104:H4 in Europe from May to July 2011 highlighted the potential of a rarely identified E. coli serogroup to cause severe disease. Prior to the outbreak, there were very few reports of disease caused by this pathogen and thus little known of its diversity and evolution. The identification of cases of HUS caused by E. coli O104:H4 in France and Turkey after the outbreak and with no clear epidemiological links raises questions about whether these sporadic cases are derived from the outbreak. Here, we report genome sequences of five independent isolates from these cases and results of a comparative analysis with historical and 2011 outbreak isolates. These analyses revealed that the five isolates are not derived from the outbreak strain; however, they are more closely related to the outbreak strain and each other than to isolates identified prior to the 2011 outbreak. Over the short time scale represented by these closely related organisms, the majority of genome variation is found within their mobile genetic elements: none of the nine O104:H4 isolates compared here contain the same set of plasmids, and their prophages and genomic islands also differ. Moreover, the presence of closely related HUS-associated E. coli O104:H4 isolates supports the contention that fully virulent O104:H4 isolates are widespread and emphasizes the possibility of future food-borne E. coli O104:H4 outbreaks.
Introduction: Advances in precision medicine for solid tumors have led to an increase in the number of molecular biomarkers and signatures associated with clinical decisions and clinical trial eligibility. In advanced disease settings, timely clinical decision making informed by robust and accurate molecular biomarker testing is increasingly important. As such, there is a need for a comprehensive, decentralized and rigorously validated diagnostic test. To address this need, we developed and analytically validated PGDx elioTM tissue complete (ETC) assay, which reports somatic single nucleotide variants (SNVs), insertions and deletions (indels), amplifications, and rearrangements, as well as microsatellite stability (MSI) and tumor mutational burden (TMB). Methods: >500 FFPE tumor tissue specimens, across 35 tumor types and 9 organ systems, were analyzed using our 500+ gene targeted panel, ETC (assay in development). Accuracy of the results was compared to orthogonal methods, such as whole exome sequencing (WES), IHC, FISH, NGS, and other on-market IVD assays. Results for all variant types were analyzed for overall percent agreement (OPA). Additional analytical studies were performed to assess precision/repeatability for detection of somatic variants, evaluated across multiple operators, instruments and days. Results: 112 unique samples were evaluated for SNVs and indels by ETC and accuracy assessed via orthogonal validated NGS assays, demonstrating an OPA >99.9%. Detection of amplification events was assessed in 176 samples and concordance with orthogonal FISH and IHC assays demonstrated >93.0 % OPA. Rearrangement accuracy was evaluated across several clinically relevant translocations, with an >94.0 % OPA when compared to FISH and an RNA-based sequencing method (n=270). 115 pan cancer samples were analyzed for microsatellite status and accuracy with PCR was determined, with an overall agreement of 100.0%. Lastly, TMB was determined in 118 samples and results compared to WES-derived TMB, displaying a high level of concordance (Pearson correlation, p=0.903) across a range of TMB scores (0.2-89.7 muts/Mbp). Conclusions: The PGDx elio tissue complete assay system, including proprietary, automated bioinformatics, provides accurate and reproducible results for the detection of clinically relevant genetic alterations across tumor types. Further verification and validation studies of this gene panel are ongoing. The PGDx elio tissue complete assay will employ a decentralized, kitted model, increasing clinical accessibility to NGS and allow for delivery of highly accurate and timely results. Citation Format: Kelly M.R. Gerding, Kenneth C Valkenburg, Christina Oliveras, James R. White, Leila Ettehadieh, Gustavo Cerquiera, Christopher Gault, James Hernandez, Eric Kong, Samuel Angiuoli, John Simmons, Isabell Loftin, Abigail McElhinny. Analytical validation of a 500+ gene comprehensive NGS assay to detect genomic alterations across 35 tumor types [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B027. doi:10.1158/1535-7163.TARG-19-B027
Introduction: Genomic analysis using next-generation sequencing (NGS) enables simultaneous detection of targetable alterations and biomarkers with emerging clinical utility in non-small cell lung cancer (NSCLC) patients. Importantly, many of these alterations are mutually exclusive and tumor tissue for molecular testing is often limited. As such, we developed PGDx elioTM tissue complete (ETC) as a comprehensive NGS assay capable of detecting somatic single nucleotide variants (SNVs), insertions and deletions (indels), amplifications, and rearrangements, as well as microsatellite stability (MSI) and tumor mutational burden (TMB). Here, we present the performance of this assay in detecting key clinical variants in NSCLC. Methods: Studies comprising >300 NSCLC specimens (FFPE tissue and characterized cell lines) were analyzed using ETC, a 500+ gene assay (under development), across key clinically relevant variants in NSCLC, including tumor mutation burden (TMB). Accuracy of the results were compared to orthogonal methods (e.g. whole-exome sequencing (WES), IHC, FISH) and analyzed for the overall percent agreement (OPA). Additionally, archival FFPE samples from 46 NSCLC patients previously found to harbor ALK translocations, MET amplifications, MET exon 14 skipping mutations, EGFR mutations, and/or ROS1 translocations were also analyzed. The results were compared to orthogonal methods and the overall genomic landscape evaluated. Results: Clinical FFPE and characterized cell line specimens were evaluated for the following alterations: EGFR mutations (L858R, T790M, and Exon 19 deletions), BRAF V600E mutations, and ALK and ROS1 translocations. Compared to orthogonal methods, the NGS assay demonstrated >93% OPA across all variants. Comparison of TMB results to WES data demonstrated high accuracy and precision, across a range of DNA inputs (50-200 ng) and tumor purities (10-30%). In the 46 retrospective NSCLC cases, the NGS assay identified 15 ALK translocations, 6 MET amplifications, 1 MET exon 14 skipping mutation, and 5 EGFR mutations, with most being mutually exclusive. The majority of cases were confirmed by orthogonal assays, with the few apparent discordances likely due to tumor heterogeneity, assay distinctions, or analyte input. Higher TMB was found in cases without targetable alterations. Conclusions: ETC provides accurate and reproducible results for the detection of clinically relevant alterations in NSCLC. Further verification and validation studies of this gene panel are ongoing. Overall NGS showed excellent concordance with orthogonal variant detection methods. Importantly, ETC demonstrated added value in assessing all genomic alteration types in a single assay, as well as reporting composite genomic scores, suggesting that NGS may offer a comprehensive solution to molecular testing. Citation Format: Elizabeth Weingartner, Kelly M.R. Gerding, Gustavo Cerquiera, Christopher Gault, James Hernandez, Kenneth Valkenburg, Laurel Keefer, Eileen Sagini, Dorhyun Johng, Caitlin Gilley, Colby Ganey, Leila Ettehadieh, Diandra Denier, Christina Oliveras, Kartikeya Joshi, Eric Kong, Eniko Papp, Amy Greer, James R. White, Donna Nichol, John Simmons. Comparison of a comprehensive NGS profiling assay and conventional molecular testing approaches for detection of clinically relevant alterations in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B029. doi:10.1158/1535-7163.TARG-19-B029
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