Novel Next-generation Sequencing Research Articles

Role of Transcriptomics in Precision Oncology

: Transcriptome profiling is one of the most widely used approaches in the field of multiomics research. It plays a crucial role in the prognostic, diagnostic, and predictive treatment of cancer patients. Novel next-generation sequencing (NGS) technologies permit the identification of cancer biomarkers, gene signatures, and their abnormal expression, affecting oncogenic and molecular targets and novel biomarkers for cancer therapies. Multiomics studies have changed the overall understanding of cancer and opened a precise perspective for tumor diagnostics and therapy. The use of these approaches has strengthened our understanding of disease pathophysiology and classifications at the molecular level, including specific interference with drug mechanisms of action. Still, it has limited added value in the clinical setting. The omics data on precision medicine include the application of data from genes, transcripts, and proteins for diagnosis, monitoring of diseases, risk factor determination, counseling, and development of novel therapeutics. Bioinformatics applications have expanded statistics-based analysis toward deriving molecular pathways and process models for characterizing phenotypes and drug action mechanisms. In this review, we will discuss transcriptomics and interference analysis that allows the identification of predictive biomarkers at the molecular level to test drug response and analyze the molecular process interface of disease progression-relevant pathophysiology and mechanism of action to propose predictive biomarkers.

Use of next-generation sequencing to detect mutations associated with antiviral drug resistance in cytomegalovirus.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality among immunocompromised hosts, including transplant recipients. Antiviral prophylaxis or treatment is used to reduce the incidence of CMV disease in this patient population; however, there is concern about increasing antiviral resistance. Detection of antiviral resistance in CMV was traditionally accomplished using Sanger sequencing of UL54 and UL97 genes, in which specific mutations may result in reduced antiviral activity. In this study, a novel next-generation sequencing (NGS) method was developed and validated to detect mutations in UL54/UL97 associated with antiviral resistance. Plasma samples (n = 27) submitted for antiviral resistance testing by Sanger sequencing were also analyzed using the NGS method. When compared to Sanger sequencing, the NGS assay demonstrated 100% (27/27) overall agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations. The limit of detection of the NGS method was determined to be 500 IU/mL, and the lower threshold for detecting mutations associated with resistance was established at 15%. The NGS assay represents a novel laboratory tool that assists healthcare providers in treating patients who are infected with CMV harboring resistance-associated mutations and who may benefit from tailored antiviral therapy.

Comprehensive genomic profiling of Taiwanese patients with breast cancer using a novel targeted panel: Preliminary analyses from a prospective triple-negative cohort.

e12553 Background: Breast cancer is one of the leading causes of cancer-related deaths in women, and there is a demand in developing an Asian-based genetic profiling database to improve treatment outcomes. This study aims to determine the molecular alterations and identify potential therapeutic targets from a cohort of Taiwanese triple negative breast cancers using a novel next-generation sequencing (NGS) targeted panel. Methods: The VGH-TAYLOR study was designed to understand the genetic profiling of different subtypes of breast cancer in Taiwan. The study population comprised both early- (first-line surgery or neoadjuvant therapy) and late-stage (early/late relapse or de novo stage IV disease) of breast cancers, and we conducted molecular profiling with the illumina TruSight Oncology 500 assay using both DNA and RNA extracted from FFPE tissues from a subset of 101 prospectively enrolled breast cancers, which were previously assayed with a commercialized panel of 161 genes. Results: A total of 100 breast cancers from VGH-TAYLOR study were successfully assayed, with the majority (n = 97) being triple-negative, two HER2 over-expressed and the remaining one being HER2-low phenotypically. The most prevalent actionable alterations (more than 5% of study cohort) came from BRCA2 (24%), BRCA1 (12%), PALB2 (5%), PIK3CA (38%, both mutations and amplifications), ERBB2 (14%, both mutations and amplifications), ERBB3 (10%) and PTEN (12%). One-quarter of assayed samples were categorized into tumor mutation burden (TMB)-high cancers ( > 10 mutations per million bases), which came from 15 out of 74 (20%) early-stage and 10 out of 26 (38%) late-stage patients (chi-square test p-value: 0.06). In general, the novel panel with 500 targeted genes reported more actionable alterations than the former one with limited interrogated genes ( BRCA2: 7%, BRCA1: 12%, PALB2: 7%, PIK3CA: 45%). Conclusions: Our study ascertained the clinical applicability and feasibility of comprehensive genomic profiling from a cohort of Taiwanese breast cancers. Overall, the successful rate for NGS with QC metrics are around 90%. The novelty benefits from more investigated genes as well as multi-gene biomarkers such as TMB and microsatellite instability (MSI) status. Patients with a positive finding from the genomic assay may benefit from an immune checkpoint inhibitor (TMB-high and MSI), poly ADP- ribose polymerase (PARP) inhibitor (homologous recombination deficiency genes: BRCA1/2 and PALB2), selective estrogen receptor degrader ( ESR1), tyrosine kinase inhibitor ( ERBB2/3), phosphoinositide 3-kinase inhibitor ( PIK3CA) and so on. Further studies with more samples enrolled and longer follow up are warranted to elucidate the true value of comprehensive genomic profiling for breast cancer.

Genotyping and Minimal Residual Disease (MRD) Assessment in cfDNA By the Euroclonality-NGS DNA Capture (EC-NDC) Panel in Mantle Cell Lymphoma (MCL)

Background Modern concepts for the treatment of mantle cell lymphomas (MCL) increasingly integrate clinical and biological variables for risk-adapted treatment strategies. Novel next-generation sequencing (NGS) technologies are highly suited for tumor genotyping as well as disease monitoring by minimal residual disease (MRD) assessment, identifying patients (pts) with a high risk of relapse. Here, we investigated whether the EuroClonality (EC)-NGS DNA Capture (EC-NDC) assay is a) applicable for genotyping of MCL in peripheral blood (PB) and plasma cell-free (cf)DNA and b) suitable for MRD assessment in plasma as compared to the amplicon-based EuroClonality Immunoglobulin-based (IG-)NGS assay. Patients and methods A total of 109 PB samples (55 baseline and 54 interim staging) from a cohort of MCL treated within the TRIANGLE trial by the European MCL network (NCT02858258) were selected according to a PB infiltration ≥10% at diagnosis. From prospectively collected PB samples in national reference labs, DNA from the cellular blood compartment (PBC) and plasma cfDNA was extracted. For targeted capture, the EC-NDC assay (Univ8® Genomics, UK) was used to simultaneously detect immunoglobulin and T cell receptor (IG/TR) rearrangements, structural variants (SV) and single nucleotide variants (SNVs) in 72 genes (Stewart, Blood Adv, 2021). For diagnostic PBC and cfDNA samples, IG clonotypes were called with ≥6 unique reads and ≥5% of annotated IG reads, SV were called with ≥4 unique reads and SNV were called with ≥6 unique reads at ≥4% variant allele frequency (VAF). For amplicon-based MRD analysis the EuroClonality IG-NGS (IG-NGS) assay was used with IG clonotypes called with ≥5% of annotated IG reads and at ≥1 log higher abundance to the polyclonal background. MRD was analyzed by IG-NGS in PBC using 8 µg PBC DNA to reach a sensitivity of 10-6. For MRD analysis in cfDNA, we aimed to use ≥5000 genome equivalents (GE) for both IG-NGS and EC-NDC. Data were analyzed by ARResT/Interrogate (Bystry, Bioinformatics, 2017) with an adapted pipeline for targeted capture in cfDNA. MRD status was defined as positive by detecting a lymphoma-specific IG clonotype and/or SV at ≥1 read, or alternatively by retrieval of ≥1 disease-specific mutation at ≥3 reads. Results Total cfDNA amounts at baseline were high (mean 92.6 ng/mL plasma) and even increasing at interim staging (mean 123.8 ng/mL) while decreasing by 10-fold in 38 samples at end of treatment (EoT)(mean 11.45 ng/mL). At baseline, a tumor-specific IGH-VJ clonotype was detected by IG-NGS in all 55 PBC and cfDNA samples. Genotyping by EC-NDC showed fully concordant results in 55 PBC and cfDNA samples with respect to IG clonotypes, SV and SNV, except for 3 PBC samples with low usable reads covering IGH-VJ regions. The presence of a t(11;14)(q13;q32) translocation was confirmed in 54/55 pts in PBC and cfDNA, while one patient with unknown t(11;14) translocation status by FISH was negative by EC-NDC. EC-NDC identified 106 putative somatic SNVs (VAF 2.7-75%, median 30%) in PBC and cfDNA, and among those were genes described as drivers in MCL (ATM (25/55), TP53 (11/55), KMT2D (9/55), SAMHD1 (5/55), CCND1 (5/55) and 18 other genes (Fig. 1). Six pts with detectable t(11;14) and IG rearrangement did not show additional SNVs. On average, 6 MRD markers/pt were identified (range: 3-10). At interim staging, MRD was analyzed in 54 PBC and 44 cfDNA samples. Twenty-five (47%) pts were MRD+ in PBC by IG-NGS. Twenty (46%) pts were MRD+ in cfDNA by EC-NDC (8 cases with ≥2 markers, 4 with 2 markers, and 8 with 1 marker). From 10/44 (23%) cfDNA samples MRD+ by IG-NGS, 9 were also MRD+ by ED-NDC. From 44 pts with parallel analysis of PBC and cfDNA, 14 (32%) were MRD+ and 18 (41%) were MRD-, while 12 (27%) showed discordant results. Conclusion To the best of our knowledge, this is the first analysis comparing targeted capture and amplicon assays for application in PBC and cfDNA in MCL. The capture-based EC-NDC assay allows a comprehensive liquid biopsy-based genotyping of MCL. In addition, both IG-NGS and EC-NDC assays are suitable for MRD assessment in cfDNA with evidence of more sensitive MRD detection EC-NDC due to multiple MRD targets detected and traced by this assay. Clinical data are still needed to explore the potential prognostic impact of MRD assessment in plasma vs PBC. Updated MRD analysis at EoT and correlation with clinical data will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 3750: Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology

Abstract Background: DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA hypermethylation events co-occur in groups of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Here we designed and performed a first step of validation of a novel next-generation sequencing (NGS) targeted methylation panel that can simultaneously evaluate the methylation levels of multiple cancer-related genes. Methods: The WG00446_MET_A AmpliSeq™ Methylation Panel for Cancer Research was designed using the Ion AmpliSeq technology to amplify 152 regions with an amplicon size range of 125-175bp, that covers a total of 1107 promoter and intragenic CpGs of 18 cancer-related genes. The performance of the panel was assessed by running in duplicate commercially available fully methylated and unmethylated control human gDNA samples and a variable mixture of them to simulate 10%, 25%, 75%, and 90% of global methylation. The libraries (manual preparation using the Ion AmpliSeq Plus Library Kit) were loaded on 530 chips and run on the GeneStudio S5 Sequencer (Thermo Fisher Scientific). The sequencing output was analyzed using the methylation_analysis plugin, available on Ion Torrent Suite v5.16 (Thermo Fisher Scientific). Results: The WG00446_MET_A panel allowed to run 6 samples per 530 chips to reach an observed mean target depth >2,000X (observed reads on-targets: >80%; average number of mapped reads: >450,000/sample). The conversion efficiency, determined by spiking-in Unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed % of global methylation for all CpGs were >98% and <2% for fully methylated and unmethylated gDNA samples, respectively. The observed results for the variable mixtures were in agreement with what expected. Conclusions: The methylation-specific NGS analysis using the WG00446_MET_A panel represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. It will offer the opportunity to design a more robust algorithm for disease prediction for those cancer patients with a low quantity of biological material available. The orthogonal testing on DNA extracted by FFPE and frozen samples is ongoing with a subset of amplicons from the WG00446_MET_A panel. Citation Format: Federico Pio Fabrizio, Stefano Castellana, Angelo Sparaneo, Tommaso Mazza, Flavia Centra, Domenico Trombetta, Vito Michele Fazio, Lucia Anna Muscarella. Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology [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 3750.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

AbstractClonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Seasonal variation and tissues specificity of endophytic fungi of Dillenia indica L. and their extracellular enzymatic activity.

Endophytes are microbes that live inside the tissues of plants without causing any disease. Many of these belonging to fungi have been exploited earlier for their biological activities. This study focused on the exploration and characterization of culturable endophytic fungi inhabiting the medicinal plant Dillenia indica L. during four different seasons (summer, monsoon, autumn, and winter) from 2018 to 2019. A total of 2360 segments from different parts (leaves, fruits, and stem) were screened to isolate endophytic fungi. During the study, 25 species of fungi belonging to 20 genera were isolated from the selected plant. The identification of these fungi was validated at morphological, microscopic, and molecular levels. Results indicate the plant has the highest affinity for Daldinia eschscholtzii, followed by Colletotrichum gloeosporioides and Cladosporium cladosporioides. Further, the percent frequency was highest in leaves, followed by stem and fruits. The results were further supported by a similar trend of colonization rate for different plant parts. The monsoon season had the highest number of isolates (312), followed by summer (208), winter (164), and autumn (114). Species diversity was highest during the monsoon season and lowest during the winter. These fungi also produce amylase, lipase, protease, asparaginase, cellulase, and ligninolytic enzymes. This study focused only on culturable fungal endophytes, yet the scope can be extended for other non-culturable microbes and their interaction by using high-throughput genomics and novel next-generation sequencing (NGS) tools. The results indicate that Dillenia indica L. harbors novel endophytic fungi having industrial applications.

Targeted Locus Amplification (TLA): A Novel Next Generation Sequencing (NGS) Technology to Detect New Molecular Markers and Monitoring Minimal Residual Disease (MRD) in Mantle Cell and Follicular Lymphoma

Background: Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell (MCL) and follicular (FL) lymphoma [Pott C., Blood 2010; Ladetto M, Blood 2013]. However, some technical limitations hamper its feasibility in daily clinical routine. First of all, current techniques allow the identification of a molecular marker only in 75-80% of MCL and 55-60% of FL patients. All the other “no marker” cases cannot be studied for MRD purposes. Next generation sequencing (NGS) tools might overcome these limitations. Among them, the recently developed targeted locus amplification (TLA) technology [de Vree P.J., Nat Biotechnol 2014] that selectively amplifies and sequences entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and gene fusions in acute lymphoblastic leukemia. [Kuiper R.P.; Vroegindeweij EM, 57th ASH Communication, Blood 2015].The aim of this study was to test the TLA approach in MCL and FL diagnostic samples, with at least one translocation confirmed by FISH and a failure in molecular marker identification, with the aim to increase the rate of success in marker screening for MRD purposes. Moreover, the performances of the newly TLA molecular markers were compared to the standardized quantitative PCR (qPCR) approach for MRD monitoring.Methods: Genomic DNA was extracted from bone marrow (BM) and peripheral blood (PB) samples of MCL and FL patients, enrolled in prospective clinical trials of the Fondazione Italiana Linfomi (FIL). All patients provided written informed consent for the research use of the biological samples. Libraries for TLA (Cergentis, Utrecht) were prepared using only one couple of antisense primers targeting the immunoglobulin heavy chain (IGH) enhancer locus, as previously described [Hottentot QP, Methods Mol Biol. 2017], and sequenced on MiSeq platform (Illumina, San Diego). MRD monitoring was carried out by qPCR designing allele specific oligonucleotides (ASO) and consensus probes based on the new breakpoint sequences obtained from TLA NGS. Finally, the efficiency of TLA novel markers to monitor MRD was compared to MRD data obtained from clonal IGH rearrangements detected and tracked by standardized ASO qPCR. [Van der Velden VHJ, Leukemia 2007].Results: TLA was firstly tested on 21 t(11;14)-positive, BCL-1/IGH major translocation cluster (MTC) negative MCL baseline samples (8 BM and 13 PB): in 86% of cases (18/21) a novel BCL-1/IGH breakpoint was identified by NGS. In these cases, TLA described new rearrangements between “no-MTC” BCL1 loci and JH1, JH4, JH5 or JH6 regions on chromosome 14. Therefore, additional 8 FL baseline BM samples, resulting t(14;18) FISH positive and BCL-2/IGH MBR/mcr/“minor rearrangements”-negative by PCR, were tested obtaining a 62% (5 out of 8 samples) success rate. Also in these cases, new and not yet described BCL2/IGH rearrangements were found. Additionally, to assess the specificity of the technology, the MBR-positive control sample (DOHH2 cell line) was tested and correctly sequenced, as well. Interestingly, among the new TLA positive FL cases, in one sample, the NGS technology highlighted the breakpoint on the DH region of chromosome 14, describing an unusual t(14;18) translocation. 5/29 cases (17%) in which TLA was not able to identify a new marker were characterized by a low tumor infiltration, mainly <5% by flow cytometry (median 4.4%, range 0.03-8%). Secondly, a validation of the NGS sequences was performed by ASO primers designed on the new TLA breakpoints and MRD was monitored by ASO qPCR. Overall, in 14 MCL cases where also a classic IGH-based marker was available, the MRD results were superimposable between the two markers (Fig1A), with good and overlapping performances in terms of overall correlation (r2=0,86; Fig 1B). In FL samples, ASO qPCR confirmed the high tumor burden of the analyzed tissues (Fig 1C). Finally, an extension of the analyzed samples series and MRD validation is currently ongoing among additional FL patients.Conclusion: The TLA NGS technology allowed to identify a translocation-based novel molecular marker in FISH positive t(11;14) and t(14;18) MCL and FL baseline samples, where the classical Sanger sequencing failed a marker identification. These new breakpoints can reliably be used for the design of ASO qPCR primers for MRD detection in previously not traceable patients [Display omitted] DisclosuresKlous:Cergentis BV: Employment. Yilmaz:Cergentis BV: Employment. Vitolo:Gilead: Honoraria; Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria. Luminari:PFIZER: Speakers Bureau; GILEAD: Speakers Bureau; TEVA: Membership on an entity's Board of Directors or advisory committees; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; ROCHE: Membership on an entity's Board of Directors or advisory committees; TAKEDA: Membership on an entity's Board of Directors or advisory committees. Federico:takeda: Honoraria, Research Funding. Splinter:Cergentis BV: Employment.

Computational based design and tracking of synthetic variants of Porcine circovirus reveal relations between silent genomic information and viral fitness

Viral genomes not only code the protein content, but also include silent, overlapping codes which are important to the regulation of the viral life cycle and affect its evolution. Due to the high density of these codes, their non-modular nature and the complex intracellular processes they encode, the ability of current approaches to decipher them is very limited. We describe the first computational-experimental pipeline for studying the effects of viral silent and non-silent information on its fitness. The pipeline was implemented to study the Porcine Circovirus type 2 (PCV2), the shortest known eukaryotic virus, and includes the following steps: (1) Based on the analyses of 2100 variants of PCV, suspected silent codes were inferred. (2) Five hundred variants of the PCV2 were designed to include various ‘smart’ silent mutations. (3) Using state of the art synthetic biology approaches, the genomes of these five hundred variants were generated. (4) Competition experiments between the variants were performed in Porcine kidney-15 (PK15) cell-lines. (5) The variant titers were analyzed based on novel next-generation sequencing (NGS) experiments. (6) The features related to the titer of the variants were inferred and their analyses enabled detection of various novel silent functional sequence and structural motifs. Furthermore, we demonstrate that 50 of the silent variants exhibit higher fitness than the wildtype in the analyzed conditions.

Development of a Whole-urine, Multiplexed, Next-generation RNA-sequencing Assay for Early Detection of Aggressive Prostate Cancer

BackgroundDespite biomarker development advances, early detection of aggressive prostate cancer (PCa) remains challenging. We previously developed a clinical-grade urine test (Michigan Prostate Score [MiPS]) for individualized aggressive PCa risk prediction. MiPS combines serum prostate-specific antigen (PSA), the TMPRSS2:ERG (T2:ERG) gene fusion, and PCA3 lncRNA in whole urine after digital rectal examination (DRE). ObjectiveTo improve on MiPS with a novel next-generation sequencing (NGS) multibiomarker urine assay for early detection of aggressive PCa. Design, setting, and participantsPreclinical development and validation of a post-DRE urine RNA NGS assay (Urine Prostate Seq [UPSeq]) assessing 84 PCa transcriptomic biomarkers, including T2:ERG, PCA3, additional PCa fusions/isoforms, mRNAs, lncRNAs, and expressed mutations. Our UPSeq model was trained on 73 patients and validated on a held-out set of 36 patients representing the spectrum of disease (benign to grade group [GG] 5 PCa). Outcome measurements and statistical analysisThe area under the receiver operating characteristic curve (AUC) of UPSeq was compared with PSA, MiPS, and other existing models/biomarkers for predicting GG ≥3 PCa. Results and limitationsUPSeq demonstrated high analytical accuracy and concordance with MiPS, and was able to detect expressed germline HOXB13 and somatic SPOP mutations. In an extreme design cohort (n = 109; benign/GG 1 vs GG ≥3 PCa, stratified to exclude GG 2 cancer in order to capture signal difference between extreme ends of disease), UPSeq showed differential expression for T2:ERG.T1E4 (1.2 vs 78.8 median normalized reads, p < 0.00001) and PCA3 (1024 vs 2521, p = 0.02), additional T2:ERG splice isoforms, and other candidate biomarkers. Using machine learning, we developed a 15-transcript model on the training set (n = 73) that outperformed serum PSA and sequencing-derived MiPS in predicting GG ≥3 PCa in the held-out validation set (n = 36; AUC 0.82 vs 0.69 and 0.69, respectively). ConclusionsThese results support the potential utility of our novel urine-based RNA NGS assay to supplement PSA for improved early detection of aggressive PCa. Patient summaryWe have developed a new urine-based test for the detection of aggressive prostate cancer, which promises improvement upon current biomarker tests.

Evolution of Drug-Resistant Mycobacterium tuberculosis Strains and Their Adaptation to the Human Lung Environment.

In the last two decades, multi (MDR), extensively (XDR), extremely (XXDR) and total (TDR) drug-resistant Mycobacterium tuberculosis (M.tb) strains have emerged as a threat to public health worldwide, stressing the need to develop new tuberculosis (TB) prevention and treatment strategies. It is estimated that in the next 35 years, drug-resistant TB will kill around 75 million people and cost the global economy $16.7 trillion. Indeed, the COVID-19 pandemic alone may contribute with the development of 6.3 million new TB cases due to lack of resources and enforced confinement in TB endemic areas. Evolution of drug-resistant M.tb depends on numerous factors, such as bacterial fitness, strain’s genetic background and its capacity to adapt to the surrounding environment, as well as host-specific and environmental factors. Whole-genome transcriptomics and genome-wide association studies in recent years have shed some insights into the complexity of M.tb drug resistance and have provided a better understanding of its underlying molecular mechanisms. In this review, we will discuss M.tb phenotypic and genotypic changes driving resistance, including changes in cell envelope components, as well as recently described intrinsic and extrinsic factors promoting resistance emergence and transmission. We will further explore how drug-resistant M.tb adapts differently than drug-susceptible strains to the lung environment at the cellular level, modulating M.tb–host interactions and disease outcome, and novel next generation sequencing (NGS) strategies to study drug-resistant TB.

Next Generation Sequencing Assay for Detection of Circulating HPV DNA (cHPV-DNA) in Patients Undergoing Radical (Chemo)Radiotherapy in Anal Squamous Cell Carcinoma (ASCC).

Background: Following chemo-radiotherapy (CRT) for human papilloma virus positive (HPV+) anal squamous cell carcinoma (ASCC), detection of residual/recurrent disease is challenging. Patients frequently undergo unnecessary repeated biopsies for abnormal MRI/clinical findings. In a pilot study we assessed the role of circulating HPV-DNA in identifying “true” residual disease.Methods: We prospectively collected plasma samples at baseline (n = 21) and 12 weeks post-CRT (n = 17). Circulating HPV-DNA (cHPV DNA) was measured using a novel next generation sequencing (NGS) assay, panHPV-detect, comprising of two primer pools covering distinct regions of eight high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52, and 58) to detect circulating HPV-DNA (cHPV DNA). cHPV-DNA levels post-CRT were correlated to disease response.Results: In pre-CRT samples, panHPV-detect demonstrated 100% sensitivity and specificity for HPV associated ASCC. PanHPV-detect was able to demonstrate cHPV-DNA in 100% (9/9) patients with T1/T2N0 cancers. cHPV-DNA was detectable 12 weeks post CRT in just 2/17 patients, both of whom relapsed. 1/16 patients who had a clinical complete response (CR) at 3 months post-CRT but relapsed at 9 months and 1/1 patient with a partial response (PR). PanHPV-detect demonstrated 100% sensitivity and specificity in predicting response to CRT.Conclusion: We demonstrate that panHPV-detect, an NSG assay is a highly sensitive and specific test for the identification of cHPV-DNA in plasma at diagnosis. cHPV-DNA post-treatment may predict clinical response to CRT.

Integrated genome-wide investigations of the housefly, a global vector of diseases reveal unique dispersal patterns and bacterial communities across farms

BackgroundHouseflies (Musca domestica L.) live in intimate association with numerous microorganisms and is a vector of human pathogens. In temperate areas, houseflies will overwinter in environments constructed by humans and recolonize surrounding areas in early summer. However, the dispersal patterns and associated bacteria across season and location are unclear. We used genotyping-by-sequencing (GBS) for the simultaneous identification and genotyping of thousands of Single Nucleotide Polymorphisms (SNPs) to establish dispersal patterns of houseflies across farms. Secondly, we used 16S rRNA gene amplicon sequencing to establish the variation and association between bacterial communities and the housefly across farms.ResultsUsing GBS we identified 18,000 SNPs across 400 individuals sampled within and between 11 dairy farms in Denmark. There was evidence for sub-structuring of Danish housefly populations and with genetic structure that differed across season and sex. Further, there was a strong isolation by distance (IBD) effect, but with large variation suggesting that other hidden geographic barriers are important. Large individual variations were observed in the community structure of the microbiome and it was found to be dependent on location, sex, and collection time. Furthermore, the relative prevalence of putative pathogens was highly dependent on location and collection time.ConclusionWe were able to identify SNPs for the determination of the spatiotemporal housefly genetic structure, and to establish the variation and association between bacterial communities and the housefly across farms using novel next-generation sequencing (NGS) techniques. These results are important for disease prevention given the fine-scale population structure and IBD for the housefly, and that individual houseflies carry location specific bacteria including putative pathogens.

A novel metabarcoding diagnostic tool to explore protozoan haemoparasite diversity in mammals: a proof-of-concept study using canines from the tropics

Haemoparasites are responsible for some of the most prevalent and debilitating canine illnesses across the globe, whilst also posing a significant zoonotic risk to humankind. Nowhere are the effects of such parasites more pronounced than in developing countries in the tropics where the abundance and diversity of ectoparasites that transmit these pathogens reaches its zenith. Here we describe the use of a novel next-generation sequencing (NGS) metabarcoding based approach to screen for a range of blood-borne apicomplexan and kinetoplastid parasites from populations of temple dogs in Bangkok, Thailand. Our methodology elucidated high rates of Hepatozoon canis and Babesia vogeli infection, whilst also being able to characterise co-infections. In addition, our approach was confirmed to be more sensitive than conventional endpoint PCR diagnostic methods. Two kinetoplastid infections were also detected, including one by Trypanosoma evansi, a pathogen that is rarely screened for in dogs and another by Parabodo caudatus, a poorly documented organism that has been previously reported inhabiting the urinary tract of a dog with haematuria. Such results demonstrate the power of NGS methodologies to unearth rare and unusual pathogens, especially in regions of the world where limited information on canine vector-borne haemoparasites exist.

Abstract 2299: Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients

Abstract Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations. Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation. Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy. Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2299.

Dynamic Changes in Circulating Tumor DNA During Chemoradiation for Locally Advanced Lung Cancer

PurposeConcurrent chemoradiation therapy (CRT) is the principal treatment modality for locally advanced lung cancer. Cell death due to CRT leads to the release of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) into the bloodstream, but the kinetics and characteristics of this process are poorly understood. We hypothesized that there could be clinically meaningful changes in cfDNA and ctDNA during a course of CRT for lung cancer. Methods and materialsMultiple samples of plasma were obtained from 24 patients treated with CRT for locally advanced lung cancer to a mean dose of 66 Gy (range, 58-74 Gy) at the following intervals: before CRT, at weeks 2 and 5 during CRT, and 6 weeks after treatment. cfDNA was quantified, and a novel next generation sequencing (NGS) technique using enhanced tagged/targeted-amplicon sequencing was performed to analyze ctDNA. ResultsPatients for whom specific mutations in ctDNA were undetectable at the baseline time point had improved survival, and potentially etiologic driver mutations could be tracked throughout the course of CRT via NGS in multiple patients. We quantified the levels of cfDNA from patients before CRT, at week 2, week 5, and at 6 weeks after treatment. No differences were observed at weeks 2 and 5 of therapy, but we noted a significant increase in cfDNA in the posttreatment follow-up samples compared with samples collected before CRT (P = .05). ConclusionsDynamic changes in both cfDNA and ctDNA were observed throughout the course of CRT in patients with locally advanced lung cancer. Specific mutations with therapeutic implications can be identified and tracked using NGS methodologies. Further work is required to characterize the changes in cfDNA and ctDNA over time in patients treated with CRT and to assess the predictive and prognostic potential of this powerful technology.

Identification of novel GNAS mutations in intramuscular myxoma using next-generation sequencing with single-molecule tagged molecular inversion probes

BackgroundIntramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas. These tumors can, especially on biopsy material, be difficult to distinguish from low-grade fibromyxoid sarcoma or low-grade myxofibrosarcoma. GNAS mutations are frequently involved in IM, in contrast to these other malignant tumors. Therefore, sensitive molecular techniques for detection of GNAS aberrations in IM, which frequently yield low amounts of DNA due to poor cellularity, will be beneficial for differential diagnosis.MethodsIn our study, a total of 34 IM samples from 33 patients were analyzed for the presence of GNAS mutations, of which 29 samples were analyzed using a gene-specific TaqMan genotyping assay for the detection of GNAS hotspot mutations c.601C > T and c602G > A in IM, and 32 samples using a novel next generation sequencing (NGS)-based approach employing single-molecule tagged molecular inversion probes (smMIP) to identify mutations in exon 8 and 9 of GNAS. Results between the two assays were compared for their ability to detect GNAS mutations with high confidence.ResultsIn total, 23 of 34 samples were successfully analyzed with both techniques showing GNAS mutations in 12 out of 23 (52%) samples. The remaining 11 samples were analyzed with either TaqMan assay or smMIP assay only. The TaqMan assay revealed GNAS mutations in 16 out of 29 samples (55%), with six samples c.601C > T (p.R201C; 38%) and ten samples c.602G > A (p.R201H; 62%) missense mutations. The smMIP assay identified mutations in 16 out of 28 samples (57%), with five samples c.601C > T (p.R201C; 31%) and seven samples c.602G > A (p.R201H; 44%) missense mutations. In addition, four samples (25%) revealed novel IM-associated mutations, including c.601C > A (p.R201S), c.602G > T (p.R201L), c.602G > C (p.R201P) and c.680A > G (p.Q227R). Combining the results of both tests, 23 out of 34 sporadic IM samples (68%) showed a GNAS mutation.ConclusionsBoth the TaqMan and the smMIP assay a show a high degree of concordance in detecting GNAS hotspot mutations in IM with comparable sensitivity. However, since the NGS-based smMIP assay permits mutation detection in whole exons of GNAS, a broader range of GNAS mutations can be identified by the smMIP approach.

Development of a Novel Next-Generation Sequencing (NGS)-Based Assay for Measurable Residual Disease (MRD) in FLT3-ITD AML and Its Potential Clinical Application in Patients Treated with Chemotherapy Plus FLT3 Inhibitors

The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.Using isolated genomic DNA from bone marrow aspirates or whole-blood samples, PCR primers flanking exons 14 and 15 of the FLT3 gene were designed and highly diverse NGS libraries were generated. These libraries were then sequenced by Illumina's sequencing-by-synthesis method. The bioinformatics approach we used identifies unique FLT3-ITD mutations of varying length along with wild type sequences and calculates a mutant allelic frequency. The assay was validated using clinical samples to assess accuracy and reproducibility. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% but was capable of detecting FLT3-ITD mutations at a level as low as 0.003%.We next validated the assay using clinical samples from patients with FLT3-ITD AML. The negative prognostic impact of FLT3-ITD mutations can be mitigated in part when an FLT3 inhibitor is administered in combination with induction chemotherapy, as demonstrated in CALGB10603/RATIFY (N Engl J Med. 2017;377:454). It was reported in this study that patients treated with an FLT3 inhibitor combined with chemotherapy followed by allogeneic transplant in first remission had better overall survival than their counterparts in the control arm. One hypothesis for this outcome is that the FLT3-inhibitor-treated patients had a lower leukemic burden prior to transplant. As a pilot test of this concept, we used our MRD assay on a series of bone marrow aspirate samples collected from 10 patients with newly diagnosed FLT3-ITD AML. The patients were selected to be as uniform as possible. All patients had intermediate-risk karyotype, a detectable FLT3-ITD mutation by conventional PCR, and mutated NPM1. All patients received cytarabine-based intensive induction and achieved morphologic first remission with a single course of chemotherapy. Finally, all patients underwent allogeneic transplant in first remission. The sample analyzed for MRD was the first collected after remission induction, 5-8 weeks after the start of therapy. The investigators performing the MRD assay were blinded to the clinical data. Four patients received chemotherapy alone, while 6 were treated with chemotherapy (7+3) plus an FLT3 inhibitor. In all patients' remission samples, the MRD assay identified the FLT3-ITD mutation that precisely matched the one observed in the original diagnostic specimen. This demonstrates the sensitivity of the assay (all samples had a detectable mutation), and the unique length of each patient's mutation confers a degree of specificity not achievable with MRD detection methods that focus on other AML-associated mutations. Supporting our hypothesis was the observation that patients treated with FLT3 inhibitors had MRD levels lower than those in patients treated with chemotherapy alone (Figure).Our results help establish the role of NGS-based MRD assays for the clinical management of FLT3-ITD AML. This assay could be used to define the depth of remission, identify persistent disease, and help guide decision making in the use of FLT3 inhibitors as continuation therapy. This study provides validation of the clinical utility of our MRD assay, which will be used to analyze the remission samples from patients in the ongoing phase 3, randomized, double-blind, placebo-controlled QuANTUM-First clinical trial, in which patients with newly diagnosed FLT3-ITD AML are randomized to receive either the highly potent and selective FLT3 inhibitor quizartinib or placebo in combination with chemotherapy, followed by single-agent quizartinib as continuation therapy. [Display omitted] DisclosuresShi:Novartis: Employment, Equity Ownership; Daiichi Sankyo: Other: Provide clinical trial testing services. Chang:Daiichi Sankyo: Employment. Laing:Novartis: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Berisha:Daiichi Sankyo: Employment. Adams:Johns Hopkins University: Employment. Ding:Navigate BP: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Nakamaru:Daiichi Sankyo: Employment. Lameh:Navigate BioPharma Inc,: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Pollner:Navigate BioPharma Inc.: Employment. Kobayashi:Daiichi Sankyo: Employment.

Incidence of HLA Loss in a Global Multicentric Cohort of Post-Transplantation Relapses: Results from the Hlaloss Collaborative Study

Introduction. Genomic loss of an HLA haplotype encoding incompatible alleles (“HLA loss”) has been described in previous single-center studies as a mechanism by which leukemic cells evade the graft-versus-leukemia effect mediated by alloreactive donor T cells and outgrow into a clinically evident relapse. HLA loss accounts for up to 30% of relapses after HLA-haploidentical transplants (Crucitti, Leukemia 2015), but the actual frequency and clinical relevance of this phenomenon in unrelated donor HSCTs, including cord blood transplants, are largely unknown. Here we present the first global collaborative study to investigate the incidence of HLA loss across different transplant settings.Methods. Twenty transplant centers from Europe (n=16), North America (n=3) and Asia (n=1) joined to form the HLALOSS consortium. To date, we collected a total of 619 cases of hematologic relapse from adult patients with acute myeloid leukemia (78.5%), acute lymphoblastic leukemia (13.9%), myelodysplastic syndromes (4%) or myeloproliferative neoplasms (1.1%) after allogeneic HSCT from HLA-haploidentical relatives (31.7%), HLA-mismatched unrelated donors (MMUD, 21.3%), 10/10-matched unrelated donors (MUD, 37.2%), or unrelated cord blood units (UCB, 9.8%). Where available, the donor and patient germlines and the patient pre-transplant disease were collected in parallel. Until today, 476 cases were analyzed using conventional HLA typing of sorted leukemic blasts, the recently developed HLA-KMR assay (Ahci and Toffalori, Blood, 2017) or a novel Next-Generation Sequencing (NGS) method. The latter was developed adapting the HLA typing strategy in use at the DKMS (Lange, BMC Genomics 2013) to the study of chimeric samples, and allowing to cover all possible HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles and to analyze at least 48 different cases in a single run.Results. Out of the 476 relapses analyzed to date, 396 (83.2%) were informative for the study of HLA loss. Of these, 155 occurred after haploidentical HSCT, 101 after MMUD HSCT, 93 after 10/10-matched, HLA-DPB1 mismatched MUD, and 47 after UCB HSCTs. Three-hundred-two (76.2%) of cases were analyzed using the NGS platform. This method resulted particularly robust, reliable and sensitive in analyzing large sample series: the mean coverage across the 6 sequenced loci was over 8500x, up to 0.5% of the HLA allele of interest could be detected in artificial chimerism curves, and relapse samples tested in parallel via the sequencing platform and HLA-KMR (n=10) showed remarkable concordance between the two methods (R2=0.86, p<0.0001). In total, we detected 51 HLA loss post-transplantation relapses out of the 396 cases analyzed (12.8%). Of these, 35 occurred after haploidentical HSCT (22.6% of relapses in this setting), 12 after MMUD HSCT (11.9%), 4 after 10/10 MUD HSCT (4.3%) and, notably, none after UCB HSCT.Conclusions. The present data, obtained from the largest collaborative study on the immunobiology of relapse to date, confirm the clinical relevance of HLA loss as a major mechanism of immune evasion and post-transplantation relapse after allogeneic HSCT, with an incidence which is proportional to the number of donor-recipient HLA mismatches. The only exception is represented by UCB HSCT which, despite being often performed across multiple major HLA incompatibilities, does not appear to be associated with this relapse modality. This finding might reflect the fact that in UCB HSCT, multiple HLA mismatches are often not encoded in cis on the same chromosome, thereby reducing the selective advantage for leukemic cells that undergo an HLA haplotype loss. This phenomenon might in turn contribute to the lower incidence of relapse reported for UCB HSCT compared to other stem cell sources. DisclosuresVago:Moderna TX: Research Funding; GENDX: Research Funding. Stoelzel:Neovii: Speakers Bureau. Gojo:Novartis: Membership on an entity's Board of Directors or advisory committees; Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Busca:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Honoraria; Pfizer Pharmaceuticals: Honoraria, Speakers Bureau; Merk: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Kobbe:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding; Roche: Honoraria, Research Funding. Kroeger:Novartis: Honoraria, Research Funding; Sanofi: Honoraria; Riemser: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria. Finke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Mohty:Takeda: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Servier: Consultancy; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Molmed: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Janssen: Honoraria, Research Funding, Speakers Bureau. Beelen:Medac: Consultancy, Other: Travel Support. Fleischhauer:GENDX: Research Funding.

154. Diagnosis and Genotyping of Coxiella burnetii Causing Endocarditis in a Patient With Prosthetic Pulmonary Valve Replacement (PVR) Using Next-Generation Sequencing (NGS) of Plasma

BackgroundIdentification of Coxiella burnetii, the etiologic agent of Q Fever, in culture-negative endocarditis (CNE) remains challenging, and strain-level information is typically unavailable through conventional testing. We used a novel next-generation sequencing (NGS) assay on plasma cell-free DNA to facilitate rapid diagnosis and genotyping in a patient with C. burnetii CNE.MethodsNGS was performed on plasma by Karius, Inc. (Redwood City, California). Human reads were removed and remaining sequences were aligned to a curated database of over 1,000 pathogens. Organisms present above a predefined significance threshold were reported. For C. burnetti strain-typing, alignments to different Coxiella strains in the pathogen database were compared by BLAST bit-score to determine the most closely related strain to the infecting organism. C. burnetii genotype group was also determined by in silico analysis of polymorphic ORF deletion markers known to distinguish groups I–VI.ResultsTwenty-nine-year-old male with history of Tetralogy of Fallot, multiple pulmonary valve replacement (PVR), and 18 months of intermittent fever and night sweats were admitted. Relevant history included travel in South and South East Asia, the use of a LivaNova 3T Heater-Cooler device during surgery (i.e., at risk for Mycobacterium chimaera), and drinking unpasteurized milk. Cardiac CT showed 2 pulmonary opacities concerning for septic emboli and echocardiography showed echodensity on pulmonic valve. Blood cultures were negative. NGS detected C. burnetii within 48 hours of sample receipt. On the basis of these results, hydroxychloroquine and doxycycline were initiated with symptomatic improvement. Strain-typing demonstrated highest relatedness to the CbuK_Q154 (group IV) strain typically seen in North America. Genotype group was independently confirmed by inference of a pattern of ORF deletion most similar to group IV (and highly related group VII). Serologic testing for C. burnetii confirmed the diagnosis. After 4 weeks of antibiotics, the patient underwent successful PVR with graft exchange.ConclusionNGS testing aided in diagnosis of C. burnetii CNE, enabling early targeted antimicrobial therapy. It also allowed inference of strain-level information, supporting further investigations regarding epidemiologic origins of this pathogen.Disclosures S. Dalai, Karius, Inc.: Consultant, Consulting fee. S. Venkatasubrahmanyam, Karius, Inc.: Employee, Salary.