Plasma Next-generation Sequencing Research Articles

Clinical features associated with NeoRAS wild-type metastatic colorectal cancer A SCRUM-Japan GOZILA substudy

“NeoRAS WT” refers to the loss of RAS mutations (MTs) following first-line treatment in metastatic colorectal cancer (mCRC). We evaluate the incidence and clinicopathological characteristics of NeoRAS WT mCRC using next-generation sequencing of plasma circulating tumor DNA. Patients with mCRC enrolled in the GOZILA study initially diagnosed with tissue RAS MT mCRC and received subsequent systemic therapy are eligible. NeoRAS WT is defined as the absence of detectable RAS MT in plasma and assessed in all eligible patients (Group A) and in a subgroup with at least one somatic alteration detected in plasma (Group B). Overall, 478 patients are included. NeoRAS WT prevalence is 19.0% (91/478) in Group A and 9.8% (42/429) in Group B. Absence of liver or lymph node metastasis and tissue RAS MTs other than KRAS exon 2 MTs are significantly associated with NeoRAS WT emergence. Overall, 1/6 and 2/6 patients with NeoRAS WT treated with anti-EGFR monoclonal antibodies (mAbs) show partial response and stable disease for ≥6 months, respectively. NeoRAS WT mCRC is observed at a meaningful prevalence, and anti-EGFR mAb-based therapy may be effective.

Liquid biopsy in patients with metastatic pancreatic cancer using an optimized targeted sequencing approach: Data from the pancreatic cancer registry PanDaDETECT.

e16303 Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) conveys a dismal prognosis, and biomarkers for clinical diagnostics and treatment monitoring are urgently needed. Detection of circulating tumor DNA (ctDNA) using liquid biopsy (LB) has been shown to correlate with treatment response in other tumor entities. Here, we evaluated the feasibility of LB by targeted next generation sequencing (NGS) in metastatic PDAC, using an NGS approach optimized for PDAC. Methods: Patients from our PanDaDETECT registry with metastatic PDAC were included. Plasma samples were collected at initial diagnosis and every three months during palliative treatment. Targeted NGS of plasma samples was performed using a small, custom-generated gene panel optimized for LB provided by Qiagen (QiaSeq Targeted DNA Panel), covering the four most frequently mutated genes in PDAC with full exon coverage (KRAS, TP53, SMAD4 and CDKN2A). Sequencing was performed on the Illumina NextSeq 550 platform. LB results were compared to matched tissue NGS data from tumor samples. Results were correlated with the serological biomarker Ca 19-9 and radiographic response data. All patients provided written informed consent. The responsible ethics committee approved the study. Results: From 2020 to 2022, 57 patients were included and underwent plasma NGS at least at initial diagnosis. For 52 of these patients a tissue NGS result was available, and in 49 patients (94,2%), at least one of the four genes covered by our LB panel was mutated in the tumor tissue. In 35 patients, our LB approach detected ctDNA in the plasma sample at initial diagnosis (61,4%). Comparing tissue and plasma sequencing data at initial diagnosis, our LB approach detected ctDNA in 32 out of 49 patients who had a known mutation in tissue NGS in at least one of the four genes covered by the LB panel (65,3%). For 22 patients with positive ctDNA detection in plasma NGS, follow-up plasma samples after three months of treatment were available. In this subgroup, detection of ctDNA showed a superior correlation with radiographic response assessed by RECIST criteria than the serological biomarker Ca 19-9. In patients with a partial response or stable disease as defined by RECIST, ctDNA levels decreased markedly, while remaining elevated in patients showing disease progression. For 11 patients, plasma NGS at 6 months of treatment or later was performed, the results at these later time points correlated well with clinical and radiographic response in 9 out of 11 patients. Conclusions: Our LB approach optimized for PDAC is feasible and shows a good correlation with serological and radiographic response. Our optimized NGS panel covering only four genes is able to detect ctDNA in a majority of PDAC patients. LB by targeted NGS has the potential to detect a broad spectrum of relevant mutations in PDAC in an automatable and cost-effective matter.

The cost-effectiveness of including liquid biopsy into molecular profiling strategies for newly diagnosed advanced non-squamous non-small cell lung cancer in an Asian population

ObjectivesLiquid biopsy is complementary to tissue biopsy for lung cancer profiling, yet evidence of the cost-effectiveness is limited. This could retard implementation and reimbursement in clinical practice. The aim of this study is to estimate the cost-effectiveness of profiling strategies that include liquid biopsy and to identify the optimal profiling approach for newly diagnosed advanced non-squamous non-small cell lung cancer (NSCLC) in an Asian population using Singapore as an example. Materials and methodsA decision tree and partitioned-survival model was developed from the Singapore healthcare system’s perspective to evaluate the cost-effectiveness of five molecular profiling strategies: either tissue or plasma next-generation sequencing (NGS) alone, a concurrent, and two sequential approaches. Model inputs were informed by local data or published literature. Sensitivity analyses and scenario analyses were undertaken to understand the robustness of the conclusions for decision making. The optimal strategy at different willingness-to-pay (WTP) thresholds was presented by cost-effectiveness acceptability frontier and the expected loss curve. ResultsThe sequential tissue-plasma NGS approach revealed an additional 0.0981 quality adjusted life years (QALYs) for an extra cost of S$3,074 over a 20-year time horizon compared to tissue NGS alone, resulting in an incremental cost-effectiveness ratio (ICER) of S$31,318/QALY and an incremental net monetary benefit of S$1,343 per patient. The findings were sensitive to the costs of pembrolizumab and osimertinib and the probabilities of re-biopsy after tissue NGS. Sequential plasma-tissue NGS and plasma NGS alone were more costly and less effective than alternatives. ConclusionThe sequential tissue-plasma NGS approach generated the highest net monetary benefit and was the optimal testing strategy when WTP was S$45,000/QALY. It retained superiority but understandably with a higher ICER when expensive, non-first line treatments were included. Overall, its routine clinical practice should be proactively considered for newly diagnosed advanced non-squamous NSCLC in an Asian population.

The plasma miRNome and venous thromboembolism in high-grade glioma: miRNA Sequencing of a nested case-control cohort.

Patients with high-grade gliomas are at high risk of venous thromboembolism (VTE). MicroRNAs (miRNAs) are small non-coding RNAs with multiple roles in tumour biology, haemostasis and platelet function. Their association with VTE risk in high-grade glioma has not been comprehensively mapped so far. We thus conducted a nested case-control study within 152 patients with WHO grade IV glioma that had been part of a prospective cohort study on VTE risk factors. At inclusion a single blood draw was taken, and patients were thereafter followed for a maximum of 2 years. During that time, 24 patients (16%) developed VTE. Of the other 128 patients, we randomly selected 24 age- and sex-matched controls. After quality control, the final group size was 21 patients with VTE during follow-up and 23 without VTE. Small RNA next-generation sequencing of plasma was performed. We observed that hsa-miR-451a was globally the most abundant miRNA. Notably, 51% of all miRNAs showed a correlation with platelet count. The analysis of miRNAs differentially regulated in VTE patients-with and without platelet adjustment-identified potential VTE biomarker candidates such as has-miR-221-3p. Therewith, we here provide one of the largest and deepest peripheral blood miRNA datasets of high-grade glioma patients so far, in which we identified first VTE biomarker candidates that can serve as the starting point for future research.

Abstract 970: Liquid biopsy-informed precision oncology clinical trial to evaluate the utility of ctDNA comprehensive genomic profiling

Abstract Background: The expanding role of next-generation sequencing (NGS) in precision oncology has led to a paradigm shift towards genotype-targeted therapies, with liquid biopsies (LBs) offering a minimally invasive means of comprehensive genomic profiling. Methods: This is an observational LB-informed precision oncology clinical trial with primary endpoints to assess feasibility and determine the prevalence of actionable mutations in LB informing genotype-targeted therapeutic recommendations by the Johns Hopkins Molecular Tumor Board (JH MTB; NCT05585684). Secondary endpoints are progression-free and overall survival, ctDNA dynamics assessment, and concordance between plasma and tumor NGS. Target enrollment is 150 patients with advanced solid tumors who have progressed on at least one line of systemic therapy. Serial LB-derived genotypes are obtained at baseline, early on therapy, and upon progression using a CLIA ultra-sensitive 33-gene panel NGS assay (Elio Plasma Resolve, Personal Genome Diagnostics/Labcorp). Variant actionability is assessed by an ensemble knowledgebase, registry, and computational characterization approach, and results are reviewed by the JH MTB, followed by treatment recommendations. Results: The trial’s pre-specified interim analysis included 30 enrolled patients with evaluable LBs; 30 baseline, 13 on-therapy, and 6 progression LBs were analyzed. Twenty-five patients had non-small cell lung cancer among which 9 were EGFR-mutated, 1 patient had small cell lung cancer, 3 patients had gastro-esophageal cancer, and 1 patient had a neuroendocrine tumor. In total, 74 variants were identified across all time points; these were cross-referenced in somatic and germline registries, variant knowledgebases, and the biomedical literature, together with computational characterization of functional consequence to assign mutation actionability. Tumor-confirmed variants comprised 36% of baseline and 42% of both on-therapy and progression variants. At baseline, 20% of the variants were putatively clonal hematopoiesis-derived with similar findings at the on-therapy timepoint. Thirty percent of baseline variants and 33% of variants at progression were deemed actionable. Upon JH MTB review, 29% of patients received a genotype-matched therapeutic recommendation based on LB results, with MTB recommendations followed in 38% of the patients. In analyzing ctDNA dynamics, of the 13 patients with on-therapy samples, 23% showed ctDNA clearance, 31% showed persistence, while 46% had undetectable ctDNA at both time points. Among patients with LBs at the time of progression, a third showed ctDNA emergence, a third had ctDNA persistence, and the remainder had undetectable ctDNA throughout the follow-up period. Conclusion: Our findings highlight the clinical relevance of liquid biopsies in guiding treatment selection and monitoring. Citation Format: Maria Fatteh, Michael Conroy, Timsy Wanchoo, Selina Shiqing Teh, Jaime Wehr, Archana Balan, Rachel Karchin, Ellen Verner, Katerina Karaindrou, Rena Xian, Christopher Gocke, Ming-Tseh Lin, Peggy Fitzpatrick, Christine L. Hann, Joseph C. Murray, Vincent K. Lam, Josephine L. Feliciano, Julie R. Brahmer, Kristen A. Marrone, Patrick M. Forde, Katie Fiallos, Dana Petry, Taxiarchis Botsis, Mark Sausen, the Johns Hopkins Molecular Tumor Board Investigators, Jenna Canzoniero, Jessica Tao, Valsamo Anagnostou. Liquid biopsy-informed precision oncology clinical trial to evaluate the utility of ctDNA comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 970.

A novel method for simultaneous detection of hematological tumors and infectious pathogens by metagenomic next generation sequencing of plasma

BackgroundMetagenomic next-generation sequencing (mNGS) is valuable for pathogen identification; however, distinguishing between infectious diseases and conditions with potentially similar clinical manifestations, including malignant tumors, is challenging. Therefore, we developed a method for simultaneous detection of infectious pathogens and cancer in blood samples. MethodsPlasma samples (n = 244) were collected from 150 and 94 patients with infections and hematological malignancies, respectively, and analyzed by mNGS for pathogen detection, alongside human tumor chromosomal copy number variation (CNV) analysis (≥5Mbp or 10Mbp CNV region). Further, an evaluation set, comprising 87 plasma samples, was analyzed by mNGS and human CNV analysis, to validate the feasibility of the method. ResultsAmong 94 patients with hematological malignancy, sensitivity values of CNV detection for tumor diagnosis were 69.15 % and 32.98 % for CNV region 5Mbp and 10Mbp, respectively, with corresponding specificities of 92.62 % and 100 % in the infection group. Area under the ROC curve (AUC) values for 5Mbp and 10Mbp region were 0.825 and 0.665, respectively, which was a significant difference of 0.160 (95 % CI: 0.110–0.210; p < 0.001), highlighting the superiority of 5Mbp output region data. Six patients with high-risk CNV results were identified in the validation study: three with history of tumor treatment, two eventually newly-diagnosed with hematological malignancies, and one with indeterminate final diagnosis. ConclusionsConcurrent CNV analysis alongside mNGS for infection diagnosis is promising for detecting malignant tumors. We recommend adopting a CNV region of 10Mbp over 5Mbp for our model, because of the lower false-positive rate (FPR).

Application of cell-free plasma next-generation sequencing technology in the diagnosis and management of pediatric meningitis

This retrospective study evaluates the clinical utility of CFPNGS in the diagnosis and management of pediatric meningitis. CFPNGS identified a causative pathogen in 36% of 28 subjects, compared to 50% for diverse conventional testing (57% combined). CFPNGS may be considered as an adjunct to standard testing.

Brief Report: Droplet Digital Polymerase Chain Reaction Versus Plasma Next-Generation Sequencing in Detecting Clearance of Plasma EGFR Mutations and Carcinoembryonic Antigen Levels as a Surrogate Measure

IntroductionTo compare the performance of droplet digital polymerase chain reaction (ddPCR) and plasma next-generation sequencing (NGS) in detecting clearance of plasma EGFR (pEGFR) mutations. MethodsPatients with treatment-naive advanced EGFR-mutated lung cancer treated with first-line tyrosine kinase inhibitors (TKIs) were included. pEGFR were measured at baseline and first response assessment using ddPCR and NGS. Clearance of pEGFR was defined as undetectable levels after a positive baseline result. Results were correlated with time-to-treatment failure (TTF). In exploratory analysis, corresponding change in carcinoembryonic antigen (CEA) levels was evaluated. ResultsBetween January 1, 2020, and December 31, 2021, 27 patients were recruited. Ex19del comprised 74% (20 of 27) and L858R 26% (seven of 27). Osimertinib was used in 59% (16 of 27), dacomitinib 4% (one of 27), and gefitinib/erlotinib 37% (10 of 27). Sensitivity of ddPCR and NGS in detecting pEGFR mutation at baseline was 70% (19 of 27) and 78% (21 of 27), respectively (p = 0.16). All patients with detectable pEGFR by ddPCR were detected by NGS.At a median of 8 (range 3–24) weeks post-TKI initiation, clearance of pEGFR was achieved in 68% (13 of 19) and 71% (15 of 21) using ddPCR and NGS, respectively. Concordance between ddPCR and NGS was 79% (kappa = 0.513, p = 0.013). Clearance of pEGFR was associated with longer median TTF (not reached versus 6 months, p = 0.03) and median decrease in CEA levels by 70% from baseline.In another cohort of 124 patients, decrease in CEA levels by greater than 70% within 90 days of TKI initiation was associated with doubling of both TTF and overall survival. ConclusionsPlasma NGS trended toward higher sensitivity than ddPCR in detecting pEGFR, although both had similar concordance in detecting pEGFR clearance. Our results support using NGS at diagnosis and interchangeability of NGS and ddPCR for monitoring, whereas CEA could be explored as a surrogate for pEGFR clearance.

Impact of tissue biomarker testing by in-house platform on result times in metastatic non-small cell lung cancer.

49 Background: Biomarker testing for actionable mutations is standard of care for patients diagnosed with metastatic non-small cell, non-squamous cell lung cancer. However, testing via third-party laboratories can take weeks to result, leading to treatment decision delays identified at our own institution. Methods: We conducted a retrospective chart review of metastatic non-small cell, non-squamous cell lung cancer patients treated at Orlando Regional Medical Center before (6/1/2021-5/31/2022) as well as after (1/1/2023-5/31/2023) the initiation of in-house next generation sequencing (NGS) tissue testing using Genexus. Endpoints included average and median time to result of plasma and tissue NGS both before and after initiation of in-house molecular testing. An independent two sample t-test was conducted with p&lt;0.05 considered significant. Results: A total of 49 and 18 patients were included pre and post initiation of in house NGS, respectively. For patients with tissue testing, the average time from collection to result was 31.3 versus 13.9 days (p&lt;0.01), median 29 versus 7 days. For patients who underwent plasma testing (n=49, n=10), average time from collection to result was 13.3 versus 9.6 days (p=0.38), median 9 versus 9 days. Conclusions: Time to comprehensive tissue testing result significantly decreased after the commencement of in-house NGS. Rapid turnaround time allows clinicians to select appropriate first-line treatment without delay in care. Further efforts are needed to expedite molecular testing in all stages of non-small cell lung cancer and evaluate sequential versus concurrent plasma and tissue testing.

A Phase II Study of Osimertinib in Patients with Advanced-Stage Non-Small Cell Lung Cancer following Prior Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR TKI) Therapy with EGFR and T790M Mutations Detected in Plasma Circulating Tumour DNA (PLASMA Study).

Epidermal growth factor receptor (EGFR) T790M mutations drive resistance in 50% of patients with advanced non-small cell lung cancer (NSCLC) who progress on first/second generation (1G/2G) EGFR tyrosine kinase inhibitors (TKIs) and are sensitive to Osimertinib. Tissue sampling is the gold-standard modality of T790M testing, but it is invasive. We evaluated the efficacy of Osimertinib in patients with EGFR mutant NSCLC and T790M in circulating tumour DNA (ctDNA). PLASMA is a prospective, open-label, multicentre single-arm Phase II study. Patients with advanced NSCLC harbouring sensitizing EGFR and T790M mutations in plasma at progression from ≥one 1G/2G TKI were treated with 80 mg of Osimertinib daily until progression. The primary endpoint was the objective response rate (ORR); the secondary endpoints included progression-free survival (PFS), overall survival (OS), disease control rate (DCR) and toxicities. Plasma next-generation sequencing was performed to determine Osimertinib resistance mechanisms and assess serial ctDNA. A total of 110 patients from eight centres in five countries were enrolled from 2017 to 2019. The median follow-up duration was 2.64 (IQR 2.44-3.12) years. The ORR was 50.9% (95% CI 41.2-60.6) and the DCR was 84.5% (95% CI 76.4-90.7). Median PFS was 7.4 (95% CI 6.0-9.3) months; median OS was 1.63 (95% CI 1.35-2.16) years. Of all of the patients, 76% had treatment-related adverse events (TRAEs), most commonly paronychia (22.7%); 11% experienced ≥ Grade 3 TRAEs. The ctDNA baseline load and dynamics were prognostic. Osimertinib is active in NSCLC harbouring sensitizing EGFR and T790M mutations in ctDNA testing post 1G/2G TKIs.

Expression of EGFR-mutant proteins and genomic evolution in EGFR-mutant transformed small cell lung cancer.

The transformation of epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) into small cell lung cancer (SCLC) accounts for 3-14% of the resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs). At present, there is no relevant research to explore the dynamic expression of EGFR-mutant proteins and genomic evolution in EGFR-mutant transformed SCLC/neuroendocrine carcinoma (NEC). Genetic analysis and protein level analysis by next-generation sequencing (NGS), Whole-exome sequencing (WES) and immunohistochemistry were performed to explore expression of EGFR-mutant proteins and genomic evolution in EGFR-mutant transformed SCLC. The research used three patient-derived organoids (PDOs) to explore the efficacy of combo [chemotherapy (chemo) plus TKI or bevacizumab] treatment. According to the subsequent treatment regimens after SCLC/NEC transformation, 35 patients were divided into chemo (n=21) and combo (n=14) groups. EGFR L858R and EGFR E746-750 del protein expression by immunohistochemistry was 80.0% (4/5) and 100% (6/6), respectively (P=0.455) in initially-transformed tissues. Meanwhile, EGFR-mutant proteins were expressed in 85.7% (6/7) of dynamic rebiopsy tissues or effusion samples after the first transformation. Then, by the pathway enrichment analysis of tissue and plasma NGS, the EGFR-related pathways were still activated after SCLC/NEC transformation. Moreover, WES analysis revealed that transformed SCLC shared a common clonal origin from the baseline LUAD. The drug sensitivity of three PDOs demonstrated potent anti-cancer activity of EGFR-TKIs plus chemo, compared with chemo or TKI alone. There were significant differences in objective response rate (ORR) between the combo and chemo groups [42.9 % vs. 4.8%, P=0.010, 95% confidence interval (CI): 1.5-145.2]. Furthermore, the median post-transformation progression-free survival (pPFS) was significantly prolonged in the combo group, with 5.4 (95% CI: 3.4-7.4) versus 3.5 (95% CI: 2.7-4.3, P=0.012) months. EGFR 19del or L858R-mutant proteins could be constantly expressed, and EGFR pathway still existed in EGFR-mutant transformed SCLC/NEC with a common clonal origin from the baseline LUAD. Taking together, these molecular characteristics potentially favored clinical efficacy in transformed SCLC/NEC treated with the combo regimen.

Phase 2 study of telisotuzumab vedotin (Teliso-V) monotherapy in patients with previously untreated MET-amplified locally advanced/metastatic non-squamous non-small cell lung cancer (NSQ NSCLC).

TPS9149 Background: Teliso-V is a first-in-class anti–c-Met antibody-drug conjugate composed of the monoclonal antibody telisotuzumab (ABT-700), a cleavable valine-citrulline (dipeptide) linker, and cytotoxic monomethylauristatin E (MMAE; potent microtubule polymerization inhibitor). Direct MMAE payload delivery to c-Met protein overexpressing (OE) cells enables Teliso-V to work independently of c-Met signaling. In NSCLC, while c-Met OE is more common than MET gene amplification (Amp), about 90% of MET Amp tumors will also be c-Met OE. In an ongoing phase 2 trial, Teliso-V showed encouraging efficacy and acceptable safety in previously treated c-Met OE NSQ NSCLC population (Camidge et al. JCO.2022.40.16_suppl.9016). From this study, a retrospective analysis of 10 patients with MET Amp (≥1.8 MET gene to chromosome 7 copy number ratio by fluorescence in situ hybridization [FISH]) demonstrated an objective response rate (ORR) of 80%. Described here is an ongoing phase 2 study evaluating efficacy and safety of Teliso-V monotherapy in patients with previously untreated MET Amp locally advanced/metastatic NSQ NSCLC. Methods: This is a phase 2, single-arm, open-label, global study (NCT05513703). Eligible patients (≥18 years) have histologically documented advanced/metastatic NSQ NSCLC, measurable disease by RECIST v1.1, confirmed MET Amp determined centrally by FISH or locally by FISH, tissue next-generation sequencing (NGS), or plasma NGS using sponsor-approved assays. Additional eligibility criteria include Eastern Cooperative Oncology Group performance status 0 or 1, no alterations in EGFR, ALK, ROS1, or BRAF that predict sensitivity to targeted therapy, no prior systemic therapy for locally advanced or metastatic NSCLC, no prior c-Met–targeted antibody therapy. Teliso-V is administered intravenously at 1.9 mg/kg every 2 weeks until disease progression, intolerable toxicity, or other study drug discontinuation criteria are met. Primary endpoint is ORR assessed by independent central review (ICR). Secondary endpoints include duration of response and disease control rate, progression-free survival per ICR, overall survival, patient-reported outcomes (PROs). Tumor assessments (per RECIST v1.1) are performed at baseline and approx. every 6 (year 1), 8 (year 2), and 12 weeks (year 3 and beyond). PROs are assessed through validated questionnaires (EORTC QLQ-LC13, EORTC QLQ-C30, EQ-5D-5L). Safety and tolerability are evaluated by adverse events, physical examinations, laboratory data, and vital signs. Pharmacokinetic and biomarker evaluations may be used for exploratory analyses. The trial intends to enroll approx. 70 efficacy-evaluable patients, with interim analyses planned after 20 and 50 patients are able to be followed for ≥6 months. The study was open to enrollment as of Nov 2022. Clinical trial information: NCT05513703 .

Which trial do we need? Plasma metagenomic next-generation sequencing to diagnose infections in patients with haematological malignancies and febrile neutropenia: proposal for a randomized-controlled trial

Which trial do we need? Plasma metagenomic next-generation sequencing to diagnose infections in patients with haematological malignancies and febrile neutropenia: proposal for a randomized-controlled trial

Metagenomic next-generation sequencing of plasma for the identification of bloodstream infectious pathogens in severe aplastic anemia

Objective: To analyze the diagnostic value of cell-free plasma metagenomic next-generation sequencing (mNGS) pathogen identification for severe aplastic anemia (SAA) bloodstream infection. Methods: From February 2021 to February 2022, mNGS and conventional detection methods (blood culture, etc.) were used to detect 33 samples from 29 consecutive AA patients admitted to the Anemia Diagnosis and Treatment Center of the Hematology Hospital of the Chinese Academy of Medical Sciences to assess the diagnostic consistency of mNGS and conventional detection, as well as the impact on clinical treatment benefits and clinical accuracy. Results: ①Among the 33 samples evaluated by mNGS and conventional detection methods, 25 cases (75.76%) carried potential pathogenic microorganisms. A total of 72 pathogenic microorganisms were identified from all cases, of which 65 (90.28%) were detected only by mNGS. ②All 33 cases were evaluated for diagnostic consistency, of which 2 cases (6.06%) were Composite, 18 cases (54.55%) were mNGS only, 2 cases (6.06%) were Conventional method only, 1 case (3.03%) was both common compliances (mNGS/Conventional testing) , and 10 cases (30.3%) were completely non-conforming (None) . ③All 33 cases were evaluated for clinical treatment benefit. Among them, 8 cases (24.24%) received Initiation of targeted treatment, 1 case (3.03%) received Treatment de-escalation, 13 cases (39.39%) received Confirmation, and the remaining 11 cases (33.33%) received No clinical benefit. ④ The sensitivity of 80.77%, specificity of 70.00%, positive predictive value of 63.64%, negative predictive value of 84.85%, positive likelihood ratio of 2.692, and negative likelihood ratio of 0.275 distinguished mNGS from conventional detection methods (21/12 vs 5/28, P<0.001) . Conclusion: mNGS can not only contribute to accurately diagnosing bloodstream infection in patients with aplastic anemia, but can also help to guide accurate anti-infection treatment, and the clinical accuracy is high.

Metagenomic Next-Generation Sequencing of Plasma for Diagnosis of COVID-19-Associated Pulmonary Aspergillosis.

Timely diagnosis remains an unmet need in non-neutropenic patients at risk for aspergillosis, including those with COVID-19-associated pulmonary aspergillosis (CAPA), which in its early stages is characterized by tissue-invasive growth of the lungs with limited angioinvasion. Currently available mycological tests show limited sensitivity when testing blood specimens. Metagenomic next-generation sequencing (mNGS) to detect microbial cell-free DNA (mcfDNA) in plasma might overcome some of the limitations of conventional diagnostics. A two-center cohort study involving 114 COVID-19 intensive care unit patients evaluated the performance of plasma mcfDNA sequencing for the diagnosis of CAPA. Classification of CAPA was performed using the European Confederation for Medical Mycology (ECMM)/International Society for Human and Animal Mycoses (ISHAM) criteria. A total of 218 plasma samples were collected between April 2020 and June 2021 and tested for mcfDNA (Karius test). Only 6 patients were classified as probable CAPA, and 2 were classified as possible, while 106 patients did not fulfill CAPA criteria. The Karius test detected DNA of mold pathogens in 12 samples from 8 patients, including Aspergillus fumigatus in 10 samples from 6 patients. Mold pathogen DNA was detected in 5 of 6 (83% sensitivity) cases with probable CAPA (A. fumigatus in 8 samples from 4 patients and Rhizopus microsporus in 1 sample), while the test did not detect molds in 103 of 106 (97% specificity) cases without CAPA. The Karius test showed promising performance for diagnosis of CAPA when testing plasma, being highly specific. The test detected molds in all but one patient with probable CAPA, including cases where other mycological tests from blood resulted continuously negative, outlining the need for validation in larger studies.

Clinical Diagnosis Application of Metagenomic Next-Generation Sequencing of Plasma in Suspected Sepsis.

We analyzed the clinical concordance of mNGS test results from blood samples and improved the clinical efficiency of mNGS in the diagnosis of suspected sepsis pathogens. In this study, 99 samples of suspected blood flow infection were included for plasma mNGS, and the correlation between mNGS results and blood culture results, serum inflammatory indices, clinical symptoms and antibiotic treatment was analyzed, as well as the comparison with the detection rate of BALF pathogens, as well as the classification of different pathogens in the mNGS results were analyzed. The mNGS pathogen detection rate was higher than that of traditional blood culture (83.02% vs 35.82%). The rate of the mNGS results being consistent with the clinical diagnosis was also higher than that of traditional blood culture (58.49% vs 20.75%). This study shows that bacteria and fungi are the main pathogens in sepsis, and viral sepsis is very rare. In this study, 32% of sepsis patients were secondary to pneumonia. Compared with the pathogen detection rate using alveolar lavage fluid, the detection rate from plasma mNGS was 62.5%. Samples were also easy to sample, noninvasive, and more convenient for clinical application. This study shows that compared with blood culture, the detection rate of mNGS pathogen that meets the diagnosis of sepsis is higher. We need a combination of multiple indicators to monitor the early diagnosis and treatment of sepsis.

The effect of a plasma next-generation sequencing test on antimicrobial management in immunocompetent and immunocompromised patients-A single-center retrospective study.

To describe the use of next-generation sequencing (NGS) and to determine whether NGS leads to changes in antimicrobial management. This retrospective cohort study included patients aged ≥18 years admitted to a single tertiary-care center in Houston, Texas, with an NGS test performed between January 1, 2017, and December 31, 2018. In total, 167 NGS tests were performed. Most patients were of non-Hispanic ethnicity (n = 129), white (n = 106), and male (n = 116), with a mean age of 52 years (SD, 16). Moreover, 61 patients were immunocompromised: solid-organ transplant (n = 30), patients with human immunodeficiency virus (n = 14), and rheumatology patients on immunosuppressive therapy (n = 12). Of the 167 NGS tests performed, 118 (71%) were positive. Test results associated with a change in antimicrobial management were found in 120 (72%) of 167 cases, with an average of 0.32 (SD, 1.57) fewer antimicrobials after the test. The largest change in antimicrobial management was in glycopeptide use (36 discontinuations) followed by antimycobacterial drug use (27 additions among 8 patients). Also, 49 patients had negative NGS results, but only 36 patients had their antibiotics discontinued. Plasma NGS testing is associated with a change in antimicrobial management in most cases. We observed a decrease in glycopeptide use after NGS results, which highlights physicians' comfort in withdrawing methicillin-resistant Staphylococcus aureus (MRSA) coverage. In addition, antimycobacterial coverage increased, corresponding with early mycobacterial detection by NGS. Further studies are needed to determine effective ways to use NGS testing as an antimicrobial stewardship tool.

Cell-free plasma next-generation sequencing assists in the evaluation of secondary pneumonia in patients with COVID-19: a case series.

Secondary pneumonia occurs in 8-24% of patients with Coronavirus 2019 (COVID-19) infection and is associated with increased morbidity and mortality. Diagnosis of secondary pneumonia can be challenging. The purpose of this study was to evaluate the use of plasma microbial cell free DNA sequencing (mcfNGS) in the evaluation of secondary pneumonia after COVID-19. We performed a single-center case series of patients with COVID-19 who underwent mcfNGS to evaluate secondary pneumonia and reported the organisms identified, concordance with available tests, clinical utility, and outcomes. In 8/13 (61%) cases, mcfNGS detected 1-6 organisms, with clinically significant organisms identified in 4 cases, including Pneumocystis jirovecii, and Legionella spp. Management was changed in 85% (11/13) of patients based on results, including initiation of targeted therapy, de-escalation of empiric antimicrobials, and avoiding contingent escalation of antifungals. mcfNGS may be helpful to identify pathogens causing secondary pneumonia, including opportunistic pathogens in immunocompromised patients with COVID-19. However, providers need to carefully interpret this test within the clinical context.

546. Resolution of a Cluster of Travel-Associated Leptospira santarosai Infection in Four Adolescents by Plasma Microbial Cell-Free DNA Detection with the Karius Test®

Abstract Background Leptospira can cause severe infections but their diagnosis can be challenging. Diagnosis is limited by nonspecific, protean clinical symptoms; the fastidious nature of Leptospira; and poor specificity, sensitivity, and long turnaround times of serologic testing. Given these limitations, identifying epidemiological clusters of leptospirosis can be challenging. Rapid, non-invasive diagnosis of leptospirosis by microbial cell-free DNA (mcfDNA) next-generation sequencing (NGS) of plasma offers a means to overcome these limitations in individual diagnosis and public health epidemiological surveillance. Methods The Karius Test® (KT) detects and quantifies mcfDNA in molecules/µL (MPM) in plasma from a curated genomic database of &amp;gt;20,000 organisms reporting on &amp;gt;1000 pathogens through the Karius CLIA certified/CAP accredited lab (Redwood City, CA). Four KT detections of Leptospira santarosai were identified April - August 2021 across four independent United States institutions. Clinical review was performed by pediatric infectious diseases consultants. Results KT detected L. santarosai in four adolescents with travel to Costa Rica or Belize (Table). A broad work-up was performed in all patients. McfDNA NGS was the first test to identify L. santarosai as the diagnosis in all cases with an average time to diagnosis of 2.5+/- 0.58 days from sample collection, enabling narrowed, targeted antibiotic treatment. L. santarosai mcfDNA was positive in three patients despite pretreatment; average L. santarosai mcfDNA concentration was 173.5+/- 186.1 MPM. Leptospira serologies were confirmatory in three patients, potential epidemiological exposures were identified in all patients, and all recovered. Conclusion KT enabled rapid, non-invasive diagnosis of diverse manifestations of L. santarosai against a competing broad differential in four adolescent travelers returning from Costa Rica or Belize. The resolution of the etiology of this cluster of patients with leptospirosis demonstrates the potential utility of mcfDNA to quickly diagnose and support case definitions of reportable diseases and the potential importance of plasma mcfDNA NGS in public health and epidemiological surveillance. Disclosures Asim A. Ahmed, MD, Karius: Employee|Karius: Stocks/Bonds Sara Saporta-Keating, MS, MD, Pfizer: Co-investigator in investigator-driven study sponsored by Pfizer.

348. Cell-free plasma next-generation sequencing assists in the evaluation of invasive mold infection in patients with COVID-19: a case series

Abstract Background Distinguishing COVID-19 Associated Pulmonary Aspergillosis (CAPA) and invasive mold infections (IMIs) from other causes of secondary pneumonia in COVID-19 can be challenging. 1,3-β-D-Glucan and galactomannan are commonly utilized biomarkers for the workup of IMIs but are limited by a lack of specificity and sensitivity respectively. Cell-free plasma next-generation sequencing (cfNGS) is a promising non-invasive approach that can provide direct detection of pathogens in patient's serum. This study explored its potential role in the evaluation of secondary pneumonia in patients with COVID-19. Methods We performed a retrospective single-center observational study from March 2020 to December 2021 at Virginia Commonwealth University Medical Center, a 811-bed tertiary care center, to evaluate patients with laboratory confirmed SARS-CoV-2 virus infection who underwent cfNGS for the evaluation of CAPA. CfNGS (Karius, Inc., Redwood City, CA) was performed at the discretion of the clinical provider and we evaluated the test indication, patient history, clinical impact, correlation with serum biomarkers, and 30 day all-cause mortality. Results Thirteen patients were evaluated and none had Aspergillus species detected. One patient had Pneumocystis jirovecii on cfNGS. There was a 76.9% (10/13) concordance rate with patients’ serum fungal biomarkers. CfNGS also detected concomitant organisms in 53.8% (7/13) of our cohort. These data assisted in changes of clinical management for 84.6% (11/13) of patients and lead to the change in antifungal usage in 69.2% (9/13). Conclusion In this study, both negative and positive cfNGS test results assisted in important clinical decision making. cfNGS may have a role in the evaluation of CAPA or other IMIs in patients with COVID-19. Disclosures Megan M. Morales, MD, Karius: Paid speaker.