Next-generation Sequencing Of Cell-free DNA Research Articles

Plasma Microbial Cell-free DNA Next-generation Sequencing Can Be a Useful Diagnostic Tool in Patients With Osteoarticular Infections.

Recent advances in shotgun metagenomic sequencing (sMGS) for detecting microbial cell-free DNA (mcfDNA) in peripheral blood have shown promise across various patient populations. This study evaluates the application of sMGS for diagnosing osteoarticular infections (OAIs), a condition with significant diagnostic challenges. We conducted a retrospective analysis on 73 patients suspected of OAIs at the Mayo Clinic from 2019 to 2023, incorporating mcfDNA sMGS (Karius test [KT]) into their diagnostic evaluation. We categorized the clinical impact of KT on OAI diagnoses and management into 4 distinct outcomes. (1) KT was able to confirm an established diagnosis, (2) KT supported noninfectious diseases diagnosis, (3) KT established an unsuspected diagnosis, (4) KT did not add relevant information. In our cohort, KT was performed in 73 patients. Among the infected individuals, KT yielded positive results in 22 of 43 (51.2%) cases. Of these 22 cases, 11 (50%) showed agreement with conventional diagnostic workup, whereas in 5 (22.7%) cases, the KT established an unsuspected diagnosis. Native vertebral osteomyelitis diagnosis (P < .001) or OAIs with concomitant presence of endocarditis or endovascular infection (P = .005) were statistically associated with a definite, probable, or possible diagnostic certainty of KT result. In complex OAIs, KT enhanced diagnostic accuracy by 11.6%, proving especially beneficial in diagnosing native vertebral osteomyelitis and infections with concurrent endocarditis or endovascular complications. Our findings underscore the utility of KT in the diagnostic workflow for challenging OAI cases, potentially altering clinical management for a significant subset of patients.

Utility of microbial cell free DNA next-generation sequencing for diagnosis and management of infectious diseases

Microbial cell-free DNA (mcfDNA) sequencing is a promising tool to identify infectious pathogens when traditional methods fail to identify the causative agent. We performed a retrospective observational cohort study to evaluate clinical outcomes among pediatric and adult patients who underwent mcfDNA testing. 127 mcfDNA tests were reviewed from 112 patients. Baseline characteristics included 61 (54.5 %) adults, 52 (40.9 %) tests were from female patients, and 67 (52.8 %) tests were obtained from patients designated as immunocompromised. Of all tests obtained, 59 (46.4 %) were deemed clinically relevant. 41 (32.3 %) of tests also led to a change in antimicrobial management for the corresponding patient. No statistically significant association was ascertained between patient-specific factors and clinically relevant test results. Testing in certain clinical scenarios or high-risk settings may be useful, however further studies are needed to assess the cost-benefit of this approach.

Classifying Glioma via Liquid Biopsy: Progress toward an Unmet Clinical Need.

The diagnosis and classification of glioma by liquid biopsy represent a critical unmet need in neuro-oncology. A recent study demonstrates targeted next-generation sequencing of cell-free DNA from cerebrospinal fluid as an evolving option for liquid biopsy in patients with glioma. See related article by Iser et al., p. 2974.

Abstract P19: Comprehensive Benchmark of Somatic Mutation Callers on Cell-Free DNA Next-Generation Sequencing Data in Cancer Liquid Biopsy

Abstract Circulating tumour DNA (ctDNA) has emerged as a rich source of biomarkers in precision oncology. However, ctDNA sequence data is often noisier than that derived from standard tissue biopsies, posing significant challenges for accurate cancer mutation detection. Besides, there is a lack of standardised and unbiased benchmark datasets to evaluate and compare performance of variant callers on ctDNA. To address this issue, we present a novel benchmarking approach and comprehensive benchmark of cancer somatic mutation callers on cell-free DNA (cfDNA) Next Generation Sequencing (NGS) data. To create a large-scale benchmark dataset, we developed a new methodology using real colorectal cancer patients plasma samples exhibiting either ultra-high and an ultra-low levels of ctDNA. This enabled the generation of dilution series of deep Whole Genome Sequencing (WGS, 150x) and the creation of high-quality consensus ground truth calls. We evaluated the performance of 9 SNV callers and 7 short INDEL callers frequently used in academic research, including 2 callers that were specifically designed for cfDNA. We further validated our findings at higher depth of coverage using deep Whole Exome Sequencing (WES, &amp;gt;2,000x) data. We further investigated each caller's features to develop practical guidelines for users depending on their application setting: unguided analysis focusing on overall accuracy vs. guided analysis focusing on sensitivity. Citation Format: Hanaé Carrié, Ngak Leng Sim, Pui Mun Wong, Anna Gan, Yi Ting Lau, Polly Poon, Saranya Thangaraju, Iain Tan, Yoon Sim Yap, Limsoon Wong, Anders Skanderup. Comprehensive Benchmark of Somatic Mutation Callers on Cell-Free DNA Next-Generation Sequencing Data in Cancer Liquid Biopsy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P19.

P20 Clinical metagenomics from cell-free DNA: overview and lessons learned from 4 years as a clinical metagenomics provider

Abstract Background Clinical metagenomics emerges more and more from academic research to clinical practice. Here, we share an overview of the field of clinical metagenomics on cell-free DNA found in the blood of patients with severe infections and share a few learning points regarding the implementation as a routine lab service and CE-IVD marked software running in the cloud. Methods We use next-generation sequencing of cell-free DNA to digitally determine relevant pathogens from patients with systemic infections in blood and other bodily fluids using our CE-IVD marked DISQVER diagnostic platform. Until now we have collected microbial data from several thousand samples within 4 years of routine service. Results The set up of the DISQVER test is within the regulatory framework of CE-IVD marking of software as a service (SaaS) and ISO 13485 accreditation. This brings practical implications on database quality, software lock-in/development/testing, change and risk management, technical documentation, requiring substantial resources. Also the wetlab process runs within tightly defined and controlled QM boundaries, pre-set by extensive analytical validations. We clinically validated our test for pathogen identification from blood within 8 clinical trials for intensive care medicine, haematology and coinfection in COVID-19, showing 2- to 3-fold higher identification rates for pathogens in comparison to standard of care blood culture. Furthermore, several case reports/ case series from different clinical backgrounds, frequently with difficult to diagnose pathogens, have been published. From the analysis of our routine processes with several thousand samples, we could show that the turn-around time of our mNGS process is very fast and can detect more species than blood culture. In particular, the relatively high proportion of fungi and dsDNA viruses indicates superiority of the method compared with the standard diagnostic pathway. Conclusions To bridge the gap between academic research and clinical implementation, many aspects of regulatory, technical and data protection requirements need to be addressed. Within the timeframe of commercial availability DISQVER from cell-free DNA found in the blood of infected patients is increasingly becoming a useful tool for infection diagnostics.

Proof-of-concept evaluation of next-generation sequencing-based liquid biopsy for non-invasive cancer detection in cats.

This proof-of-concept evaluation demonstrates that next-generation sequencing-based liquid biopsy can detect genomic alterations in the blood of cats with cancer and the absence of such alterations in the blood of presumably cancer-free cats. Two cats with cytologically confirmed lymphoma and nine presumably cancer-free cats were included in this analysis. Whole blood was collected from each subject and samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Both cancer-diagnosed subjects had somatic copy number variants (a "cancer signal") identified in cell-free DNA, suggesting the current presence of cancer in these subjects. All nine presumably cancer-free subjects had unremarkable genomic profiles, suggesting the absence of cancer in these subjects. Liquid biopsy using next-generation sequencing of cell-free DNA allows for blood-based detection of cancer-associated genomic alterations in cats. Such technology has the potential to offer considerable utility in veterinary medicine, particularly for the non-invasive prioritization of small cell intestinal lymphoma versus inflammatory bowel disease in cats with gastrointestinal signs. This study lays the foundation for future studies to fully validate this type of testing for use in clinical practice.

Detection of invasive Bartonella infections with next-generation sequencing of microbial cell-free DNA.

We report 9 patients with invasive Bartonella infections, including 5 with endocarditis, who were diagnosed with microbial cell-free DNA next-generation sequencing and Bartonella serology studies. Diagnosis with plasma mcfDNA NGS enabled a faster clinical and laboratory diagnosis in 8 patients. Prompt diagnosis impacted antibiotic management in all 9 patients.

Metagenomic next-generation sequencing of cell-free DNA for the identification of viruses causing central nervous system infections.

This study provides significant new data on the application of metagenomic next-generation sequencing (mNGS) to clinical diagnostics of central nervous system (CNS) viral infections, which can have high mortality rates and severe sequelae. Conventional diagnostic procedures for identifying viruses can be inefficient and rely on preconceived assumptions about the pathogen, making mNGS an appealing alternative. However, the effectiveness of mNGS is affected by the presence of human DNA contamination, which can be minimized by using cell-free DNA (cfDNA) instead of whole-cell DNA (wcDNA). This multi-center retrospective study of patients with suspected viral CNS infection found that mNGS using cfDNA had a significantly lower proportion of human DNA and higher sensitivity for detecting viruses than mNGS using wcDNA. Herpesviruses, particularly VZV, were found to be the most common DNA viruses in these patients. Overall, mNGS using cfDNA is a promising complementary diagnostic method for detecting CNS viral infections.

Detection of Isolated Extramedullary AML Using Next-Generation Sequencing of Cell-Free DNA

Introduction Isolated extramedullary (IEM) manifestation of AML is a recurrent event especially after allogenic hematopoietic cell transplantation (alloHCT). To date, measurable residual disease (MRD) assessment for early detection and monitoring of this difficult-to-treat patient cohort has not been established. Using cell-free DNA (cfDNA) from plasma or serum for MRD measurement shows comparable sensitivity to bone marrow mononuclear cells (BMMCs) and improved sensitivity over peripheral blood mononuclear cells (PBMCs) in medullary AML (Nakamura et al., Blood, 2019). Here, we evaluate next-generation sequencing (NGS) for MRD assessment of cfDNA in a cohort of patients with IEM AML. Methods We retrospectively collected tumor tissue and matched BMMC/PBMCs and cfDNA serum or plasma samples from IEM AML patients at our institution. BMMC/PBMCs and IEM tumor tissue were sequenced with 35- to 48-gene panels at diagnosis and relapse. Amplicon-based NGS-MRD (sensitivity 0.01%) of matched cfDNA and BMMC/PBMCs was quantified at time of IEM manifestation and in all available samples collected up to 12 months before IEM manifestation using the known mutations from IEM AML tissue. Amplicon-based NGS and bioinformatic error-correction was conducted and analyzed as previously described (Thol et al., Blood, 2018). Results We identified a cohort of 15 IEM AML patients at our institution. One patient was diagnosed with IEM AML at first diagnosis, 14 patients suffered from IEM relapse. For three patients, matched BMMC/PBMCs and cfDNA samples were collected from a second IEM AML relapse during the disease course. 15 of 18 IEM AML episodes (83%) occurred as relapse after alloHCT. 42 AML-related mutations were identified in the IEM AML tissue with a median of three mutations per patient (range 1-5). 33 of 39 mutations (85%) found in IEM relapse tissue had been already present at first diagnosis. All mutations were then evaluated in cfDNA and BMMCs at the time of the IEM manifestation by sensitive NGS-MRD analysis for all 18 IEM AML episodes. For two IEM episodes, BMMCs were not available and PBMCs were used instead. 17 of 18 (94%) cfDNA samples were NGS-MRD positive at the time of IEM manifestation, while only 8 of 18 (44%) BMMC/PBMC samples were positive. 20 of 53 mutations (38%) at first and second IEM episode could only be detected using cfDNA, 15 mutations (28%) were detected in both cfDNA and BMMC/PBMCs and only one mutation was solely found in BMMCs. 17 mutations (32%) could not be detected by either method (Figure 1). The variant allele frequency (VAF) of mutations detected by both methods was significantly higher using cfDNA with a median VAF of 2.45% (range 0.12 - 24%) compared to BMMC/PBMCs with a median VAF of 0.048% (range 0.13 - 10%; p &amp;lt; 0.01). Median VAF of mutations that were only found in cfDNA was 0.63% (range 0.017 - 39.6) and did not significantly differ from the cfDNA VAF of mutations that were found by both methods (p=0.43), indicating that these mutations represented the major clone. However, no correlation was found between IEM tumor VAF and cfDNA VAF (r=0.01; p=0.95). For ten IEM episodes from nine patients at least one cfDNA sample was available within 12 months before IEM relapse. In six IEM episodes (60%) cfDNA samples were MRD positive, one at 6 months, one at 5 months, two at 3 months and two at 2 months before IEM relapse. In four IEM episodes (40%) cfDNA samples were MRD negative, one at 10 months, one at 5 months and two at 2 months before IEM relapse. Conclusion In summary, cfDNA analysis from serum or plasma of AML patients can identify the molecular characteristics of a large proportion of patients with IEM AML. More than one third of clinically relevant mutations could only be detected using cfDNA and were missed by NGS-MRD of BMMC/PBMCs. Our data suggest that MRD monitoring from cfDNA detects IEM relapse earlier than routine monitoring from PB or BM in a significant proportion of patients.

556. Commercial Microbial Cell-Free DNA Next-Generation Sequencing in a Pediatric Quaternary Care Facility Yields Limited Diagnostic Utility

Abstract Background Microbial cell-free DNA next-generation sequencing (mcfNGS) is a novel diagnostic tool increasingly utilized as a rapid, noninvasive approach to aid in the diagnosis of infectious diseases. However, its utility in pediatric patients is not well established. This study aimed to explore the diagnostic utility of commercial microbial cell-free DNA sequencing in a pediatric quaternary care facility. Methods This retrospective study included 15 patients with mcfNGS results between June 2018 and April 2023. Chart abstraction was performed to determine changes in clinical management. Change in management was defined as any clinical decision implemented based on mcfNGS results. Overall clinical impact was defined as either a beneficial or deleterious change in patient outcome resulting from clinical management changes made based on mcfNGS results. Results Of the 15 patients, a total of 14 valid results were reported, 13 (86.7%) of which were from the first attempt. At least one microbial target was detected in 10/14 (71.4%) patients. Standard microbiology testing (SMT) revealed a pathogen or diagnostic marker of such in 8/14 (57.1%) patients. Notably, there were no instances in which mcfNGS detected a true pathogen in the context of negative SMT. Of the 14 patients with valid reports, clinical management was changed based on mcfNGS in 3 (21.4%) patients. This included addition of anti-fungal therapy, de-escalation of antimicrobials, and initiation of immunosuppressive corticosteroid therapy in the context of a negative mcfNGS result. Of the 10 patients in which at least one microorganism was detected, 70% were determined to be either contamination or of unclear clinical significance. There was only one instance in which mcfNGS alone led to a beneficial clinical impact as determined by de-escalation of anti-fungal therapy. Figure 1. mcfNGS was performed on 15 pediatric patients between July 2018 and April 2023. Pie chart represents categorization of mcfNGS results and their clinical impact. Conclusion While infectious disease metagenomic NGS holds great promise, the utility of currently available commercial assays for non-invasive specimens remains poorly understood. Here we report that in a small cohort of pediatric patients, commercial mcfNGS added minimal value over standard microbiological testing. Additional investigation is required to determine the ideal clinical picture warranting mcfNGS. Disclosures Jennifer Dien Bard, PhD, Abbott Molecular: Grant/Research Support|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|BioMerieux: Honoraria|Genetic Signature: Advisor/Consultant|Genetic Signature: Grant/Research Support|Luminex: Grant/Research Support|Salve: Stocks/Bonds|Thermo Fisher: Honoraria

Identification of Potential Genomic Alterations Using Pan-Cancer Cell-Free DNA Next-Generation Sequencing in Patients With Gastric Cancer.

Molecular cancer profiling may lead to appropriate trials for molecularly targeted therapies. Cell-free DNA (cfDNA) is a promising diagnostic and/or prognostic biomarker in gastric cancer (GC). We characterized somatic genomic alterations in cfDNA of patients with GC. Medical records and cfDNA data of 81 patients diagnosed as having GC were reviewed. Forty-nine and 32 patients were tested using the Oncomine Pan-Cancer Cell-Free Assay on the Ion Torrent platform and AlphaLiquid 100 kit on the Illumina platform, respectively. Tier I or II alterations were detected in 64.2% (52/81) of patients. Biomarkers for potential targeted therapy were detected in 55.6% of patients (45/81), and clinical trials are underway. ERBB2 amplification is actionable and was detected in 4.9% of patients (4/81). Among biomarkers showing potential for possible targeted therapy, TP53 mutation (38.3%, 35 variants in 31 patients, 31/81) and FGFR2 amplification (6.2%, 5/81) were detected the most. Next-generation sequencing of cfDNA is a promising technique for the molecular profiling of GC. Evidence suggests that cfDNA analysis can provide accurate and reliable information on somatic genomic alterations in patients with GC, potentially replacing tissue biopsy as a diagnostic and prognostic tool. Through cfDNA analysis for molecular profiling, it may be possible to translate the molecular classification into therapeutic targets and predictive biomarkers, leading to personalized treatment options for patients with GC in the future.

Validation of targeted next-generation sequencing of cell-free DNA from archival cerebrospinal fluid specimens for the detection of somatic variants in cancer involving the leptomeninges: Cytopathologic and radiographic correlation.

Leptomeningeal metastases occur across multiple solid and lymphoid cancers, and patients typically undergo cytopathologic assessment of cerebrospinal fluid (CSF) in this setting. For patients diagnosed with metastatic cancer, the detection of actionable somatic mutations in CSF can provide clinically valuable information for treatment without the need for additional tissue collection. The authors validated a targeted next-generation sequencing assay for the detection of somatic variants in cancer (OncoPanel) on cell-free DNA (cfDNA) isolated from archival CSF specimens in a cohort of 25 patients who had undergone molecular testing of a prior tumor specimen. CSF storage time and volume had no impact on cfDNA concentration or mean target coverage of the assay. Previously identified somatic variants in CSF cfDNA were detected in 88%, 50%, and 27% of specimens diagnosed cytologically as positive, suspicious/atypical, and negative for malignancy, respectively. Somatic variants were identified in 81% of CSF specimens from patients who had leptomeningeal enhancement on magnetic resonance imaging compared with 31% from patients without such enhancement. These data highlight the stability of cfDNA in CSF, which allows for cytopathologic evaluation before triage for next-generation sequencing assays. For a subset of cases in which clinical suspicion is high but cytologic or radiographic studies are inconclusive, the detection of pathogenic somatic variants in CSF cfDNA may aid in the diagnosis of leptomeningeal metastases.

Identification of an optimal mutant allele frequency to detect activating KRAS, NRAS, and BRAF mutations in a commercial cell-free DNA next-generation sequencing assay in colorectal and pancreatic adenocarcinomas.

Evaluation for activating mutations in KRAS, NRAS, and BRAF in colorectal cancer (CRC) and in KRAS in pancreatic ductal adenocarcinoma (PDAC) is essential for clinical care. Plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) allows convenient assessment of a tumor's molecular profile, however low tumor DNA shedding limits sensitivity. We investigated mutant allele frequency (MAF) of other oncogenic dominant genes to identify a threshold for accurate detection of KRAS, NRAS, and BRAF (RAS/RAF) mutations in cfDNA. Molecular and clinical data were obtained from the Duke Molecular Registry of Tumors and the SCRUM-Japan GOZILA study. Patients with CRC or PDAC and a KRAS, NRAS, or BRAF activating single nucleotide variant (SNV) present on tissue NGS and with available cfDNA assays were included. Recursive partitioning and Wilcoxon-rank statistics methods identified potential cut-points for discriminative MAF values. One hundred and thirty-five CRC and 30 PDAC cases with 198 total cfDNA assays met criteria. Greatest non-RAS/RAF dominant gene MAF of 0.34% provided maximum discrimination for predicting RAS/RAF SNV detection. Sensitivity for RAS/RAF SNVs increased with dominant gene MAF, with MAF ≥1% predicting sensitivity >98%, MAF between 0.34 and 1% predicting sensitivity of 84.0%, and MAF £0.34% predicting sensitivity of 50%. For 43 cfDNA assays that did not detect RAS/RAF SNVs, 18 assays detected 34 other oncogenic variants, of which 80.6% were not also detected on tissue. Non-RAS/RAF dominant oncogenic mutation MAF ≥1% on cfDNA NGS predicts high sensitivity to detect RAS/RAF oncogenic SNVs in CRC and PDAC. MAF £0.34% indicates an assay may not reliably detect RAS/RAF SNVs, despite detection on tissue testing. Most variants from assays that did not detect RAS/RAF had MAF <1% and were not detected on tissue, suggesting potential confounding. These data suggest a practical approach to determining cfDNA assay adequacy, with implications for guiding clinical decisions in CRC and PDAC.

Methylated DNA biomarkers and incident cancer in the American Cancer Society (ACS) Cancer Prevention Study-3 (CPS-3) cohort.

3004 Background: Multi-cancer early detection (MCED) tests are being developed to complement current single-cancer screening, and are based on next-generation sequencing of cell-free DNA in blood plasma with subsequent analysis of genomic features by machine-learning algorithms. To evaluate the detectability of cancer signals in individuals without known cancer at the time of blood draw, we assessed pre-diagnostic signals using a previously validated locked, methylation-based assay in plasma samples from the ACS CPS-3 cohort. Methods: CPS-3 is a prospective cohort study of cancer incidence and mortality. Participants (N=294,000) donated a blood sample (36 mL) at enrollment (2006-2013) and are followed for cancer outcomes via routine linkages with population-based cancer and death registries. Selected participants diagnosed with cancer within 3 years (Y) of blood donation and an equivalent number of matched non-cancer participants were chosen for processing. Despite differences in sample collection (tube, protocol) and long storage time in freezer, standard requirements for MCED sample quality were applied. Primary outcome: demonstrate that cancer signal was detectable via the MCED test up to 3Y prior to conventional diagnosis. Secondary outcomes: assess detectability based on time from blood draw and other clinical factors. Sampling weights were considered in the statistical analyses. Results: Of 268,726 CPS-3 participants, 2,850 were selected and assayed by cohort sampling, and 2,754 were included in the primary analysis set. CPS-3 cohort demographics and cancer incidence patterns compared to Surveillance, Epidemiology, and End Results suggested that this cohort is younger and healthier than the U.S. population. The test negative rate was 100% among non-cancers. The test positive rate (TPR) stratified by time between blood draw and clinical diagnosis was 2.3% (3Y), 6.0% (2Y), 15.2% (1Y). For eventually fatal cancers, TPR was 5.1% (3Y), 21.6% (2Y), 55.1% (1Y) vs 1.8%, 4.1%, 10.2% for non-fatal cancers. By stage, within 1Y, TPR was 73.7% for Distant at 4-6 months (M) and 94.1% at 1-3M vs 26.7% for Regional at 4-6M and 71.0% at 1-3M. The overall accuracy of predicting cancer signal origin was 95% (1Y). Many cancers were detected 3Y prior to clinical diagnosis: for example, a case of colon/rectum cancer at Regional stage was detected at 991 days. Conclusions: The ACS CPS-3 cohort includes persons at lower-than-average risk of cancer but offered a biospecimen repository with sufficient plasma samples to assess the preclinical detectability of cancer signals by retrospective evaluation. Rapid changes in detectability over time underscore the short preclinical time scales for aggressive cancers to develop, and therefore to be detected. These insights into cancer biology reinforce the importance of pre-diagnostic samples in the study of cancer signals for early detection.

FRI-536 - Microbial cell-free DNA next-generation sequencing of ascites in acutely decompensated cirrhosis patients: a proof-of-concept study

FRI-536 - Microbial cell-free DNA next-generation sequencing of ascites in acutely decompensated cirrhosis patients: a proof-of-concept study

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

Tackling Osimertinib Resistance in EGFR-Mutant Non-Small Cell Lung Cancer.

The current landscape of targeted therapies directed against oncogenic driver alterations in non-small cell lung cancer (NSCLC) is expanding. Patients with EGFR-mutant NSCLC can derive significant benefit from EGFR tyrosine kinase inhibitor (TKI) therapy, including the third-generation EGFR TKI osimertinib. However, invariably, all patients will experience disease progression with this therapy mainly due to the adaptation of cancer cells through primary or secondary molecular mechanisms of resistance. The comprehension and access to tissue and cell-free DNA next-generation sequencing have fueled the development of innovative therapeutic strategies to prevent and overcome resistance to osimertinib in the clinical setting. Herein, we review the biological and clinical implications of molecular mechanisms of osimertinib resistance and the ongoing development of therapeutic strategies to overcome or prevent resistance.

Baseline circulating tumour DNA and total metabolic tumour volume as early outcome predictors in aggressive large B-cell lymphoma. A real-world 112-patient cohort.

Approximately 20%-50% of patients with large B-cell lymphoma (LBCL) experience poor outcomes. We aimed to evaluate the combined prognostic value of circulating tumour DNA (ctDNA) and total metabolic tumour volume (TMTV) in LBCL. This observational single-centre study included 112 newly diagnosed LBCL patients, receiving R-CHOP/R-CHOP-like chemotherapies. CtDNA load was calculated following next-generation sequencing of cell-free DNA (cfDNA) using a targeted 40-gene lymphopanel. TMTV was measured using a fully automated artificial intelligence-based method for lymphoma lesion segmentation. CtDNA was detected in cfDNA samples from 95 patients with a median concentration of 3.15 log haploid genome equivalents per mL. TMTV measurements were available for 102 patients. The median TMTV was 501 mL. High ctDNA load (>3.57 log hGE/mL) or high TMTV (>200 mL) were associated with shorter 1-year PFS (44% vs. 83%, p < 0.001 and 64% vs. 97%, p = 0.002, respectively). When combined, three prognostic groups were identified. The shortest PFS was observed when both TMTV and ctDNA load were high (p < 0.001). Even with a short follow up, combining ctDNA load with TMTV improved the risk stratification of patients with aggressive LBCL. In the near future, very high-risk patients could benefit from CAR T-cell therapy or bispecific antibodies as first-line treatments.

Safety and efficacy of the erdafitinib (erda) intravesical delivery system, TAR-210, in patients (pts) with non–muscle-invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC) harboring select FGFR mutations or fusions: Phase 1 first-in-human study.

TPS583 Background: Treatment options are limited for pts with NMIBC and MIBC who experience disease recurrence or who are ineligible for or refuse standard of care. Erda, an oral selective pan-FGFR tyrosine kinase inhibitor, is approved in adults with locally advanced or metastatic urothelial cancer with select FGFR3/2 alterations ( alt) who have progressed during or after ≥1 line of platinum-containing chemotherapy. FGFRalt are among the most common oncogenic drivers detected in NMIBC and MIBC, and are more prevalent in NMIBC. TAR-210 is an intravesical drug delivery system designed to provide local, continuous release of erda within the bladder, thus limiting systemic toxicity. This study evaluates the safety, pharmacokinetics (PK), and efficacy of TAR-210 in pts with NMIBC or MIBC with select FGFRalt. Methods: Open-label, multicenter phase 1 study of TAR-210 in pts with recurrent NMIBC or MIBC (NCT05316155). Eligible pts are aged ≥18 yrs with adequate organ function and tumors with select FGFRalt. A flexible molecular eligibility strategy is used to allow for local or central fresh/archival tissue-based FGFR testing by next-generation sequencing (NGS) or PCR, or urine cell-free DNA NGS testing. Four cohorts will be enrolled: pts with recurrent, bacillus Calmette-Guerin (BCG)-experienced papillary-only high-risk (HR) NMIBC (high-grade Ta/T1) refusing or ineligible for radical cystectomy (RC) (Cohort 1) or scheduled for RC (Cohort 2); pts with recurrent, intermediate-risk NMIBC (Ta/T1) with a history of low-grade disease (Cohort 3); pts with cT2-T3a MIBC scheduled for RC refusing or ineligible for neoadjuvant cisplatin (Cohort 4). Pts in Cohorts 1 and 2 will have TURBT with resection of all visible disease prior to dosing, whereas pts in Cohort 3 must have visible disease prior to dosing. The primary end point is safety (adverse events, including dose-limiting toxicity). Secondary end points include PK, recurrence-free survival (Cohorts 1 and 2), complete response (CR) rate and duration of CR (Cohort 3), and pathologic CR rate, pT0 rate, and rate of downstaging to &lt;pT2 (Cohort 4). Dose escalation (Part 1; n≈12, Cohorts 1 and 3 only) will be followed by dose expansion (Part 2; n≈50-80). Cohorts 1 and 3: response assessment will be after a 3-mo dosing cycle; pts with CR may receive ≤3 additional 3-mo dosing cycles if no recurrence, progression, or unacceptable toxicity. Cohorts 2 and 4: response assessment will be at RC after 8 wks of dosing. Follow-up disease surveillance (cystoscopy, urine cytology, imaging) will be every 3 mos to end of Yr 2 and every 6 mos in Yr 3 in Cohorts 1 and 3 and at 3 mos post-RC in Cohorts 2 and 4. Four pts were enrolled since April 2022 (1 in Cohort 1, 3 in Cohort 3); enrollment for Cohorts 2 and 4 is planned soon. Clinical trial information: NCT05316155 .

Karius With a Q: Role for Microbial Cell-Free DNA Next-Generation Sequencing in Diagnosis of Acute Q Fever.

The diagnosis of Q fever can be challenging and a high index of suspicion is necessary. Within this case series, we highlight the utility of the microbial cell-free DNA next-generation sequencing or Karius Test in the timely diagnosis and management of acute Q fever.