Next-generation Sequencing Testing Research Articles

Seven-year performance of a clinical metagenomic next-generation sequencing test for diagnosis of central nervous system infections.

Metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) is an agnostic method for broad-based diagnosis of central nervous system (CNS) infections. Here we analyzed the 7-year performance of clinical CSF mNGS testing of 4,828 samples from June 2016 to April 2023 performed by the University of California, San Francisco (UCSF) clinical microbiology laboratory. Overall, mNGS testing detected 797 organisms from 697 (14.4%) of 4,828 samples, consisting of 363 (45.5%) DNA viruses, 211 (26.4%) RNA viruses, 132 (16.6%) bacteria, 68 (8.5%) fungi and 23 (2.9%) parasites. We also extracted clinical and laboratory metadata from a subset of the samples (n = 1,164) from 1,053 UCSF patients. Among the 220 infectious diagnoses in this subset, 48 (21.8%) were identified by mNGS alone. The sensitivity, specificity and accuracy of mNGS testing for CNS infections were 63.1%, 99.6% and 92.9%, respectively. mNGS testing exhibited higher sensitivity (63.1%) than indirect serologic testing (28.8%) and direct detection testing from both CSF (45.9%) and non-CSF (15.0%) samples (P < 0.001 for all three comparisons). When only considering diagnoses made by CSF direct detection testing, the sensitivity of mNGS testing increased to 86%. These results justify the routine use of diagnostic mNGS testing for hospitalized patients with suspected CNS infection.

BIOM-55. SENSITIVITY OF IMMUNOHISTOCHEMISTRY WITH BRAF VE1 ANTIBODY FOR BRAF V600E IN PRIMARY CENTRAL NERVOUS SYSTEM TUMORS

Abstract BACKGROUND BRAF gene encodes for a key protein of the RAS-RAF-MEK-ERK pathway. BRAF alterations are found in a variety of primary central nervous system (CNS) tumors ranging from 3% of glioblastomas to 60% of pleomorphic xanthoastrocytomas. BRAF V600E mutation is the most common BRAF alteration found in CNS malignancies, and it has been successfully targeted with BRAF/MEK inhibitors. Novel BRAF inhibitors, still in development, have shown promising antitumor activity. Next-generation sequencing (NGS) has become a standard test in the era of histo-molecular diagnosis, but it can be unavailable or inaccessible, and results usually take longer. Immunohistochemistry (ICH) with VE1 antibody for BRAF V600E protein has shown high discordant results with sequencing in other tumor types. We aimed to establish the concordance of BRAF VE1 and NGS sequencing in CNS tumors. METHODS Single-center retrospective analysis of patients with BRAF V600E mutant CNS tumors by NGS testing. RESULTS 30 patients with BRAF V600E mutant primary CNS tumors by NGS were included, 18 were male. 15 were classified as IDH-wildtype gliomas, 4 low grade neuroepithelial gliomas, 8 pleomorphic xanthoastrocytoma, 2 pilocytic astrocytomas and 1 papillary craniopharyngioma. IHC testing with BRAF VE1 antibody was done at the initial pathological classification in 23 of the cases being positive in 15 and negative in 8 cases. In 7 cases IHC was not done but has been requested for retrospective analysis and results are pending. Sensitivity for BRAF VE1 IHC was 65%. CONCLUSION When compared to NGS testing, ICH with BRAF VE1 antibody for BRAF V600E in CNS tumors shows modest sensitivity, being a limited tool when results are needed promptly or NGS is inaccessible. Determining specificity of the test will be crucial to establish its reliability for patient selection. Sequencing should be obtained when possible and remains the more reliable method for detecting V600E.

RADT-23. MOLECULAR PROFILING OF GLIOMAS: INSIGHTS INTO RADIONECROSIS AND PSEUDOPROGRESSION THROUGH NEXT-GENERATION SEQUENCING

Abstract BACKGROUND Gliomas, the most common primary tumors of the CNS, presenting diagnostic and therapeutic challenges due to their heterogeneity. Next-generation sequencing (NGS) enhances diagnostic precision by identifying genetic alterations, guiding accurate diagnosis and personalized treatment strategies. Post-treatment complications such as radionecrosis and pseudoprogression present additional diagnostic difficulties, often mimicking tumor recurrence. This study aimed to evaluate the association between genetic alterations detected by NGS and treatment-related complications. METHODS This retrospective study analyzed data from electronic medical records at a Brazilian Radiation Therapy Department (2018-2023). The study included patients with gliomas who underwent surgical resection followed by adjuvant radiochemotherapy and subsequently developed complications such as radionecrosis and pseudoprogression. Genetic alterations detected by the HSL 500 (NGS) test were evaluated for their association with these complications using Fisher’s Exact Test. RESULTS Out of 192 patients treated for glioma, 46 (23.96%) experienced treatment-related complications, with 20 patients having available NGS test results. Among these, eight cases were identified as pseudoprogression, fifteen as radionecrosis, with three patients presenting both conditions. All 20 patients with NGS results were diagnosed with high-grade gliomas, eight of whom underwent reirradiation, resulting in two cases of radionecrosis post-reirradiation. Although no statistically significant association was found between genetic alterations and radionecrosis or pseudoprogression, specific mutations were prevalent in each group. In radionecrosis cases, the most common mutations were TERT promoter (c.1-124C&amp;gt;T) (n=9, p=0.613) and EGFR amplification (n=8, p=1.0). In pseudoprogression, frequent mutations included TERT promoter (c.1-124C&amp;gt;T) (n=6, p=0.642), EGFR (n=5, p=0.67), PTEN (n=4, p=1.0), NF1 (n=2, p=0.537), and P53 (n=2, p=0.197). CONCLUSION Molecular testing for glioma characterization is crucial for diagnosis and prognosis, aiding in understanding treatment-related complications. Strategies to avoid radionecrosis and pseudoprogression are vital to improve patient quality of life, especially in high-grade gliomas. Further studies are needed to confirm these trends and explore clinical relevance of mutations.

Molecular and clinical profiles of pediatric monogenic diabetes subtypes: comprehensive genetic analysis of 138 patients.

Single gene variants that give rise to neonatal diabetes mellitus (NDM), maturity onset diabetes of the young (MODY) and syndromic forms of diabetes mellitus (SDM) are responsible for 3.1-4.2% of all diabetes cases. This single-center study with a relatively larger sample size aimed to evaluate the clinical and genetic characteristics of Chinese children with suspected monogenic diabetes (MD) using next generation sequencing (NGS) methods. Data were collected from 1550 consecutive children diagnosed with diabetes/hyperglycemia at the Endocrinology Department of Children's Hospital of Nanjing Medical University from 2012 to 2023. The genotype and phenotype of 138 children with suspected MD were retrospectively analyzed. Among 138 children, 16, 97, and 25 patients with NDM, suspected MODY and SDM were assessed by NGS, with a pick-up rate of 87.5%, 57.8%, and 56%, respectively. In total, there was a high pick-up rate of MD, with 58% (80 of 138) among antibody-negative pediatric patients. Pathogenic variants were found in GCK, HNF1A, INS, KCNJ11, INSR, HNF4A, ABCC8, WFS1, ALMS1, HNF1B, BLK and ZFP57 genes with 13 novel variants in addition to 4 patients with CNVs. In this cohort, GCK-MODY was the leading cause and the mildest type of MODY. GCK-MODY displayed favorable lipid profile when compared to non-GCK-MODY and MODYX, which might be cardioprotective. Following an accurate genetic diagnosis of diabetes, 19 patients switched from insulin therapy to oral agents or lifestyle interventions. NGS tests helped to identify the precise etiology of monogenic diabetic patients which has implications for better individualized management.

Boundary-work of genomic medicine: safeguarding the future of diagnostic next-generation sequencing in the clinic

Next-generation sequencing (NGS) technologies – which allows to look at large parts or even the whole genome at once - are making their way into diagnostic clinical care. With trends towards ‘mainstreaming’ genetic services into general medicine, significant ethical challenges, and a disputed clinical utility and cost-benefit ratio, genomic medicine’s autonomy and dominance in defining and offering NGS care may come under increased pressure from the outside (e.g., regulators, other healthcare providers and facilities, ethicists, and patients). In this paper, we show how the field of genomic medicine engages in substantial boundary work in reaction to these circumstances. Building on multi-sited fieldwork in two centres for human genetics in Belgium and the Netherlands, we show how acts of demarcation serve to uphold an image of expertise and authority which helps to maintain the field’s autonomy and dominance. Through examining the delineations put forward in interviews, practice (based on observations in multidisciplinary meetings and consultations), and grey and academic literature, we show the politics involved in moving NGS forward fairly seamlessly in a way that suits the field. First, we show how genetic healthcare professionals have redefined what makes a genetic test ‘valuable’ so that it underlines its current value. Secondly, we examine how a genetic imaginary is put forward that both emphasizes the extraordinary character of genomic medicine and the normalcy of NGS testing. By underlining the need for their expertise whilst simultaneously normalizing the ethical challenges and positioning themselves as most capable of reflecting on these, the field minimized external regulation and kept a close grip on defining ethical issues and policy. Despite their current dominance in shaping the future of genomic care, we argue that the closedness of the field hinders it in benefiting from external expertise, reflection and monitoring to ensure enduring and broad support for this future.

Novel postzygotic RASA1 mutation in a patient with Parkes Weber syndrome: A case report and literature review.

Not only germline but also postzygotic mutations in the RASA1 or EPHB4 genes can lead to capillary malformation-arteriovenous malformation (CM-AVM) syndrome. As it is not always possible to clinically distinguish between constitutional variants and postzygotic mosaicism, a sufficiently high sequencing depth must be used in genetic diagnostics to detect both. Capillary malformation-arteriovenous malformation (CM-AVM) syndrome, with or without Parkes Weber syndrome, is a rare autosomal dominant disease caused by pathogenic RASA1 or EPHB4 variants. Up to 80% of CM-AVM cases have an affected parent. Gene panel sequencing was performed for a 4-year-old girl with multiple CMs, two capillary stains on the left leg, and associated overgrowth of the second toe. We also reviewed published cases with mosaic RASA1 and EPHB4 mutations. A mosaic RASA1 loss-of-function mutation was detected with a variant allele frequency (VAF) of 20% in the blood and oral epithelial cells of the index patient. The literature review illustrates that the severity of the clinical phenotype does not correlate with the VAF. We also identified a germline nonsense variant in the patient's TEK gene. However, inactivating TEK variants do not cause a vascular phenotype but can confer an increased risk for primary congenital glaucoma with variable expressivity. The case presented here illustrates that the choice of the sequencing depth of a diagnostic next-generation sequencing test for CM-AVM patients should always take mosaicism into account and that a good knowledge of the sequenced genes and associated disease mechanisms is necessary for adequate genetic counseling.

The Impact of Next-Generation Sequencing Workflows on Outcomes in Advanced Lung Cancer: A Retrospective Analysis at One Academic Health System.

Broad-based molecular testing with next-generation sequencing (NGS) is now the standard of care in advanced non-small cell lung cancer (NSCLC). Two approaches to molecular testing are (1) reflexive testing at pathologic NSCLC confirmation, often using an in-house molecular panel, and (2) send-out testing to private vendors, ordered by a clinician. This study explored the outcomes with reflex versus send-out testing. A retrospective chart review was conducted of patients diagnosed with de novo stage IV NSCLC in 2019 and 2020 at three hospitals in the same system, one academic hospital (Northwestern Memorial Hospital, or NMH) utilizing reflex, in-house NGS, and two community-based hospitals (Central DuPage Hospital, or CDH, and Delnor, or D) sending out tissue samples for testing. The outcomes assessed were the time from biopsy to results, biopsy to treatment, the incidence of first-line targetable mutations and the use of first-line targeted therapies, and overall survival. In total, 191 patients met the inclusion criteria, 85 at NMH, 106 at CDH + D, and in total, 131 in 2019 and 60 in 2020. The time to results was significantly shorter with reflexive NGS when compared with send-out testing; the time to treatment was also shorter but not statistically significant. At CDH + D, the time to results was significantly shorter with a limited panel than with comprehensive testing, but the time to treatment was similar. NGS testing rates were 95% at NMH and 84.5% at CDH + D (p = 0.009), with 31.0% at NMH receiving 1L targeted therapies versus 20.8% at CDH + D (p = 0.08). In 2019, the median time from biopsy to treatment was 35 days at NMH and 38 days at CDH and Delnor; in 2020, time to treatment was 26 days and 37 days, respectively. Overall survival trended longer in 2020 relative to 2019 independent of site. Reflexive NGS testing is associated with a shorter time to actionable results and higher rates of first-line targetable mutations than send-out testing. In practices with send-out testing, limited panels had slightly faster turnaround times but no difference in time to treatment. If resources allow, reflexive NGS should be considered in healthcare systems for patients with NSCLC.

The discrepancy of somatic BRCA1/2 pathogenic variants from two different platforms in epithelial ovarian, fallopian tube, and peritoneal cancer

The somatic BRCA1 or BRCA2 Pathogenic Variant (PV)/Likely PV (LPV) from Next Generation Sequencing (NGS) is the most important biomarker for PARP inhibitor use and maintenance-targeted therapies. A discrepancy in the detection rates of BRCA1 and BRCA2 PV/LPV was identified among the NGS platforms. The objective of this study was to compare the somatic BRCA results from two distinct platforms using the same cohort and to identify the causes of these differences. Patients with epithelial ovarian cancer who concurrently underwent tumor NGS using two different platforms between January 2022 and June 2023 were included in this study. The two platforms used were in-house tumor NGS (Illumina NextSeq 550Dx, SureSelectXT library kit, and datasets from 1000 Genomes, ESP6500, ExAC, and ClinVar) and GreenPlan homologous recombination deficiency (HRD) test (Illumina NextSeq 550Dx, customized Twist Bioscience library kit, and datasets from COSMIC and OncoKB). The results of somatic mutations in BRCA1 and BRCA2 were compared between the two platforms. Of the 118 patients, 11.9% (n = 14) exhibited a discordant interpretation of BRCA1 or BRCA2 between the two platforms. Eleven patients (9.3%) exhibited negative results in the in-house platform but positive results (eight seven as PV of BRCA1, one as PV of BRCA2, one as LPV of BRCA1, and two as LPV of BRCA2) in the GreenPlan HRD test, while three patients (2.6%) had positive BRCA pathogenic variants (two as PV of BRCA1 [c.3340G > T, c.5152 + 3 A > C], one as LPV of BRCA2 [c.8174G > T], and one as LPV of BRCA1 [c.5017_5019delCAC]) in the in-house platform but a negative result in the GreenPlan HRD test. The discordance rate of somatic BRCA1 or BRCA2 mutations from different platforms was approximately 12%. In the case of the strong implication of BRCA PV/LPV with a negative result with one genetic test, different platforms could be considered in limited cases. Careful interpretation and further studies for the cross-validation of gene analysis platforms are needed.

Optimization criteria for ordering myeloid neoplasm next‐generation sequencing

AbstractIntroductionMyeloid neoplasms (MNs) frequently harbor pathogenic mutations not detected by karyotyping and fluorescence in situ hybridization; hence, next‐generation sequencing (NGS) is necessary for diagnosis, risk stratification, and therapy. If, however, NGS is not clinically indicated but still performed, the results may promote futile avenues of investigation, heighten patient distress, and increase cost.MethodsWe created criteria to approve NGS testing for MN (MN‐NGS) with the goal of maximizing actionable results. These actionable results include making a new MN diagnosis, characterizing a MN with baseline mutational status, and altering treatment plans. Approval criteria included clinical suspicion of new, relapsed, or worsening disease and end‐of‐induction chemotherapy. Cancellation criteria included the suspicion of non‐myeloid disease only, no suspicion of progression of a known MN, no evidence for recurrence post‐transplant, a diagnosis of chronic myeloid leukemia, and cases using blood when a concurrent bone marrow NGS is being performed. We applied these criteria to NGS tests ordered at our institution between August and December 2018 and determined whether any tests meeting our cancelation criteria yielded actionable results.ResultsConsecutive MN‐NGS orders (n = 174) were retrospectively categorized as appropriate (Group A, n = 115), inappropriate (Group B, n = 29), and appropriately canceled (group C, n = 30). Seventy‐five of the 115 (65%) Group A tests and none of the 29 (0%) Group B tests yielded actionable results (p &lt; 0.0001).ConclusionApproximately one third (59/174) of MN‐NGS test orders can be safely canceled using these criteria, which would result in $150,370 of Centers for Medicare and Medicaid Services‐reimbursed savings annually.

Precision Oncology in Older Cancer Patients: A Single-Center Experience

In the last two decades, next-generation sequencing (NGS) has facilitated enormous progress in cancer medicine, in both diagnosis and treatment. However, the usefulness of NGS in older cancer patients is unclear. To determine the role of NGS in older cancer patients, we retrospectively assessed demographic, clinicopathologic, and disease-specific data from 100 randomly selected cancer patients (any subtype/stage) who underwent NGS testing in 2020 at our institution and compared the treatment outcomes (progression-free survival [PFS] and overall survival [OS]) in the younger and older patient cohorts (A [n = 34] and B [n = 66]: age &lt;70 and ≥70 years, respectively). Overall, 27% had targetable mutations, and 8% received NGS-determined targeted therapy (45% and 19% of patients with a targetable mutation in cohorts A and B, respectively; p = 0.2), of whom 38% (3% of the whole cohort) benefited from the therapy (PFS &gt; 6 months). The median OS (from diagnosis) was 192 and 197 weeks in cohorts A and B, respectively (p = 0.08). This pilot study revealed no significant age-stratified difference in the diagnostic approach and treatment strategy. A small, but relevant, proportion of the cohort (3%) benefited from NGS-determined treatment. Nevertheless, older cancer patients with targetable mutations less frequently received targetable therapies.

Reflex Testing of Endometrial Carcinoma For Next Generation Sequencing: An Institutional Experience

Abstract Introduction/Objective Endometrioid carcinoma is now separated into four subgroups based on molecular profiling. These subgroups (POLE mutant [POLEmut], mismatch repair-deficient [MMRd], p53 abnormal [p53abnl], and no specific molecular profile [NSMP]) have been shown to be diagnostically and prognostically relevant. This is especially highlighted by the inclusion of molecular classification into the 2023 FIGO staging for EMCA. In conjunction with our gynecologic oncologists (gynonc) and colleagues in molecular pathology, we developed and implemented a reflex process by which next generation sequencing (NGS) is done on all hysterectomies performed at our institution for EMCA. Methods/Case Report In collaboration between gynonc, molecular diagnostics, surgical pathology and operations personnel, an order set was built within our LIS. At the time of sign-out, the gynecologic pathologist (GYNP) selects the block for NGS testing and orders the cuts for NGS, digital scanning of the corresponding H&amp;E and the NGS testing. Histology in turn processes the cuts, scans the H&amp;E and transfers the material for NGS. The digital slides are stored in an accessible repository and annotated by the GYNP. Results of NGS testing are released into the patient’s EMR and molecular/histologic correlation is subsequently performed by the GYNPs. Results (if a Case Study enter NA) In the period between 1/1/23 and 2/28/24, we processed 131 EMCA cases through this pathway. Of 103 endometrioid-type EMCA reflexed during this period, 7 were classified as POLEmut, 23 as MMRd, 13 as p53abnl, and 60 as NSMP. 16 cases required re-review of IHC and/or NGS results due to discrepancies. 4 p53abnl cases required upstaging according to the new FIGO, while no POLEmut cases required downstaging as they were all stage I. Conclusion Overall, our pilot pathway was considered a success and continues. We believe our process can serve as a framework for other institutions and can be applied to other malignancies.

Resolving Ambiguities in Deceased Donor HLA Typing: Next Generation Sequencing’s Role in DPB1 Allele Discrimination and Immunological Implications

Abstract HLA typing is crucial for assessing immunological compatibility between donors and recipients before transplantation. Deceased donor (DD) typing, typically via real-time PCR (rtPCR), provides rapid results vital for organ allocation. rtPCR primarily targets exons encoding the highly polymorphic peptide binding region (PBR) of HLA molecules. rtPCR cannot target all polymorphic sequences. Consequently, several possibilities of allele combinations are provided, leading to ambiguous typing results. As recent evidence suggests antibody responses can target regions in exons outside the PBR, notably in Exon 3 of the DPB1 locus, typing ambiguities can lead to the inability to assess histocompatibility between a donor and recipient. Herein, the aim was to investigate the utility of Next Generation Sequencing (NGS) in resolving DPB1 ambiguities in DD typing. A retrospective analysis of DD typing from February 2022 to January 2024 examined instances where NGS was used to resolve ambiguities reported via rtPCR. Each case was reviewed to understand the reason for resorting to NGS typing. The study focused on DPB1 ambiguities and assessed whether NGS effectively reduced ambiguity and differentiated alleles with polymorphisms outside the PBR. In total, 29 DD cases were found to have NGS testing results. In 14 cases, NGS was specifically performed to resolve DPB1 ambiguities. The ambiguities were significantly reduced from hundreds detected in rtPCR to 4 or fewer possible allele combinations with NGS. Eleven cases demonstrated polymorphism in exons outside the PBRs. Of these, only 2 cases showed no typing ambiguity with NGS. In the remaining 9 cases, though polymorphisms could be resolved in the PBR, ambiguities remained in exon 3, specifically at codon 96. In these cases, NGS could not distinguish lysine (K) from arginine (R). For example, DPB1*835:01 with the 96R motif could not be distinguished from DPB1*34:01with the 96K motif. The current investigation found that NGS cannot resolve all ambiguities at position 96, likely due, at least in part, to persistent cis-trans ambiguities. Our results suggest that although NGS drastically limited the number of ambiguities in 9 of 11 cases, it still could not adequately distinguish all immunologically relevant alleles. Inability to resolve the 96R/K epitope, which has been shown to be immunogenic, could impact solid organ transplantation, potentially leading to rejection in mismatched recipient-donor pairs at this position. Sequencing methods that generate longer reads, such as nanopore, may help resolve some of these cases if they are indeed due to cis-trans ambiguities.

Targetable molecular algorithm and training platform development for the treatment of non-small cell lung cancer.

Over the last decade, treatment of patients with advanced non-small cell lung cancer (NSCLC) has become dependent on tissue and/or blood biomarkers to guide treatment decisions. Timely access to comprehensive biomarker and tumor signature information is crucial for diagnostic testing. With the rapid development and implementation of complex biomarker testing, comprehensive molecular profiling with in-depth analysis of DNA, RNA, and proteins can be easily performed. Initial data from the MedStar Health system showed considerable disparities in the use of next generation sequencing (NGS) between hospitals, and there is a clear need to improve education and understanding regarding which cases are appropriate for NGS, as well as the use of protocols to make those decisions in an expeditious manner. Clinical pathways are systems-based tools that aim to create greater transparency around care decision making, therapeutic selection, and care delivery. They enhance quality and efficiency by reducing non-value-added intra-provider variability in care. We aimed to create a comprehensive clinical pathway system, the Targetable Molecular Algorithm (TMA), to increase the understanding and use of NGS in NSCLC by physicians in training, oncology nurse navigators, nurse practitioners, and general oncologists. We provide an overview of the implementation of the platform along with navigation guide. A realistic case study-a typical clinical workflow for a patient requiring NGS testing with an EGFR mutation-is also reviewed, demonstrating how the TMA platform can be applied. Additionally, we highlight the importance of the resource, and discuss its strengths, weaknesses, and potential future applications. This new and innovative pathway system will make decision-making easier for clinicians trying to understand the appropriate tests and treatment algorithms for their patients. Our aim is to increase the appropriate and timely use of NGS among health-system providers with the hope that this system will empower physicians to provide better care by providing a quick, simple, user-friendly tool for comprehensive patient care.

Association between genetic information provision and decisional conflict in patients with cancer.

227 Background: The decisional conflict scale (DCS) measures personal perception of uncertainty when facing a decision. In patients with cancer, decision conflict is likely when considering uncertainty in oncologic systemic treatment outcomes. Cancer genomic information can introduce more uncertainty and complexity in care. This study aims to evaluate patients’ DCS score pre or post discussing next generation sequencing (NGS) results with a medical oncologist, and assess factors that correlate with high decision conflict. Methods: Patients diagnosed with incurable/metastatic cancer who underwent molecular characterization with tissue+/- liquid NGS testing were enrolled. Survey instruments included the validated DCS (five subscales: informed, values clarity, support, uncertainty, effective decision), and EQ-5D-5L. DCS was administered pre or post medical oncology consultation that included review of tissue +/- liquid NGS results and treatment options. Higher DCS score correlates with higher perception of uncertainty. Descriptive statistics were used to assess clinical and demographic risk factors. Multivariable logistic regression analysis estimated the correlation between high DCS and clinical factors. Results: 335 DCS surveys were completed by 227 patients: 188 before and 147 after discussing tissue+/- liquid NGS results with a medical oncologist. Baseline characteristics: 56% female, median age 65, ECOG 0-1 58%, median EQ5D VAS score 68, GI/lung/gyne/breast/other 42/32/10/5/11%, white/Asian/other/unknown 66/12/5/17%. Tier 1 variants were identified in 35% of patients. Patients reported decreased decision conflict after consultation. Multivariable logistic regression analysis including sex, age, race and tumor group did not predict for high DCS pre-consultation. MVA post NGS results that also included Tier 1 variants (present / absent) did not predict for high DSC post consultation. Conclusions: Communication regarding genomic-based tumor assessment can positively support patients in decision conflict, and improve their confidence in their treatment choice, regardless of the findings of the report. Low DCS after consultation supports the goal of shared decision making for cancer treatment. Clinical trial information: NCT05057234 . DCS subscale Pre-consultation Post-consultation p value Informed 49.3 37.5 &lt;0.001 Values clarity 45.3 35.5 &lt;0.001 Support 34.4 28.2 0.005 Uncertainty 46.8 36.1 &lt;0.001 Effective decision 40.0 31.4 &lt;0.001 TOTAL 42.9 33.6 &lt;0.001

Comparing the diagnostic value of targeted with metagenomic next-generation sequencing in immunocompromised patients with lower respiratory tract infection

BackgroundAccurate identification of the etiology of lower respiratory tract infections (LRTI) is crucial, particularly for immunocompromised patients with more complex etiologies. The advent of next-generation sequencing (NGS) has enhanced the effectiveness of pathogen detection. However, assessments of the clinical diagnostic value of targeted NGS (tNGS) in immunocompromised patients with LRTI are limited.MethodsTo evaluate the diagnostic value of tNGS in immunocompromised patients with LRTI, a total of 88 patients, of whom 54 were immunocompromised, were enrolled. These patients underwent tNGS testing of bronchoalveolar lavage fluid (BALF). Results from both metagenomic next-generation sequencing (mNGS) and conventional microbiological tests (CMT) were also available for all participants. The performance of tNGS was assessed by comparing its findings against mNGS, CMT, and the clinical composite diagnosis.ResultsIn the cohort of 88 patients, tNGS showed comparable diagnostic value to mNGS and was significantly superior to CMT. Compared to CMT and composite reference standard, tNGS showed sensitivity of 94.55% and 90.48%, respectively. In immunocompromised patients, despite a more diverse pathogen variety, tNGS maintained similar sensitivity to mNGS and outperformed CMT. tNGS positively influenced etiologic diagnosis and antibiotic decision-making in 72.72% of cases, leading to a change in antibiotic regimen in 17.05% of cases. We also compared the detection of microbial nucleic acids by tNGS with mNGS and found that tNGS could identify 87.99% of the microbial nucleic acids identified by mNGS.ConclusionIn summary, our study demonstrated that tNGS offers promising clinical diagnostic accuracy in immunocompromised patients, as evidenced by its favorable comparison with CMT, the composite reference standard, and mNGS.

Cost-effectiveness of next-generation sequencing for advanced EGFR/ALK-negative non-small cell lung cancer

ObjectivesThis study aimed to evaluate the cost-effectiveness of next-generation sequencing (NGS) versus sequential single-gene testing (SGT), including the long-term costs and survival outcomes of relevant treatments for advanced EGFR/ALK-negative non-small cell lung cancer (NSCLC). Materials and MethodsWe developed a decision tree linked to a partitioned survival model to estimate the clinical outcomes and costs over the five-year analysis period. The decision tree consisted of treatment types based on molecular biomarker (ROS1, BRAF, NTRK, MET, RET, and KRAS alterations) test results. The probability of receiving each targeted therapy was estimated based on 1) the testing rate, 2) the proportion of alterations detected, and 3) the proportion of patients receiving treatment consistent with the testing results. We estimated the long-term overall survival and progression-free survival for each treatment using parametric estimation by reconstructing patient-level data from clinical trials. The costs of testing, drugs, administration, physician visits, monitoring, adverse events, post-progression, and end-of-life care were included. The utility values were obtained from a previous study. The incremental cost-effectiveness ratio (ICER) was used to evaluate the cost-effectiveness of NGS within a threshold of $38,701 (50,000,000 KRW) per quality-adjusted life year (QALY). ResultsThe incremental life-years (LYs) and QALYs for the NGS group versus the SGT group were 0.028 and 0.023, respectively. The total medical cost for the NGS group was $8,375 higher than that for the SGT group. The difference in drug costs accounted for most of the differences in total medical costs. NGS was not cost-effective compared to sequential SGT, with an ICER of $300,233/LY and $359,405/QALY, respectively. ConclusionsNGS is not cost-effective for advanced EGFR/ALK-negative NSCLC, but has a survival benefit over sequential SGT. Our findings provide a basis for decision-making regarding the coverage and clinical utilization of NGS in regions where EGFR alterations are prevalent.

Economic effects of next-generation sequencing diagnostics in unspecific sepsis patients - a budget impact analysis from the healthcare providers' perspective in Germany.

Next-generation sequencing (NGS) tools have clinical advantages over blood culture but are more expensive. This study assesses the budget impact and break-even point of NGS testing costs from a healthcare provider's perspective in Germany. The budget impact was calculated based on aggregated data of German post-operative surgery cases. Simulated cost savings were calculated based on a simulated reduction in hospital length of stay (LOS) of four or eight days with a positivity rate of 71% and compared to the costs of one (scenario A) or two tests (scenario B) per case. Furthermore, the break-even point of the cost of two tests compared to saved costs through shortened LOS was conducted. For 9,450 cases, an average budget impact for scenario A and scenario B of €1,290.41 [95% CI €1,119.64 - €1,461.19] and - €208.59 [95% CI - €379.36 - - €37.81] was identified for gastrointestinal and kidney surgery cases, and €1,355.58 [95% CI €1,049.62 - €1,661.55] and €18.72 [95% CI - €324.69 - €287.24] for vascular artery surgery cases, respectively. The break-even analysis showed that using two tests per case could achieve a minimum positive contribution margin with an average of 1.9 tests per case across the study population. The results revealed a positive budget impact for one NGS test and a slightly negative budget impact for two NGS tests per case. Findings suggest that largest cost savings are generated for more severe cases and are highly dependent on the patient population.

Pathogen detection by targeted next-generation sequencing test in adult hematological malignancies patients with suspected infections.

Infections in patients with hematological malignancies (HM) are a significant cause of morbidity and mortality. Timely and effective empirical anti-infective treatment can reduce the infection-related mortality rate. Targeted next-generation sequencing (tNGS) offers a rapid diagnostic approach for identifying diverse pathogens in these patients. However, relevant research is still limited to adult patients with HM. We conducted a retrospective analysis of adult HM patients admitted to our hospital from March 2023 to September 2023, focusing on their clinical characteristics and the results of both tNGS and conventional microbiological tests (CMTs). We evaluated the performance of tNGS and CMTs in pathogenic diagnosis and described the distribution characteristics of pathogens in adult HM patients with infections. The study included 209 samples collected from 137 patients. Results showed that the overall pathogen detection rate differed significantly between tNGS and CMTs (60.3% vs. 24.4%, p < 0.001). The sensitivity (69.7% vs. 35.9%), negative predictive value (NPV) (48.2% vs. 42.4%), and accuracy (66.5% vs. 56.5%) of pathogen detection were notably superior with tNGS compared to CMTs. Among the 142 samples with clinically diagnosed infections, tNGS combined with CMTs identified a definite or probable microbial etiology in 114 samples (80.3%). Of the 36 samples with concordant positive results from both tNGS and CMTs, 72.2% (26/36) exhibited full or partial agreement. Our study further showed the highest detection rate for viral infections (57.0%), predominantly for Epstein-Barr virus (DNA-V, 18.3%). Followed by bacterial infections (46.5%), the detection rate of Gram-negative bacteria (G+, 35.9%) was higher than that of Gram-positive bacteria (G-, 21.8%) in this study. Klebsiella pneumoniae (G-, 12.7%) had the highest detection rate among these emerging bacteria, followed by Pseudomonas aeruginosa (G-, 10.6%) and Enterococcus faecium (G+, 7.7%). Bacterial-viral coinfections were the most common type of mixed infection (35.5%). In conclusion, tNGS outperforms CMTs in both sensitivity and pathogen spectrum. Therefore, it can serve as an adjunct to CMTs to facilitate the precise adjustment of anti-infective regimens for adult HM patients. Our findings establish a basis for formulating empirical anti-infective therapy strategies tailored to the pathogen distribution in this patient population.

Artificial intelligence algorithm for neoplastic cell percentage estimation and its application to copy number variation in urinary tract cancer.

Bladder cancer is characterized by frequent mutations, which provide potential therapeutic targets for most patients. The effectiveness of emerging personalized therapies depends on an accurate molecular diagnosis, for which the accurate estimation of the neoplastic cell percentage (NCP) is a crucial initial step. However, the established method for determining the NCP, manual counting by a pathologist, is time-consuming and not easily executable. To address this, artificial intelligence (AI) models were developed to estimate the NCP using nine convolutional neural networks and the scanned images of 39 cases of urinary tract cancer. The performance of the AI models was compared to that of six pathologists for 119 cases in the validation cohort. The ground truth value was obtained through multiplexed immunofluorescence. The AI model was then applied to 41 cases in the application cohort that underwent next-generation sequencing testing, and its impact on the copy number variation (CNV) was analyzed. Each AI model demonstrated high reliability, with intraclass correlation coefficients (ICCs) ranging from 0.82 to 0.88. These values were comparable or better to those of pathologists, whose ICCs ranged from 0.78 to 0.91 in urothelial carcinoma cases, both with and without divergent differentiation/ subtypes. After applying AI-driven NCP, 190 CNV (24.2%) were reclassified with 66 (8.4%) and 78 (9.9%) moved to amplification and loss, respectively, from neutral/minor CNV. The neutral/minor CNV proportion decreased by 6%. These results suggest that AI models could assist human pathologists in repetitive and cumbersome NCP calculations.

Impact of next-generation sequencing vs polymerase chain reaction testing on payer costs and clinical outcomes throughout the treatment journeys of patients with metastatic non-small cell lung cancer.

For patients with metastatic non-small cell lung cancer (mNSCLC), next-generation sequencing (NGS) biomarker testing has been associated with a faster time to appropriate targeted therapy and more comprehensive testing relative to polymerase chain reaction (PCR) testing. However, the impact on payer costs and clinical outcomes during patients' treatment journeys has not been fully characterized. To assess the costs and clinical outcomes of NGS vs PCR biomarker testing among patients with newly diagnosed de novo mNSCLC from a US payers' perspective. A Markov model assessed costs and clinical outcomes of NGS vs PCR testing from the start of testing up to 3 years after. Patients entered the model after receiving biomarker test results and then initiated first-line (1L) targeted or nontargeted therapy (immunotherapy and/or chemotherapy) depending on actionable mutation detection. A few patients with an actionable mutation were not detected by PCR and inappropriately initiated 1L nontargeted therapy. At each 1-month cycle, patients could remain on treatment with 1L, progress to second line or later (2L+), or die. Literature-based inputs included the rates of progression-free survival (PFS) and overall survival (OS), targeted and nontargeted therapy costs, total costs of testing, and medical costs of 1L, 2L+, and death. Per patient average PFS and OS as well as cumulative costs were reported for NGS and PCR testing. In a modeled population of 100 patients (75% commercial and 25% Medicare), 45.9% of NGS and 40.0% of PCR patients tested positive for an actionable mutation. Relative to PCR, NGS was associated with $7,386 in savings per patient (NGS = $326,154; PCR = $333,540) at 1 year, driven by lower costs of testing, including estimated costs of delayed care and nontargeted therapy initiation before receiving test results (NGS = $8,866; PCR = $16,373). Treatment costs were similar (NGS = $305,644; PCR = $305,283). In the PCR cohort, the per patient costs of inappropriate 1L nontargeted therapy owing to undetected mutations were $6,455, $6,566, and $6,569 over the first 1, 2, and 3 years, respectively. Relative to PCR testing, NGS was associated with $4,060 in savings at 2 years and $1,092 at 3 years. Patients who initiated 1L targeted therapy had an additional 5.4, 8.8, and 10.4 months of PFS and an additional 1.4, 3.6, and 5.3 months of OS over the first 1, 2, and 3 years, respectively, relative to those who inappropriately initiated 1L nontargeted therapy. In this Markov model, as early as year 1, and over 3 years following biomarker testing, patients with newly diagnosed de novo mNSCLC undergoing NGS testing are projected to have cost savings and longer PFS and OS relative to those tested with PCR.