TLE3 Is a Novel Fusion Partner of JAK2 in Myeloid/Lymphoid Neoplasm With Eosinophilia Responding to JAK2 Inhibition.

Chromosomal rearrangements involving Janus kinase 2 (JAK2) are rare but recurrent findings in lymphoid or myeloid neoplasia. Detection of JAK2 fusion genes is important as patients with aberrantly activated JAK2 may benefit from treatment with tyrosine kinase inhibitors such as ruxolitinib. Here, we report a novel fusion gene between the transcriptional co-repressor-encoding gene transducin-like enhancer of split 3 (TLE3) and JAK2 in a patient initially diagnosed with chronic eosinophilic leukemia with additional mutations in PTPN11 and NRAS. The patient was successfully treated with the JAK2 inhibitor ruxolitinib for 8 months before additional somatic mutations were acquired and the disease progressed into an acute lymphoblastic T-cell leukemia/lymphoma. The present case shows similarities to previously reported cases with PCM1::JAK2 and BCR::JAK2 with regard to disease phenotype and response to ruxolitinib, and importantly, provides an example that also patients harboring other JAK2 fusion genes may benefit from treatment with JAK2 inhibitors.

Precision Diagnostics in Myeloid Malignancies: Development and Validation of a National Capture-Based Gene Panel.

Gene panel sequencing has become a common diagnostic tool for detecting somatically acquired mutations in myeloid neoplasms. However, many panels have restricted content, provide insufficient sensitivity levels, or lack clinically validated workflows. We here describe the development and validation of the Genomic Medicine Sweden myeloid gene panel (GMS-MGP), a capture-based 191 gene panel including mandatory genes in contemporary guidelines as well as emerging candidates. The GMS-MGP displayed uniform coverage across all targets, including recognized difficult GC-rich areas. The validation of 117 previously described somatic variants showed a 100% concordance with a limit-of-detection of a 0.5% variant allele frequency (VAF), achieved by utilizing error correction and filtering against a panel-of-normals. A national interlaboratory comparison investigating 56 somatic variants demonstrated highly concordant results in both detection rate and reported VAFs. In addition, prospective analysis of 323 patients analyzed with the GMS-MGP as part of standard-of-care identified clinically significant genes as well as recurrent mutations in less well-studied genes. In conclusion, the GMS-MGP workflow supports sensitive detection of all clinically relevant genes, facilitates novel findings, and is, based on the capture-based design, easy to update once new guidelines become available. The GMS-MGP provides an important step toward nationally harmonized precision diagnostics of myeloid malignancies.

Gut microbiota associations with chronic kidney disease: insights into nutritional and inflammatory parameters.

Introduction: The gut barrier, comprising gut microbiota, plays a pivotal role in chronic kidney disease (CKD) progression and nutritional status. This study aimed to explore gut barrier alterations in hemodialyzed (HD) patients, non-HD (NHD) CKD patients, and healthy volunteers. Methods: Our cross-sectional study enrolled 22 HD patients, 11 NHD patients, and 11 healthy volunteers. We evaluated fecal microbiota composition (assessed via bacterial 16S rRNA gene sequencing), fecal IgA levels, surrogate markers of gut permeability, serum cytokines, appetite mediators, nutritional status, physical activity, and quality of life. Results: HD patients exhibited significant alterations in fecal microbiota composition compared to healthy volunteers, with observed shifts in taxa known to be associated with dietary patterns or producing metabolites acting on human host. In comparison to healthy volunteers, individuals with HD patients exhibited elevated levels of inflammatory markers (CRP, IL-6 and TNF-α), glucagon-like peptide-2, and potential anorexigenic markers (including leptin and peptide YY). NHD patients had increased levels of CRP and peptide YY. Overall fecal microbiota composition was associated with height, soft lean mass, resting energy expenditure, handgrip strength, bone mineral content and plasma albumin and TNF-α. Discussion: Compared to healthy volunteers, HD patients have an altered fecal microbiota composition, a higher systemic inflammation, and a modification in plasma levels of appetite mediators. While some differences align with previous findings, heterogeneity exists likely due to various factors including lifestyle and comorbidities. Despite limitations such as sample size, our study underscores the multifaceted interplay between gut microbiota, physiological markers, and kidney function, warranting further investigation in larger cohorts.

Cytarabine dose intensification improves survival in older patients with secondary/high-risk acute myeloid leukemia in matched real-world versus clinical trial data.

Since 1980's, the established/standard treatment of acute myeloid leukemia (AML) is cytarabine infusion with anthracycline (7 + 3 regimen). We compared the 7 + 3 regimen in older secondary/high-risk AML patients from a clinical trial with a matched population from the Swedish AML Registry treated with an increased cytarabine dose in induction and consolidation as recommended in the Swedish National Guidelines since 2005. After successful propensity score matching, 104 patients per group were included. The primary outcome was overall survival (OS), and standard dosed patients had a median OS of 6.4 versus 10.7 months with increased dose intensity (hazard ratio: 0.69, p = 0.012), with 5-year OS of 8.7% and 18.1%, and remission rates of 36% and 60%, respectively (p < 0.001). Median OS after allogeneic hematopoietic cell transplantation (in 27.9% per group) was 10.4 and 20.7 months, respectively. We conclude that the more intensive cytarabine schedule seems to provide improved outcomes inthe investigated AML patient group.

Case report: Chronic Candida albicans meningitis: a rare entity diagnosed by metagenomic next-generation sequencing.

The aetiology of chronic aseptic meningitis is difficult to establish. Candida meningitis in particular is often diagnosed late, as cerebrospinal fluid (CSF) work-up and imaging findings are nonspecific. A 35-year-old patient with chronic aseptic meningitis, for which repeated microbiological testing of CSF was unrevealing, was finally diagnosed with Candida albicans (C. albicans) meningitis with cauda equina involvement using metagenomic next-generation sequencing (mNGS). This report highlights the diagnostic challenges and the difficulties of treating shunt-associated fungal meningitis.

Metagenomics for the microbiological diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia (HAP/VAP) in intensive care unit (ICU): a proof-of-concept study.

BACKGROUND: Hospital-acquired and ventilator-associated-pneumonia (HAP/VAP) are one of the most prevalent health-care associated infections in the intensive care unit (ICU). Culture-independent methods were therefore developed to provide faster route to diagnosis and treatment. Among these, metagenomic next-generation sequencing (mNGS) has shown considerable promise. METHODS: This proof-of-concept study describes the technical feasibility and evaluates the clinical validity of the mNGS for the detection and characterization of the etiologic agents causing hospital-acquired and ventilator-associated pneumonia. We performed a prospective study of all patients with HAP/VAP hospitalized in our intensive care unit for whom a bronchoalveolar lavage (BAL) was performed between July 2017 and November 2018. We compared BAL fluid culture and mNGS results of these patients. RESULTS: A total of 32 BAL fluids were fully analyzed. Of these, 22 (69%) were positive by culture and all pathogens identified were also reported by mNGS. Among the culture-positive BAL samples, additional bacterial species were revealed by mNGS for 12 patients, raising the issue of their pathogenic role (colonization versus coinfection). Among BALF with culture-negative test, 5 were positive in mNGS test. CONCLUSIONS: This study revealed concordant results for pneumonia panel pathogens between mNGS and culture-positive tests and identified additional pathogens potentially implicated in pneumonia without etiologic diagnosis by culture. mNGS has emerged as a promising methodology for infectious disease diagnoses to support conventional methods. Prospective studies with real-time mNGS are warranted to examine the impact on antimicrobial decision-making and clinical outcome.

Mycoplasma genitalium Endocarditis in Prosthetic Aortic Valve.

We report a case of Mycoplasma genitalium endocarditis in a prosthetic heart valve of a woman who sought care in Switzerland for acute aortic valve dysfunction 3 years after valve replacement. This unusual manifestation of infection with this bacterium was diagnosed using broad-range PCR despite suspicion of a mechanical disinsertion.

Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity.

Acute myeloid leukemia (AML) is initiated and propagated by leukemia stem cells (LSCs), a self-renewing population of leukemia cells responsible for therapy resistance. Hence, there is an urgent need to identify new therapeutic opportunities targeting LSCs. Here, we performed an in vivo CRISPR knockout screen to identify potential therapeutic targets by interrogating cell surface dependencies of LSCs. The facilitated glucose transporter type 1 (GLUT1) emerged as a critical in vivo metabolic dependency for LSCs in a murine MLL::AF9-driven model of AML. GLUT1 disruption by genetic ablation or pharmacological inhibition led to suppression of leukemia progression and improved survival of mice that received transplantation with LSCs. Metabolic profiling revealed that Glut1 inhibition suppressed glycolysis, decreased levels of tricarboxylic acid cycle intermediates and increased the levels of amino acids. This metabolic reprogramming was accompanied by an increase in autophagic activity and apoptosis. Moreover, Glut1 disruption caused transcriptional, morphological, and immunophenotypic changes, consistent with differentiation of AML cells. Notably, dual inhibition of GLUT1 and oxidative phosphorylation (OXPHOS) exhibited synergistic antileukemic effects in the majority of tested primary AML patient samples through restraining of their metabolic plasticity. In particular, RUNX1-mutated primary leukemia cells displayed striking sensitivity to the combination treatment compared with normal CD34+ bone marrow and cord blood cells. Collectively, our study reveals a GLUT1 dependency of murine LSCs in the bone marrow microenvironment and demonstrates that dual inhibition of GLUT1 and OXPHOS is a promising therapeutic approach for AML.

The Swiss Pathogen Surveillance Platform - towards a nation-wide One Health data exchange platform for bacterial, viral and fungal genomics and associated metadata.

The Swiss Pathogen Surveillance Platform (SPSP) is a shared secure surveillance platform between human and veterinary medicine, to also include environmental and foodborne isolates. It enables rapid and detailed transmission monitoring and outbreak surveillance of pathogens using whole genome sequencing data and associated metadata. It features controlled data access, complex dynamic queries, dedicated dashboards and automated data sharing with international repositories, providing actionable results for public health and the vision to improve societal well-being and health.

Postpartum septic arthritis of pubic symphysis due to Sneathia sanguinegens, Sneathia vaginalis, and Mageeibacillus indolicus: Contribution of clinicalmetagenomics.

Sneathia sanguinegens, Sneathia vaginalis, and Mageeibacillus indolicus have been recently described in the female genital tract. We present the first case of a postpartum septic arthritis of the pubic symphysis due to these organisms, identified by next generation sequencing.

Draft Genome Sequence of a Mixed-Serogroup W/Y Invasive Neisseria meningitidis Strain.

We report the genome of a Neisseria meningitidis strain (GE-156) that was isolated in Switzerland from a patient diagnosed with bacteremia. The strain belongs to a rare mixed serogroup W/Y and sequence type 11847 (clonal complex 167), as revealed by both routine laboratory examination and genomic sequencing.

The complement receptor C3AR constitutes a novel therapeutic target in NPM1-mutated AML.

Mutated nucleophosmin 1 (NPM1) is the most common genetic alteration in acute myeloid leukemia (AML), found in ∼30% of cases. Although mutations in this gene are considered favorable according to current risk stratification guidelines, a large fraction of patients will experience relapse, demonstrating the urgent need for new treatment options. Therefore, we aimed to identify cell surface proteins specifically expressed on NPM1-mutated AML cells, allowing for potential targeting with antibody-based therapies. Herein, we report on an arrayed flow cytometry-based screen directed to 362 cell surface markers. In comparing the cell surface expression on NPM1-mutated AML cells with primitive (CD34+ CD38-) normal bone marrow cells, we identified the complement receptor C3AR as being specifically expressed in NPM1-mutated AML. By flow cytometry and single-cell RNA sequencing, we further show that normal hematopoietic stem and progenitor cells lack detectable C3AR gene and protein expression, making it particularly suitable as a target for antibody therapy. We also demonstrate that C3AR in combination with GPR56 distinguishes the leukemic stem cells (LSCs) in NPM1-mutated AML from the normal hematopoietic stem cells, defining the LSC population, as shown by transplantation into immunodeficient mice. Mechanistically, the stimulation of C3AR-expressing cells with C3a, the ligand of C3AR, leads to the activation of ERK1/2 and increased survival of AML cells, suggesting that this is an important signaling axis in this subtype of AML. Finally, we show that antibodies directed against C3AR efficiently elicit natural killer cell-mediated killing of primary AML cells ex vivo, highlighting C3AR as a candidate therapeutic target in NPM1-mutated AML.

Characterization of therapy-related acute myeloid leukemia: increasing incidence and prognostic implications.

Studies of therapy-related AML (t-AML) are usually performed in selected cohorts and reliable incidence rates are lacking. In this study, we characterized, defined the incidence over time and studied prognostic implications in all t-AML patients diagnosed in Sweden between 1997 and 2015. Data were retrieved from nationwide population-based registries. In total, 6,779 AML patients were included in the study, of whom 686 (10%) had t-AML. The median age for t-AML was 71 years and 392 (57%) patients were females. During the study period, the incidence of t-AML almost doubled with a yearly increase in t-AML of 4.5% (95% confidence interval: 2.8%-6.2%), which contributed significantly to the general increase in AML incidence over the study period. t-AML solidly constituted over 10% of all AML cases during the later period of the study. Primary diagnoses with the largest increase in incidence and decrease in mortality rate during the study period (i.e., breast and prostate cancer) contributed significantly to the increased incidence of t-AML. In multivariable analysis, t-AML was associated with poorer outcome in cytogenetically intermediate- and adverse-risk cases but t-AML had no significant impact on outcome in favorable-risk AML, including core binding leukemias, acute promyelocytic leukemia and AML with mutated NPM1 without FLT3-ITD. We conclude that there is a strong increase in incidence in t-AML over time and that t-AML constitutes a successively larger proportion of the AML cases. Furthermore, we conclude that t-AML confers a poor prognosis in cytogenetically intermediate- and adverse-risk, but not in favorable-risk AML.

Contribution of Clinical Metagenomics to the Diagnosis of Bone and Joint Infections.

Bone and joint infections (BJIs) are complex infections that require precise microbiological documentation to optimize antibiotic therapy. Currently, diagnosis is based on microbiological culture, sometimes complemented by amplification and sequencing of the 16S rDNA gene. Clinical metagenomics (CMg), that is, the sequencing of the entire nucleic acids in a sample, was previously shown to identify bacteria not detected by conventional methods, but its actual contribution to the diagnosis remains to be assessed, especially with regard to 16S rDNA sequencing. In the present study, we tested the performance of CMg in 34 patients (94 samples) with suspected BJIs, as compared to culture and 16S rDNA sequencing. A total of 94 samples from 34 patients with suspicion of BJIs, recruited from two sites, were analyzed by (i) conventional culture, (ii) 16S rDNA sequencing (Sanger method), and (iii) CMg (Illumina Technology). Two negative controls were also sequenced by CMg for contamination assessment. Based on the sequencing results of negative controls, 414 out of 539 (76.7%) bacterial species detected by CMg were considered as contaminants and 125 (23.2%) as truly present. For monomicrobial infections (13 patients), the sensitivity of CMg was 83.3% as compared to culture, and 100% as compared to 16S rDNA. For polymicrobial infections (13 patients), the sensitivity of CMg was 50% compared to culture, and 100% compared to 16S rDNA. For samples negative in culture (8 patients, 21 samples), CMg detected 11 bacteria in 10 samples from 5 different patients. In 5/34 patients, CMg brought a microbiological diagnosis where conventional methods failed, and in 16/34 patients, CMg provided additional information. Finally, 99 antibiotic resistance genes were detected in 24 patients (56 samples). Provided sufficient genome coverage (87.5%), a correct inference of antibiotic susceptibility was achieved in 8/8 bacteria (100%). In conclusion, our study demonstrated that the CMg provides complementary and potentially valuable data to conventional methods of BJIs diagnosis.

A Study Protocol for Validation and Implementation of Whole-Genome and -Transcriptome Sequencing as a Comprehensive Precision Diagnostic Test in Acute Leukemias.

Background: Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed. Methods and Analysis: The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact. Discussion: Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care. Clinical Trial Registration: [https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

Changes in the gut bacterial communities in colon cancer surgery patients: an observational study.

BACKGROUND: Colon surgery has been shown to modulate the intestinal microbiota. Our objective was to characterize these changes using state-of-the-art next generation sequencing techniques. METHODS: We performed a single-centre prospective observational cohort study to evaluate the changes in the gut microbiota, i.e., taxon distribution, before and after elective oncologic colon surgery in adult patients with different antimicrobial prophylaxis regimens (standard prophylaxis with cefuroxime/metronidazole versus carbapenems for extended-spectrum beta-lactamase-producing Enterobacterales [ESBL-E] carriers). We obtained rectal samples on the day of surgery, intraoperative luminal samples, and rectal or stoma samples 3 days after surgery. We performed metataxonomic analysis based on sequencing of the bacterial 16S rRNA gene marker. Similarities and differences between bacterial communities were assessed using Bray-Curtis similarity, visualised using principal coordinates analysis and statistically tested by PERMANOVA. Comparison of taxa relative abundance was performed using ANCOM. RESULTS: We included 27 patients between March 27, 2019 and September 17, 2019. The median age was 63.6 years (IQR 56.4-76.3) and 44% were females. Most (81%) patients received standard perioperative prophylaxis as they were not ESBL carriers. There was no significant association between ESBL carriage and differences in gut microbiome. We observed large and significant increases in the genus Enterococcus between the preoperative/intraoperative samples and the postoperative sample, mainly driven by Enterococcus faecalis. There were significant differences in the postoperative microbiome between patients who received standard prophylaxis and carbapenems, specifically in the family Erysipelotrichaceae. CONCLUSION: This hypothesis-generating study showed rapid changes in the rectal microbiota following colon cancer surgery.