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Background Recurrent somatic mutations in blood stem cells cause the emergence of mutated blood cell clones, known as clonal hematopoiesis (CH). Mutations in the most prevalent driver genes DNMT3A and TET2 with a variant allele frequency (VAF)≥2% have been associated with atherosclerosis and chronic heart failure (CHF). However, the effects of mutations in other driver genes for CH with low VAF ( Purpose To assess the potential prognostic significance of mutations in distinct genes other than DNMT3A and TET2 causing CH at low VAF in patients with CHF due to post-ischemic origin. Methods We analyzed mononuclear bone marrow and peripheral blood cells from 400 CHF patients by deep error-corrected targeted amplicon sequencing allowing for the detection of very low VAF of ≥0.5% in 56 CH driver genes and associated such with the long-term mortality in these patients. Results Median age of the patients was 63 and median follow-up time was 4.8 years. We detected 1116 mutations with a VAF≥0.5% in 348 of 400 patients (87%). 21% of the patients carried 1 mutation, whereas 66% showed 2 and more mutations. Only 52 (13%) patients had no detectable mutation at all. Despite a high prevalence of mutations in the most frequently mutated driver genes DNMT3A (165 patients) and TET2 (107 pts), mutations in the genes CBL (8 pts), CEBPA (10 pts), PHF6 (22 pts), SMC1A (18 pts) and SRSF2 (14 pts) were associated with increased death compared to the average death rate of all patients (18.8%). To avoid confounding effects caused by the presence of DNMT3A and TET2 CHIP mutations, we excluded patients with DNMT3A-, TET2- and other CHIP-related mutations with a VAF≥2% for further analyses. Kaplan-Meier survival curve analyses revealed a significantly higher mortality in patients with mutations in either of the five genes, combined as the CH risk gene set for CHF (Fig. 1). Patients with mutations in the risk gene set (44 pts) did not differ from patients with mutations in other CH driver genes or without mutations with respect to age, cardiovascular risk factors, disease severity or CH clone size. By multivariate Cox proportional regression analysis, including the Seattle heart failure model (SHFM) score (as tertiles), death remained independently associated with the presence of mutations in the risk gene set (HR, 2.57; 95% CI, 1.30–5.10; p=0.007), in addition to SHFM score tertiles (tertile 2, HR, 2.36; 95% CI, 0.73–7.65 and tertile 3, HR, 7.96; 95% CI, 2.77–22.90). Conclusions Somatic mutations with low VAF in a distinct set of genes, namely in CBL, CEBPA, PHF6, SMC1A and SRSF2, are significantly associated with mortality in CHF, independently of the classical CHIP-driver mutations DNMT3A and TET2. Mutations in the CH risk gene set are prevalent in young CHF patients and comprise an independent risk factor for the outcome of CHF, potentially providing a novel tool for cardiovascular risk assessment and precision medicine in CHF. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

Aims Somatic mutations of the epigenetic regulators DNMT3A and TET2 causing clonal expansion of haematopoietic cells (clonal haematopoiesis; CH) were shown to be associated with poor prognosis in chronic ischaemic heart failure (CHF). The aim of our analysis was to define a threshold of variant allele frequency (VAF) for the prognostic significance of CH in CHF. Methods and results We analysed bone marrow and peripheral blood-derived cells from 419 patients with CHF by error-corrected amplicon sequencing. Cut-off VAFs were optimized by maximizing sensitivity plus specificity from a time-dependent receiver operating characteristic (ROC) curve analysis from censored data. 56.2% of patients were carriers of a DNMT3A- (N = 173) or a TET2- (N = 113) mutation with a VAF >0.5%, with 59 patients harbouring mutations in both genes. Survival ROC analyses revealed an optimized cut-off value of 0.73% for TET2- and 1.15% for DNMT3A-CH-driver mutations. Five-year-mortality was 18% in patients without any detected DNMT3A- or TET2 mutation (VAF Conclusion The present study defines novel threshold levels for clone size caused by acquired somatic mutations in the CH-driver genes DNMT3A and TET2 that are associated with worse outcome in patients with CHF.

Somatic mutations in hematopoietic stem cells can lead to the expansion of mutated blood cells, known as clonal hematopoiesis (CH). Mutations in the most prevalent genes DNMT3A and TET2 with a variant allele frequency (VAF)≥2% (CHIP) have been associated with atherosclerosis and chronic heart failure (CHF). However, the mutational landscape in CHF and the effects in CH-driver genes other than DNMT3A and TET2 with low VAF≤2% on CHF are still unknown. Therefore we analyzed bone marrow and blood cells from 399 CHF patients by deep error-corrected targeted sequencing of 56 genes and associated mutations with the long-term mortality (3.95 years median follow-up). We detected 1113 mutations with a VAF≥0.5% in 347 patients (87%). Despite a high prevalence of mutations in the most frequently mutated genes DNMT3A and TET2, mutations in the genes CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A and SRSF2 were associated with increased death compared to the average death rate of all patients (20%). To avoid confounding effects caused by the presence of DNMT3A and TET2 CHIP mutations, we excluded patients with CHIP-related mutations (VAF≥2%) for further analyses. Kaplan-Meier survival analyses revealed a significantly higher mortality in patients with mutations in the seven genes (53 patients), combined as the CH risk gene set for CHF. These patients showed no significant differences in any parameter including patient age, confounding diseases or blood parameters except for a reduced number of platelets. However, carrying a mutation in any of the risk genes remained significant after multivariate cox regression analysis (HR, 3.1; 95% CI, 1.8-5.4; p Somatic mutations with low VAF in these risk genes are prevalent in young CHF patients and comprise an independent risk factor for the outcome, potentially providing a novel tool for risk assessment in CHF.

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Neoangiogenesis is a pivotal therapeutic target in glioblastoma. Tumor monitoring requires imaging methods to assess treatment effects and disease progression. Until now mapping of the tumor vasculature has been difficult. We have developed a combined magnetic resonance and optical toolkit to study neoangiogenesis in glioma models. We use in vivo magnetic resonance imaging (MRI) and correlative ultramicroscopy (UM) of ex vivo cleared whole brains to track neovascularization. T2* imaging allows the identification of single vessels in glioma development and the quantification of neovessels over time. Pharmacological VEGF inhibition leads to partial vascular normalization with decreased vessel caliber, density, and permeability. To further resolve the tumor microvasculature, we performed correlated UM of fluorescently labeled microvessels in cleared brains. UM resolved typical features of neoangiogenesis and tumor cell invasion with a spatial resolution of ~5 µm. MR-UM can be used as a platform for three-dimensional mapping and high-resolution quantification of tumor angiogenesis. DOI: http://dx.doi.org/10.7554/eLife.11712.001, eLife digest Blood vessels are the body’s highways that allow blood to transport oxygen, nutrients, hormones and waste products quickly and efficiently around the body. Tumors are made up of particularly active cells and so their growth heavily depends on blood vessels. Indeed, a fundamental hallmark of tumor progression is for nearby blood vessels to form more quickly. Tumor blood vessels also differ in structure from their normal counterparts for reasons that need to be investigated in more detail. Compounds that block the formation of blood vessels have been developed for treating highly malignant brain tumors called gliomas. However, although many of these compounds show promising effects in preclinical trials, clinical trials on humans have been less successful. Having the ability to image the blood vessels in high detail during preclinical trials would help to reveal how treatments that inhibit blood vessel formation work and how tumors might develop resistance to these drugs. However, studying tumor blood vessels remains a challenge due to technical restrictions: techniques that are able to capture how the vessels change over time are unable to show individual cells in much detail, and vice versa. Magnetic resonance imaging is a versatile tool that can monitor how the blood vessel system of a tumor changes over time in living animals. On the other hand, ultramicroscopy is able to determine the structure of single cells of a particular type. By combining these techniques, Breckwoldt, Bode et al. have now developed a imaging platform that allows the formation of tumor blood vessels to be precisely mapped in the setting of a preclinical study. It also enables detailed investigations into how the structure of the blood vessels is altered by treatments that aim to inhibit the formation and growth of new vessels. Using this approach on mice with gliomas, Breckwoldt, Bode et al. demonstrated that drugs that inhibit the formation of the blood vessels that supply tumors also cause the blood vessels to take on a more normal structure. Furthermore, treating the mice with a single inhibitory drug was unable to stop tumor growth, mirroring the situation in humans. Currently, new inhibitors are being developed, offering the possibility of combined treatments that may be more effective than using a single drug on its own. The imaging platform developed by Breckwoldt, Bode et al. will allow the therapeutic effects obtained by these new treatments to be analyzed in detail during preclinical studies. DOI: http://dx.doi.org/10.7554/eLife.11712.002