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Liquid profiling uses circulating tumor DNA (ctDNA) for minimal invasive tumor mutational profiling from peripheral blood. The presence of somatic mutations in peripheral blood cells without further evidence of a hematologic neoplasm defines clonal hematopoiesis of indeterminate potential (CHIP). CHIP-mutations can be found in the cell-free DNA (cfDNA) of plasma, are a potential cause of false positive results in liquid profiling, and thus limit its usage in screening settings. Various strategies are in place to mitigate the effect of CHIP on the performance of ctDNA assays, but the detection of CHIP also represents a clinically significant incidental finding. The sequelae of CHIP comprise the risk of progression to a hematologic neoplasm including therapy-related myeloid neoplasms. While the hematological risk increases with the co-occurrence of unexplained blood count abnormalities, a number of non-hematologic diseases have independently been associated with CHIP. In particular, CHIP represents a major risk factor for cardiovascular disease such as atherosclerosis or heart failure. The management of CHIP requires an interdisciplinary setting and represents a new topic in the field of cardio-oncology. In the future, the information on CHIP may be taken into account for personalized therapy of cancer patients.

The optimal procedures to prevent, identify, monitor, and treat long-term pulmonary sequelae of COVID-19 are elusive. Here, we characterized the kinetics of respiratory and symptom recovery following COVID-19. We conducted a longitudinal, multicenter observational study in ambulatory and hospitalized COVID-19 patients recruited in early 2020 (n = 145). Pulmonary computed tomography (CT) and lung function (LF) readouts, symptom prevalence, and clinical and laboratory parameters were collected during acute COVID-19 and at 60, 100, and 180 days follow-up visits. Recovery kinetics and risk factors were investigated by logistic regression. Classification of clinical features and participants was accomplished by unsupervised and semi-supervised multiparameter clustering and machine learning. At the 6-month follow-up, 49% of participants reported persistent symptoms. The frequency of structural lung CT abnormalities ranged from 18% in the mild outpatient cases to 76% in the intensive care unit (ICU) convalescents. Prevalence of impaired LF ranged from 14% in the mild outpatient cases to 50% in the ICU survivors. Incomplete radiological lung recovery was associated with increased anti-S1/S2 antibody titer, IL-6, and CRP levels at the early follow-up. We demonstrated that the risk of perturbed pulmonary recovery could be robustly estimated at early follow-up by clustering and machine learning classifiers employing solely non-CT and non-LF parameters. The severity of acute COVID-19 and protracted systemic inflammation is strongly linked to persistent structural and functional lung abnormality. Automated screening of multiparameter health record data may assist in the prediction of incomplete pulmonary recovery and optimize COVID-19 follow-up management. The State of Tyrol (GZ 71934), Boehringer Ingelheim/Investigator initiated study (IIS 1199-0424). ClinicalTrials.gov: NCT04416100.

The analytically sensitive detection of D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR ( < 0.001). Moreover, dPCR for D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; < 0.001). Moreover, dPCR confirmed the prognostic significance of a high D816V VAF regarding survival ( < 0.001). dPCR for D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of D816V allele burden measurement. Thus, dPCR is suitable as a new method for D816V testing in patients with mastocytosis.

To describe the prevalence and spectrum of cardio-pathogenic variants in singleton fetuses after unexplained intrauterine fetal death (IUFD). DNA from post-mortem fibroblastic tissue samples of 16 fetuses after unexplained IUFD was retrieved at two tertiary university hospitals for clinical exome sequencing with subsequent filtering of 122 cardio-specific genes to elucidate underlying cardio-pathogenic variants. In total, we included 12 (75%) male and four (25%) female fetuses who were stillborn at a median gestational age of 34 +6 (23 +2 –40 +5 ) weeks. In two (12.5%) fetuses no cardio-pathogenic variants were found. In 14 (87.5%) fetuses, overall 33 variants were detected in 22 cardio-specific genes, involving 14 (63.63%) genes associated with cardiomyopathy, six (27.27%) arrhythmogenic susceptibility genes and two (9.09%) arrhythmia and cardiomyopathy associated genes. Among the 33 variants, five (15.2%) were classified as likely benign according to the American College of Medical Genetics and Genomics ; 28 (84.8%) variants were considered as variants of uncertain significance . Compared to a cohort of explained IUFDs, the cases with and without fetal variants in cardiac genes differed not significantly regarding maternal age, previous history of stillbirth, time of stillbirth or fetal sex. Unexplained stillbirth may be caused by cardio-genetic pathologies, yet a high number of variants of uncertain significance merit a more detailed post-mortem examination including family segregation analysis.

The term myeloproliferative neoplasms (MPN) refers to a heterogeneous group of diseases including not only polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), but also chronic myeloid leukemia (CML), and systemic mastocytosis (SM). Despite the clinical and biological differences between these diseases, common pathophysiological mechanisms have been identified in MPN. First, aberrant tyrosine kinase signaling due to somatic mutations in certain driver genes is common to these MPN. Second, alterations of the bone marrow microenvironment are found in all MPN types and have been implicated in the pathogenesis of the diseases. Finally, elevated levels of proinflammatory and microenvironment-regulating cytokines are commonly found in all MPN-variants. In this paper, we review the effects of MPN-related oncogenes on cytokine expression and release and describe common as well as distinct pathogenetic mechanisms underlying microenvironmental changes in various MPN. Furthermore, targeting of the microenvironment in MPN is discussed. Such novel therapies may enhance the efficacy and may overcome resistance to established tyrosine kinase inhibitor treatment in these patients. Nevertheless, additional basic studies on the complex interplay of neoplastic and stromal cells are required in order to optimize targeting strategies and to translate these concepts into clinical application.

Mast cell leukemia (MCL) is a highly fatal malignancy characterized by devastating expansion of immature mast cells in various organs. Although considered a stem cell disease, little is known about MCL-propagating neoplastic stem cells. We here describe that leukemic stem cells (LSCs) in MCL reside within a CD34 + /CD38 − fraction of the clone. Whereas highly purified CD34 + /CD38 ─ cells engrafted NSG hSCF mice with fully manifesting MCL, no MCL was produced by CD34 + /CD38 + progenitors or the bulk of KIT + /CD34 − mast cells. CD34 + /CD38 – MCL cells invariably expressed CD13 and CD133, and often also IL-1RAP, but did not express CD25, CD26 or CLL-1. CD34 + /CD38 – MCL cells also displayed several surface targets, including CD33, which was homogenously expressed on MCL LSCs in all cases, and the D816V mutant form of KIT. Although CD34 + /CD38 − cells were resistant against single drugs, exposure to combinations of CD33-targeting and KIT-targeting drugs resulted in LSC-depletion and markedly reduced engraftment in NSG hSCF mice. Together, MCL LSCs are CD34 + /CD38 − cells that express distinct profiles of markers and target antigens. Characterization of MCL LSCs should facilitate their purification and should support the development of LSC-eradicating curative treatment approaches in this fatal type of leukemia.

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4 + and CD8 + T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8 + T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8 + T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.

Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. We recently identified LIM domain only 3 (LMO3) in human mature visceral adipocytes; however, its function in these cells is currently unknown. The aim of this study was to determine the potential involvement of LMO3-dependent pathways in the modulation of key functions of mature adipocytes during obesity. Based on a recently engineered hybrid rAAV serotype Rec2 shown to efficiently transduce both brown adipose tissue (BAT) and white adipose tissue (WAT), we delivered YFP or Lmo3 to epididymal WAT (eWAT) of C57Bl6/J mice on a high-fat diet (HFD). The effects of eWAT transduction on metabolic parameters were evaluated 10 weeks later. To further define the role of LMO3 in insulin-stimulated glucose uptake, insulin signaling, adipocyte bioenergetics, as well as endocrine function, experiments were conducted in 3T3-L1 adipocytes and newly differentiated human primary mature adipocytes, engineered for transient gain or loss of LMO3 expression, respectively. AAV transduction of eWAT results in strong and stable Lmo3 expression specifically in the adipocyte fraction over a course of 10 weeks with HFD feeding. LMO3 expression in eWAT significantly improved insulin sensitivity and healthy visceral adipose tissue expansion in diet-induced obesity, paralleled by increased serum adiponectin. In vitro, LMO3 expression in 3T3-L1 adipocytes increased PPARγ transcriptional activity, insulin-stimulated GLUT4 translocation and glucose uptake, as well as mitochondrial oxidative capacity in addition to fatty acid oxidation. Mechanistically, LMO3 induced the PPARγ coregulator Ncoa1, which was required for LMO3 to enhance glucose uptake and mitochondrial oxidative gene expression. In human mature adipocytes, LMO3 overexpression promoted, while silencing of LMO3 suppressed mitochondrial oxidative capacity. LMO3 expression in visceral adipose tissue regulates multiple genes that preserve adipose tissue functionality during obesity, such as glucose metabolism, insulin sensitivity, mitochondrial function, and adiponectin secretion. Together with increased PPARγ activity and Ncoa1 expression, these gene expression changes promote insulin-induced GLUT4 translocation, glucose uptake in addition to increased mitochondrial oxidative capacity, limiting HFD-induced adipose dysfunction. These data add LMO3 as a novel regulator improving visceral adipose tissue function during obesity.Key messagesLMO3 increases beneficial visceral adipose tissue expansion and insulin sensitivity in vivo.LMO3 increases glucose uptake and oxidative mitochondrial activity in adipocytes.LMO3 increases nuclear coactivator 1 (Ncoa1).LMO3-enhanced glucose uptake and mitochondrial gene expression requires Ncoa1.