Benchmarking MicrobIEM - a user-friendly tool for decontamination of microbiome sequencing data.

BACKGROUND: Microbiome analysis is becoming a standard component in many scientific studies, but also requires extensive quality control of the 16S rRNA gene sequencing data prior to analysis. In particular, when investigating low-biomass microbial environments such as human skin, contaminants distort the true microbiome sample composition and need to be removed bioinformatically. We introduce MicrobIEM, a novel tool to bioinformatically remove contaminants using negative controls. RESULTS: We benchmarked MicrobIEM against five established decontamination approaches in four 16S rRNA amplicon sequencing datasets: three serially diluted mock communities (108-103 cells, 0.4-80% contamination) with even or staggered taxon compositions and a skin microbiome dataset. Results depended strongly on user-selected algorithm parameters. Overall, sample-based algorithms separated mock and contaminant sequences best in the even mock, whereas control-based algorithms performed better in the two staggered mocks, particularly in low-biomass samples (≤ 106 cells). We show that a correct decontamination benchmarking requires realistic staggered mock communities and unbiased evaluation measures such as Youden's index. In the skin dataset, the Decontam prevalence filter and MicrobIEM's ratio filter effectively reduced common contaminants while keeping skin-associated genera. CONCLUSIONS: MicrobIEM's ratio filter for decontamination performs better or as good as established bioinformatic decontamination tools. In contrast to established tools, MicrobIEM additionally provides interactive plots and supports selecting appropriate filtering parameters via a user-friendly graphical user interface. Therefore, MicrobIEM is the first quality control tool for microbiome experts without coding experience.

Skin microbiome and its association with host cofactors in determining atopic dermatitis severity.

BACKGROUND: Atopic dermatitis (AD) is a heterogeneous, chronic inflammatory skin disease linked to skin microbiome dysbiosis with reduced bacterial diversity and elevated relative abundance of Staphylococcus aureus (S. aureus). OBJECTIVES: We aimed to characterize the yet incompletely understood association between the skin microbiome and patients' demographic and clinical cofactors in relation to AD severity. METHODS: The skin microbiome in 48 adult moderate-to-severe AD patients was investigated using next-generation deep sequencing (16S rRNA gene, V1-V3 region) followed by denoising (DADA2) to obtain amplicon sequence variant (ASV) composition. RESULTS: In lesional skin, AD severity was associated with S. aureus relative abundance (rS  = 0.53, p < 0.001) and slightly better with the microbiome diversity measure Evenness (rS  = -0.58, p < 0.001), but not with Richness. Multiple regression confirmed the association of AD severity with microbiome diversity, including Shannon (in lesional skin, p < 0.001), Evenness (in non-lesional skin, p = 0.015) or S. aureus relative abundance (p < 0.012), and with patient's IgE levels (p < 0.001), race (p < 0.032), age (p < 0.034) and sex (p = 0.012). The lesional model explained 62% of the variation in AD severity, and the non-lesional model 50% of the variation. CONCLUSIONS: Our results specify the frequently reported "reduced diversity" of the AD-related skin microbiome to reduced Evenness, which was in turn mainly driven by S. aureus relative abundance, rather than to a reduced microbiome Richness. Finding associations between AD severity, the skin microbiome and patient's cofactors is a key aspect in developing new personalized AD treatments, particularly those targeting the AD microbiome.

Differential effects of lung inflammation on insulin resistance in humans and mice.

BACKGROUND: The rates of obesity, its associated diseases, and allergies are raising at alarming rates in most countries. House dust mites (HDM) are highly allergenic and exposure often associates with an urban sedentary indoor lifestyle, also resulting in obesity. The aim of this study was to investigate the epidemiological association and physiological impact of lung inflammation on obesity and glucose homeostasis. METHODS: Epidemiological data from 2207 adults of the population-based KORA FF4 cohort were used to test associations between asthma and rhinitis with metrics of body weight and insulin sensitivity. To obtain functional insights, C57BL/6J mice were intranasally sensitized and challenged with HDM and simultaneously fed with either low-fat or high-fat diet for 12 weeks followed by a detailed metabolic and biochemical phenotyping of the lung, liver, and adipose tissues. RESULTS: We found a direct association of asthma with insulin resistance but not body weight in humans. In mice, co-development of obesity and HDM-induced lung inflammation attenuated inflammation in lung and perigonadal fat, with little impact on body weight, but small shifts in the composition of gut microbiota. Exposure to HDM improved glucose tolerance, reduced hepatosteatosis, and increased energy expenditure and basal metabolic rate. These effects associate with increased activity of thermogenic adipose tissues independent of uncoupling protein 1. CONCLUSIONS: Asthma associates with insulin resistance in humans, but HDM challenge results in opposing effects on glucose homeostasis in mice due to increased energy expenditure, reduced adipose inflammation, and hepatosteatosis.

Distinct cytokine profiles associated with COVID-19 severity and mortality.

BACKGROUND: Markedly elevated levels of proinflammatory cytokines and defective type-I interferon responses were reported in patients with coronavirus disease 2019 (COVID-19). OBJECTIVE: We sought to determine whether particular cytokine profiles are associated with COVID-19 severity and mortality. METHODS: Cytokine concentrations and severe acute respiratory syndrome coronavirus 2 antigen were measured at hospital admission in serum of symptomatic patients with COVID-19 (N = 115), classified at hospitalization into 3 respiratory severity groups: no need for mechanical ventilatory support (No-MVS), intermediate severity requiring mechanical ventilatory support (MVS), and critical severity requiring extracorporeal membrane oxygenation (ECMO). Principal-component analysis was used to characterize cytokine profiles associated with severity and mortality. The results were thereafter confirmed in an independent validation cohort (N = 86). RESULTS: At time of hospitalization, ECMO patients presented a dominant proinflammatory response with elevated levels of TNF-α, IL-6, IL-8, and IL-10. In contrast, an elevated type-I interferon response involving IFN-α and IFN-ß was characteristic of No-MVS patients, whereas MVS patients exhibited both profiles. Mortality at 1 month was associated with higher levels of proinflammatory cytokines in ECMO patients, higher levels of type-I interferons in No-MVS patients, and their combination in MVS patients, resulting in a combined mortality prediction accuracy of 88.5% (risk ratio, 24.3; P < .0001). Severe acute respiratory syndrome coronavirus 2 antigen levels correlated with type-I interferon levels and were associated with mortality, but not with proinflammatory response or severity. CONCLUSIONS: Distinct cytokine profiles are observed in association with COVID-19 severity and are differentially predictive of mortality according to oxygen support modalities. These results warrant personalized treatment of COVID-19 patients based on cytokine profiling.

The power and potential of BIOMAP to elucidate host-microbiome interplay in skin inflammatory diseases.

The two most common chronic inflammatory skin diseases are atopic dermatitis (AD) and psoriasis. The underpinnings of the remarkable degree of clinical heterogeneity of AD and psoriasis are poorly understood and, as a consequence, disease onset and progression are unpredictable and the optimal type and time point for intervention are as yet unknown. The BIOMAP project is the first IMI (Innovative Medicines Initiative) project dedicated to investigating the causes and mechanisms of AD and psoriasis and to identify potential biomarkers responsible for the variation in disease outcome. The consortium includes 7 large pharmaceutical companies and 25 non-industry partners including academia. Since there is mounting evidence supporting an important role for microbial exposures and our microbiota as factors mediating immune polarization and AD and psoriasis pathogenesis, an entire work package is dedicated to the investigation of skin and gut microbiome linked to AD or psoriasis. The large collaborative BIOMAP project will enable the integration of patient cohorts, data and knowledge in unprecedented proportions. The project has a unique opportunity with a potential to bridge and fill the gaps between current problems and solutions. This review highlights the power and potential of the BIOMAP project in the investigation of microbe-host interplay in AD and psoriasis.

Defining biomarkers to predict symptoms in subjects with and without allergy under natural pollen exposure.

BACKGROUND: Pollen exposure induces local and systemic allergic immune responses in sensitized individuals, but nonsensitized individuals also are exposed to pollen. The kinetics of symptom expression under natural pollen exposure have never been systematically studied, especially in subjects without allergy. OBJECTIVE: We monitored the humoral immune response under natural pollen exposure to potentially uncover nasal biomarkers for in-season symptom severity and identify protective factors. METHODS: We compared humoral immune response kinetics in a panel study of subjects with seasonal allergic rhinitis (SAR) and subjects without allergy and tested for cross-sectional and interseasonal differences in levels of serum and nasal, total, and Betula verrucosa 1-specific immunoglobulin isotypes; immunoglobulin free light chains; cytokines; and chemokines. Nonsupervised principal component analysis was performed for all nasal immune variables, and single immune variables were correlated with in-season symptom severity by Spearman test. RESULTS: Symptoms followed airborne pollen concentrations in subjects with SAR, with a time lag between 0 and 13 days depending on the pollen type. Of the 7 subjects with nonallergy, 4 also exhibited in-season symptoms whereas 3 did not. Cumulative symptoms in those without allergy were lower than in those with SAR but followed the pollen exposure with similar kinetics. Nasal eotaxin-2, CCL22/MDC, and monocyte chemoattactant protein-1 (MCP-1) levels were higher in subjects with SAR, whereas IL-8 levels were higher in subjects without allergy. Principal component analysis and Spearman correlations identified nasal levels of IL-8, IL-33, and Betula verrucosa 1-specific IgG4 (sIgG4) and Betula verrucosa 1-specific IgE (sIgE) antibodies as predictive for seasonal symptom severity. CONCLUSIONS: Nasal pollen-specific IgA and IgG isotypes are potentially protective within the humoral compartment. Nasal levels of IL-8, IL-33, sIgG4 and sIgE could be predictive biomarkers for pollen-specific symptom expression, irrespective of atopy.

Skin pH-dependent Staphylococcus aureus abundance as predictor for increasing atopic dermatitis severity.

BACKGROUND: Atopic eczema (atopic dermatitis, AD) is characterized by disrupted skin barrier associated with elevated skin pH and skin microbiome dysbiosis, due to high Staphylococcus aureus loads, especially during flares. Since S aureus shows optimal growth at neutral pH, we investigated the longitudinal interplay between these factors and AD severity in a pilot study. METHOD: Emollient (with either basic pH 8.5 or pH 5.5) was applied double-blinded twice daily to 6 AD patients and 6 healthy (HE) controls for 8 weeks. Weekly, skin swabs for microbiome analysis (deep sequencing) were taken, AD severity was assessed, and skin physiology (pH, hydration, transepidermal water loss) was measured. RESULTS: Physiological, microbiome, and clinical results were not robustly related to the pH of applied emollient. In contrast to longitudinally stable microbiome in HE, S aureus frequency significantly increased in AD over 8 weeks. High S aureus abundance was associated with skin pH 5.7-6.2. High baseline S aureus frequency predicted both increase in S aureus and in AD severity (EASI and local SCORAD) after 8 weeks. CONCLUSION: Skin pH is tightly regulated by intrinsic factors and limits the abundance of S aureus. High baseline S aureus abundance in turn predicts an increase in AD severity over the study period. This underlines the importance and potential of sustained intervention regarding the skin pH and urges for larger studies linking skin pH and skin S aureus abundance to understand driving factors of disease progression.

Distribution of ACE2, CD147, CD26, and other SARS-CoV-2 associated molecules in tissues and immune cells in health and in asthma, COPD, obesity, hypertension, and COVID-19 risk factors.

BACKGROUND: Morbidity and mortality from COVID-19 caused by novel coronavirus SARS-CoV-2 is accelerating worldwide, and novel clinical presentations of COVID-19 are often reported. The range of human cells and tissues targeted by SARS-CoV-2, its potential receptors and associated regulating factors are still largely unknown. The aim of our study was to analyze the expression of known and potential SARS-CoV-2 receptors and related molecules in the extensive collection of primary human cells and tissues from healthy subjects of different age and from patients with risk factors and known comorbidities of COVID-19. METHODS: We performed RNA sequencing and explored available RNA-Seq databases to study gene expression and co-expression of ACE2, CD147 (BSG), and CD26 (DPP4) and their direct and indirect molecular partners in primary human bronchial epithelial cells, bronchial and skin biopsies, bronchoalveolar lavage fluid, whole blood, peripheral blood mononuclear cells (PBMCs), monocytes, neutrophils, DCs, NK cells, ILC1, ILC2, ILC3, CD4+ and CD8+ T cells, B cells, and plasmablasts. We analyzed the material from healthy children and adults, and from adults in relation to their disease or COVID-19 risk factor status. RESULTS: ACE2 and TMPRSS2 were coexpressed at the epithelial sites of the lung and skin, whereas CD147 (BSG), cyclophilins (PPIA andPPIB), CD26 (DPP4), and related molecules were expressed in both epithelium and in immune cells. We also observed a distinct age-related expression profile of these genes in the PBMCs and T cells from healthy children and adults. Asthma, COPD, hypertension, smoking, obesity, and male gender status generally led to the higher expression of ACE2- and CD147-related genes in the bronchial biopsy, BAL, or blood. Additionally, CD147-related genes correlated positively with age and BMI. Interestingly, we also observed higher expression of CD147-related genes in the lesional skin of patients with atopic dermatitis. CONCLUSIONS: Our data suggest different receptor repertoire potentially involved in the SARS-CoV-2 infection at the epithelial barriers and in the immune cells. Altered expression of these receptors related to age, gender, obesity and smoking, as well as with the disease status, might contribute to COVID-19 morbidity and severity patterns.

Pollen exposure weakens innate defense against respiratory viruses.

BACKGROUND: Hundreds of plant species release their pollen into the air every year during early spring. During that period, pollen allergic as well as non-allergic patients frequently present to doctors with severe respiratory tract infections. Our objective was therefore to assess whether pollen may interfere with antiviral immunity. METHODS: We combined data from real-life human exposure cohorts, a mouse model and human cell culture to test our hypothesis. RESULTS: Pollen significantly diminished interferon-λ and pro-inflammatory chemokine responses of airway epithelia to rhinovirus and viral mimics and decreased nuclear translocation of interferon regulatory factors. In mice infected with respiratory syncytial virus, co-exposure to pollen caused attenuated antiviral gene expression and increased pulmonary viral titers. In non-allergic human volunteers, nasal symptoms were positively correlated with airborne birch pollen abundance, and nasal birch pollen challenge led to downregulation of type I and -III interferons in nasal mucosa. In a large patient cohort, numbers of rhinoviruspositive cases were correlated with airborne birch pollen concentrations. CONCLUSION: The ability of pollen to suppress innate antiviral immunity, independent of allergy, suggests that high-risk population groups should avoid extensive outdoor activities when pollen and respiratory virus seasons coincide.

Baseline IL-22 expression in patients with atopic dermatitis stratifies tissue responses to fezakinumab.

BACKGROUND: IL-22 is potentially a pathogenic cytokine in patients with atopic dermatitis (AD), but the molecular effects of IL-22 antagonism have not been defined in human subjects. OBJECTIVE: We sought to evaluate the cellular and molecular effects of IL-22 blockade in tissues from patients with moderate-to-severe AD. METHODS: We assessed lesional and nonlesional skin from 59 patients with moderate-to-severe AD treated with anti-IL-22 (fezakinumab) versus placebo (2:1) using transcriptomic and immunohistochemistry analyses. RESULTS: Greater reversal of the AD genomic profile was seen with fezakinumab versus placebo, namely 25.3% versus 10.5% at 4 weeks (P = 1.7 × 10-5) and 65.5% versus 13.9% at 12 weeks (P = 9.5 × 10-19), respectively. Because IL-22 blockade showed clinical efficacy only in patients with severe AD, we used baseline median IL-22 mRNA expression to stratify for high (n = 30) and low (n = 29) IL-22 expression groups. Much stronger mean transcriptomic improvements were seen with fezakinumab in the IL-22-high drug-treated group (82.8% and 139.4% at 4 and 12 weeks, respectively) than in the respective IL-22-high placebo-treated group (39.6% and 56.3% at 4 and 12 weeks) or the IL-22-low groups. Significant downregulations of multiple immune pathways, including TH1/CXCL9, TH2/CCL18/CCL22, TH17/CCL20/DEFB4A, and TH22/IL22/S100A's, were restricted to the IL-22-high drug group (P < .05). Consistently, tissue predictors of clinical response were mostly genes involved in T-cell and dendritic cell activation and differentiation. CONCLUSIONS: This is the first report showing a profound effect of IL-22 blockade on multiple inflammatory pathways in AD. These data, supported by robust effects in patients with high IL-22 baseline expression, suggest a central role for IL-22 in AD, indicating the need for a precision medicine approach for improving therapeutic outcomes in patients with AD.

Correction: Novel decay dynamics revealed for virus-mediated drug activation in cytomegalovirus infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006299.].

Novel decay dynamics revealed for virus-mediated drug activation in cytomegalovirus infection.

Human cytomegalovirus (CMV) infection is a substantial cause of morbidity and mortality in immunocompromised hosts and globally is one of the most important congenital infections. The nucleoside analogue ganciclovir (GCV), which requires initial phosphorylation by the viral UL97 kinase, is the mainstay for treatment. To date, CMV decay kinetics during GCV therapy have not been extensively investigated and its clinical implications not fully appreciated. We measured CMV DNA levels in the blood of 92 solid organ transplant recipients with CMV disease over the initial 21 days of ganciclovir therapy and identified four distinct decay patterns, including a new pattern exhibiting a transient viral rebound (Hump) following initial decline. Since current viral dynamics models were unable to account for this Hump profile, we developed a novel multi-level model, which includes the intracellular role of UL97 in the continued activation of ganciclovir, that successfully described all the decline patterns observed. Fitting the data allowed us to estimate ganciclovir effectiveness in vivo (mean 92%), infected cell half-life (mean 0.7 days), and other viral dynamics parameters that determine which of the four kinetic patterns will ensue. An important clinical implication of our results is that the virological efficacy of GCV operates over a broad dose range. The model also raises the possibility that GCV can drive replication to a new lower steady state but ultimately cannot fully eradicate it. This model is likely to be generalizable to other anti-CMV nucleoside analogs that require activation by viral enzymes such as UL97 or its homologues.

Differential T cell response against BK virus regulatory and structural antigens: A viral dynamics modelling approach.

BK virus (BKV) associated nephropathy affects 1-10% of kidney transplant recipients, leading to graft failure in about 50% of cases. Immune responses against different BKV antigens have been shown to have a prognostic value for disease development. Data currently suggest that the structural antigens and regulatory antigens of BKV might each trigger a different mode of action of the immune response. To study the influence of different modes of action of the cellular immune response on BKV clearance dynamics, we have analysed the kinetics of BKV plasma load and anti-BKV T cell response (Elispot) in six patients with BKV associated nephropathy using ODE modelling. The results show that only a small number of hypotheses on the mode of action are compatible with the empirical data. The hypothesis with the highest empirical support is that structural antigens trigger blocking of virus production from infected cells, whereas regulatory antigens trigger an acceleration of death of infected cells. These differential modes of action could be important for our understanding of BKV resolution, as according to the hypothesis, only regulatory antigens would trigger a fast and continuous clearance of the viral load. Other hypotheses showed a lower degree of empirical support, but could potentially explain the clearing mechanisms of individual patients. Our results highlight the heterogeneity of the dynamics, including the delay between immune response against structural versus regulatory antigens, and its relevance for BKV clearance. Our modelling approach is the first that studies the process of BKV clearance by bringing together viral and immune kinetics and can provide a framework for personalised hypotheses generation on the interrelations between cellular immunity and viral dynamics.

Efficacy and safety of fezakinumab (an IL-22 monoclonal antibody) in adults with moderate-to-severe atopic dermatitis inadequately controlled by conventional treatments: A randomized, double-blind, phase 2a trial.

BACKGROUND: Interleukin 22 promotes epidermal hyperplasia and inhibits skin barrier function. OBJECTIVE: Evaluate interleukin 22 blockade in adults with moderate-to-severe atopic dermatitis (AD). METHODS: We performed a randomized, double-blind, placebo-controlled trial with intravenous fezakinumab monotherapy every 2 weeks for 10 weeks, with follow-up assessments until 20 weeks. The change in SCOring AD (SCORAD) score from baseline at 12 weeks served as the primary end point. RESULTS: At 12 weeks, the mean declines in SCORAD for the entire study population were 13.8 ± 2.7 in the fezakinumab arm and 8.0 ± 3.1 in the placebo arm (P = .134). In the severe AD patient subset (with a baseline SCORAD of ≥50), SCORAD decline was significantly stronger in the drug-treated patients than placebo-treated patients at 12 weeks (21.6 ± 3.8 vs 9.6 ± 4.2, P = .029) and 20 weeks (27.4 ± 3.9 vs 11.5 ± 5.1, P = .010). At 12 weeks, improvements in body surface area involvement in the entire population were significantly stronger in the drug-treated than placebo-treated patients (12.4% ± 2.4 vs 6.2% ± 2.7; P = .009), and in the severe AD subset, the decline in Investigator Global Assessment was significantly higher in the drug-treated than placebo-treated patients (0.7 ± 0.2 vs 0.3 ± 0.1; P = .034). All scores showed progressive improvements after last dosing (10 weeks) until end of study (20 weeks). Common adverse events were upper respiratory tract infections. LIMITATIONS: The limited sample size and lack of assessment with Eczema Area and Severity Index and a pruritus numerical rating scale were limiting factors. Significance was primarily obtained in severe AD. CONCLUSION: Fezakinumab was well-tolerated, with sustained clinical improvements after last drug dosing.

IMSEQ--a fast and error aware approach to immunogenetic sequence analysis.

MOTIVATION: Recombined T- and B-cell receptor repertoires are increasingly being studied using next generation sequencing (NGS) in order to interrogate the repertoire composition as well as changes in the distribution of receptor clones under different physiological and disease states. This type of analysis requires efficient and unambiguous clonotype assignment to a large number of NGS read sequences, including the identification of the incorporated V and J gene segments and the CDR3 sequence. Current tools have deficits with respect to performance, accuracy and documentation of their underlying algorithms and usage. RESULTS: We present IMSEQ, a method to derive clonotype repertoires from NGS data with sophisticated routines for handling errors stemming from PCR and sequencing artefacts. The application can handle different kinds of input data originating from single- or paired-end sequencing in different configurations and is generic regarding the species and gene of interest. We have carefully evaluated our method with simulated and real world data and show that IMSEQ is superior to other tools with respect to its clonotyping as well as standalone error correction and runtime performance. AVAILABILITY AND IMPLEMENTATION: IMSEQ was implemented in C++ using the SeqAn library for efficient sequence analysis. It is freely available under the GPLv2 open source license and can be downloaded at www.imtools.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: lkuchenb@inf.fu-berlin.de or peter.robinson@charite.de.

A revised strategy for monitoring BKV-specific cellular immunity in kidney transplant patients.

Reactivation of Polyomavirus BKV is a severe complication in kidney transplant patients. Current treatment requires close monitoring, and modification of immunosuppressive drugs. As an important additional tool, the monitoring of BKV immunity has been based on detection of cytokine-secreting T cells upon BKV-antigen challenge. However, low frequent BKV-specific T cells are often barely detectable and their roles in BKV clearance remain unclear. Here, we analyzed the effects of immunosuppressive agents on BKV-specific T cells in vitro. Significant reductions in expression of several markers, and reduced killing functions upon treatment with calcineurin but not mTOR inhibitors were detected. However, effects of these drugs on expression of surface markers and GranzymeB were substantially less striking than effects on cytokine expression. Consequently, we applied a novel detection strategy for BKV-specific T cells in immunosuppressed kidney transplant patients using these more robust markers, and showed significantly improved sensitivity compared with the conventional IFNγ-based method. Using this strategy and 17-color flow cytometry, we found BKV-specific helper and cytolytic CD4+ T-cell subsets that differed in their memory phenotype, which corresponded with BKV clearance in kidney transplant patients. Thus, our results offer an improved detection strategy for BKV-specific T cells in kidney transplant patients, and shed light on the contributions of these cells to BKV clearance.

Contrast-enhanced spectral mammography: Does mammography provide additional clinical benefits or can some radiation exposure be avoided?

The purpose of this study was to compare contrast-enhanced spectral mammography (CESM) with mammography (MG) and combined CESM + MG in terms of detection and size estimation of histologically proven breast cancers in order to assess the potential to reduce radiation exposure. A total of 118 patients underwent MG and CESM and had final histological results. CESM was performed as a bilateral examination starting 2 min after injection of iodinated contrast medium. Three independent blinded radiologists read the CESM, MG, and CESM + MG images with an interval of at least 4 weeks to avoid case memorization. Sensitivity and size measurement correlation and differences were calculated, average glandular dose (AGD) levels were compared, and breast densities were reported. Fisher's exact and Wilcoxon tests were performed. A total of 107 imaging pairs were available for analysis. Densities were ACR1: 2, ACR2: 45, ACR3: 42, and ACR4: 18. Mean AGD was 1.89 mGy for CESM alone, 1.78 mGy for MG, and 3.67 mGy for the combination. In very dense breasts, AGD of CESM was significantly lower than MG. Sensitivity across readers was 77.9 % for MG alone, 94.7 % for CESM, and 95 % for CESM + MG. Average tumor size measurement error compared to postsurgical pathology was -0.6 mm for MG, +0.6 mm for CESM, and +4.5 mm for CESM + MG (p < 0.001 for CESM + MG vs. both modalities). CESM alone has the same sensitivity and better size assessment as CESM + MG and was significantly better than MG with only 6.2 % increase in AGD. The combination of CESM + MG led to systematic size overestimation. When a CESM examination is planned, additional MG can be avoided, with the possibility of saving up to 61 % of radiation dose, especially in patients with dense breasts.