BACKGROUND:Epithelial barrier dysfunction is a pivotal feature of asthma, and it also commonly occurs in other inflammatory conditions such as atopic dermatitis (AD) and ulcerative colitis (UC). However, the core regulatory mechanisms underlying epithelial barrier dysfunction-especially whether shared mechanisms exist across these diseases-remain unclear. METHODS:Gene expression profiles of patients with asthma, AD, and UC were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the limma package. Common DEGs among the three diseases were identified via a Venn diagram, followed by correlation analysis with junction molecules to screen for key genes. Interaction networks (protein-protein, transcription factor-gene, miRNA-gene, chemical-gene) of the key genes were constructed, and their correlations with asthma clinical features [Asthma Control Questionnaire (ACQ) score, Inhaled Corticosteroid (ICS) dose, lung function parameters] were analyzed. The effects of the key genes on epithelial barrier function were assessed in airway, epidermal, and intestinal epithelial cells. RESULTS:A total of eight common DEGs exhibited consistent upregulation in the epithelial tissues of patients with asthma, AD, and UC. Four key genes-CDC7, PXDN, TCN1, and TIMP1-were identified; they were upregulated in the epithelium of all three diseases, significantly elevated in IL-13-stimulated airway epithelial cells, and negatively correlated with junction molecules that were downregulated in the three diseases. Furthermore, the expression of these key genes was associated with the severity of asthma. Correlation analysis between key gene expression and asthma clinical features revealed that PXDN expression was significantly negatively correlated with the lung function parameter FEV1 (forced expiratory volume in 1 s), while TCN1 expression showed a significant negative correlation with FEV1/FVC (forced expiratory volume in 1 s/forced vital capacity). Chemical-gene interaction analysis revealed that benzo[a]pyrene could induce the expression of these four key genes. Subsequent experiments confirmed that stimulation of airway epithelial cells with benzo[a]pyrene significantly upregulated the expression of these key genes. Finally, targeting these key molecules was found to alleviate the IL-13-induced reduction in CLDN1 expression in airway epithelial cells. In in vitro collagen-coated transwell assays, knocking down either CDC7 or TCN1 significantly attenuated IL-13-induced epithelial barrier disruption in airway, epidermal, and intestinal epithelia. CONCLUSION:Our findings confirm that targeting CDC7 and TCN1 facilitates the improvement of barrier function in airway epithelial cells, epidermal keratinocytes, and intestinal epithelial cells in vitro, thereby providing promising therapeutic targets for diseases characterized by epithelial barrier dysfunction.

Early-life environmental exposures are critical determinants of the onset of childhood asthma, with risk patterns demonstrating significant heterogeneity across geographical and socioeconomic landscapes. While the “hygiene hypothesis” has long framed the rural-urban disparity—where urban children typically show higher asthma prevalence than their rural counterparts—this dichotomy is being reshaped by rapid globalization and urbanization. Furthermore, this pattern differs markedly between high-income countries (HICs) and low- and middle-income countries (LMICs). In HICs, the environmental distinction is sharp, with urban environments often characterized by a scarcity of protective microbial factors coupled with an accumulation of anthropogenic pollutants. In contrast, within developing economies undergoing rapid transition, the boundaries between rural and urban areas are increasingly blurred, and environmental exposures often involve a complex interaction among protective and risk factors. These divergent pathways are further complicated by socioeconomic inequities. This review summarizes the global burden of asthma, compares the multi-dimensional determinants across rural and urban environments in HICs (the West) and LMICs (the East), and constructs a refined risk map. It also seeks to identify key characteristics with potential for public health intervention, thereby providing a scientific basis for developing targeted asthma prevention strategies tailored to varying socioeconomic contexts.

Reducing the allergenicity of milk-derived proteins is essential for the development of hypoallergenic dairy products. This study was designed to test the hypothesis that myricetin attenuates the allergenicity of whey protein by masking immunoreactive epitopes and modulating immune responses In this study, the effects of myricetin on whey protein allergenicity were evaluated using in vitro and in vivo models. Twenty-seven dietary polyphenols were screened for their ability to inhibit β-lactoglobulin antigenicity using an indirect enzyme-linked immunosorbent assay, and myricetin was identified as the most effective compound. Molecular docking analysis and free thiol group determination suggested that myricetin preferentially interacted with the free thiol group of cysteine 121, suggesting epitope masking as a potential mechanism. In a BALB/c mouse model of whey protein-induced allergy, dietary administration of whey protein combined with myricetin reduced serum mast cell protease-1, antigen-specific immunoglobulin G, and type 2 helper T-cell-associated cytokines, compared with whey protein alone. In addition, myricetin supplementation altered the composition of the intestinal microbiota. These findings support our hypothesis that myricetin can reduce the allergenicity of whey protein, likely through epitope masking and immune-modulatory effects involving the gut microbiota, and highlight its potential application as a natural strategy for producing dairy products with reduced allergenic potential.

Fruit allergy is a significant and increasingly recognized global health concern: once reported as a problem in Europe, it is now relevant in many countries, especially China, as consumption in both variety and quantity has increased dramatically. It is often associated with pollen allergy, with clinical manifestations ranging from localized oral allergy syndrome (OAS) to severe systemic reactions such as anaphylaxis. The majority of fruit allergens belong to a limited number of protein families each exhibiting distinct biochemical properties and clinical relevance. Allergy diagnosis relies on a detailed clinical history supported by in vivo and/or in vitro measurements. Component-resolved diagnosis (CRD) with purified allergen molecules provides insight into the primary sensitizing molecules and a more precise and standardized identification of sensitization patterns than possible with extract-based tests. Allergy management focuses on strict avoidance of trigger fruits, while prevention involves breeding hypoallergenic cultivars with lower allergen content and food processing technologies to reduce allergenicity. Allergen immunotherapy (AIT) has been explored as a treatment for fruit allergy with indications of improved tolerance. Emerging approaches combining AIT with biologic agents may further enhance treatment outcomes, although these novel strategies require additional research.

Background: chronic obstructive pulmonary disease (COPD) is a common respiratory disease caused by genetic and environmental factors, but the contribution of urine volatile organic compounds (VOCs) to the risk of COPD remains unclear. This study aims to use the NHANES data to explore the potential value of urine VOCs in predicting COPD. Methods: an epidemiological study including 782 participants from the National Health and Nutrition Examination Survey (NHANES) 2013-2018 was performed to evaluate the association between urine VOCs and COPD. Receiver operating characteristic (ROC) and the area under the ROC curve (AUC) analysis were used to evaluate the diagnostic performance of urine VOCs on COPD. Results: seven urine VOCs were associated with an increased risk of COPD [odds ratio (OR>1; p < 0.05)]. The dose-response relationship was also statistically significant between them. Meanwhile, urine VOCs can lead to the occurrence of COPD through the inflammatory effects. The area under the ROC curves for the combined urine VOC models as a predictor for COPD was 0.90. Conclusions: association between urine VOCs and an increased risk of COPD was found in the NHANES data. Inflammatory factors play an important role in the association of urine VOCs and COPD. In addition, urine VOCs could be useful in predicting COPD risk. More studies are needed to elucidate the mechanisms and clinical application values underlying the association between urine VOCs and COPD.

BACKGROUND AND OBJECTIVE:Myeloid-derived suppressor cells (MDSCs) participate in the progression of many diseases including chronic lung diseases. However, whether MDSCs are accumulated in the lung and how MDSCs orchestrate the pulmonary microenvironment in bronchiectasis remains unknown. Here, we aim to test a hypothesis that PMN-MDSCs are accumulated in the lung and play a role in creating an airway immunosuppressive milieu, thereby relating to clinical outcomes in bronchiectasis. METHODS:Flow cytometry and immunofluorescence staining were performed for analysing the frequencies and presence of PMN-MDSCs, LOX-1+ neutrophils, and ARG-1+ PMN-MDSCs in PBMCs, sputum, and lung tissues. T-cell proliferation assays were established for evaluating the immunosuppressive activities of PMN-MDSCs. RNA sequencing was performed to investigate the underlying mechanism of PMN-MDSCs-mediated immunosuppression. The relationship of PMN-MDSCs with the time to next exacerbation and treatment response to antibiotic therapy was analysed. RESULTS:PMN-MDSCs are accumulated in the lung and blood in bronchiectasis patients compared to healthy individuals. The majority of neutrophils in the lung of bronchiectasis patients are LOX-1+ PMN-MDSCs. Mechanistically, PMN-MDSCs suppress T cell proliferation via secreting high levels of the enzyme arginase-1 (ARG-1). Notably, the frequencies of PMN-MDSCs in sputum negatively correlate with the time to next exacerbation in bronchiectasis patients. Additionally, antibiotic therapy dramatically decreases PMN-MDSCs frequencies in the airway of bronchiectasis patients. CONCLUSION:These findings suggest that PMN-MDSCs accumulate and establish an immunosuppressive microenvironment in the lung via ARG-1 in bronchiectasis, which is associated with clinical outcome and response to antibiotic treatment, highlighting a potential role of PMN-MDSCs in bronchiectasis progression.

BACKGROUND:Growing up on traditional farms protects children from the development of asthma and allergies. However, we have identified distinct asthma-protective factors, such as poultry exposure. This study aims to examine the biological effect of rural exposure in China. METHODS:We recruited 67 rural children (7.4 ± 0.9 years) and 79 urban children (6.8 ± 0.6 years). Depending on the personal history of exposure to domestic poultry (DP), rural children were further divided into those with DP exposure (DP+ , n = 30) and those without (DP- , n = 37). Blood samples were collected to assess differential cell counts and expression of immune-related genes. Dust samples were collected from poultry stables inside rural households. In vivo activities of nasal administration of DP dust extracts were tested in an ovalbumin-induced asthma model. RESULTS:There was a stepwise increase in the percentage of eosinophils (%) from rural DP+ children (median = 1.65, IQR = [1.28, 3.75]) to rural DP- children (3.40, [1.70, 6.50]; DP+ vs. DP- , p = .087) and to the highest of their urban counterparts (4.00, [2.00, 7.25]; urban vs. DP+ , p = .017). Similarly, rural children exhibited reduced mRNA expression of immune markers, both at baseline and following lipopolysaccharide (LPS) stimulation. Whereas LPS stimulation induced increased secretion of Th1 and proinflammatory cytokines in rural DP+ children compared to rural DP- children and urban children. Bronchoalveolar lavage of mice with intranasal instillation of dust extracts from DP household showed a significant decrease in eosinophils as compared to those of control mice (p < .05). Furthermore, DP dust strongly inhibited gene expression of Th2 signature cytokines and induced IL-17 expression in the murine asthma model. CONCLUSIONS:Immune responses of rural children were dampened compared to urban children and those exposed to DP had further downregulated immune responsiveness. DP dust extracts ameliorated Th2-driven allergic airway inflammation in mice. Determining active protective components in the rural environment may provide directions for the development of primary prevention of asthma.

Cough is one of the most common symptoms observed in patients presenting with COVID-19, persisting for an extended duration following SARS-CoV-2 infection. We aim to describe the distribution of airway microbiota and explore its role in patients with post-COVID-19 chronic cough. A total of 57 patients experiencing persistent cough after infection were recruited during the Omicron wave of SARS-CoV-2 in China. Airway microbiota profiling is assessed in nasopharyngeal swab, nasal lavage, and induced sputum samples at 4 and 8 weeks after SARS-CoV-2 infection. Our findings reveal that bacterial families Staphylococcaceae, Corynebacteriaceae, and Enterobacteriaceae are the most prevalent in the upper airway, while Streptococcaceae, Lachnospiraceae, and Prevotellaceae emerge as the most prevalent bacterial families in the lower airway. An increase in the abundance of Staphylococcus in nasopharyngeal swab samples and of Streptococcus in induced sputum samples is observed after one month. Furthermore, the abundance of Staphylococcus identified in nasopharyngeal swab samples at the baseline period emerges as an insightful predictor for improvement in cough severity. In conclusion, dynamic alterations in the airway microbial composition may contribute to the post-COVID-19 chronic cough progression, while the compositional signatures of nasopharyngeal microbiota could reflect the improvement of this disease.

The gut-lung axis, pivotal for respiratory health, is inadequately explored in pulmonary and critical care medicine (PCCM) inpatients. Examining PCCM inpatients from three medical university-affiliated hospitals, we conducted 16S ribosomal RNA sequencing on stool samples (inpatients, n = 374; healthy controls, n = 105). We conducted statistical analyses to examine the gut microbiota composition in PCCM inpatients, comparing it to that of healthy controls. Additionally, we explored the associations between gut microbiota composition and various clinical factors, including age, white blood cell count, neutrophil count, platelet count, albumin level, hemoglobin level, length of hospital stay, and medical costs. PCCM inpatients exhibited lower gut microbiota diversity than healthy controls. Principal Coordinates Analysis revealed marked overall microbiota structure differences. Four enterotypes, including the exclusive Enterococcaceae enterotype in inpatients, were identified. Although no distinctions were found at the phylum level, 15 bacterial families exhibited varying abundances. Specifically, the inpatient population from PCCM showed a significantly higher abundance of Enterococcaceae, Lactobacillaceae, Erysipelatoclostridiaceae, Clostridiaceae, and Tannerellaceae. Using random forest analyses, we calculated the areas under the receiver operating characteristic curves (AUCs) to be 0.75 (95

Epithelial cells play a crucial role in asthma, contributing to chronic inflammation and airway hyperresponsiveness. m6A modification, which involves key proteins such as the demethylase fat mass and obesity-associated protein (FTO), is crucial in the regulation of various diseases, including asthma. However, the role of FTO in epithelial cells and the development of asthma remains unclear. In this study, we investigated the demethylase activity of FTO using a small-molecule inhibitor FB23 in epithelial cells and allergic inflammation in vivo and in vitro. We examined the FTO-regulated transcriptome-wide m6A profiling by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq under FB23 treatment and allergic inflammation conditions. Immunofluorescence staining was performed to assess the tissue-specific expression of FTO in asthmatic bronchial mucosa. We demonstrated that FB23 alleviated allergic inflammation in IL-4/IL-13-treated epithelial cells and house dust mite (HDM)-induced allergic airway inflammation mouse model. The demethylase activity of FTO contributed to the regulation of TNF-α signaling via NF-κB and epithelial-mesenchymal transition-related pathways under allergic inflammation conditions in epithelial cells. FTO was expressed in epithelial, submucosal gland, and smooth muscle cells in human bronchial mucosa. In conclusion, FB23-induced inhibition of FTO alleviates allergic inflammation in epithelial cells and HDM-induced mice, potentially through diverse cellular processes and epithelial-mesenchymal transition signaling pathways, suggesting that FTO is a potential therapeutic target in asthma management.

BACKGROUND:Copy number variation (CNV) is a key genetic characteristic for cancer diagnostics and can be used as a biomarker for the selection of therapeutic treatments. Using data sets established in our previous study, we benchmark the performance of cancer CNV calling by six most recent and commonly used software tools on their detection accuracy, sensitivity, and reproducibility. In comparison to other orthogonal methods, such as microarray and Bionano, we also explore the consistency of CNV calling across different technologies on a challenging genome. RESULTS:While consistent results are observed for copy gain, loss, and loss of heterozygosity (LOH) calls across sequencing centers, CNV callers, and different technologies, variation of CNV calls are mostly affected by the determination of genome ploidy. Using consensus results from six CNV callers and confirmation from three orthogonal methods, we establish a high confident CNV call set for the reference cancer cell line (HCC1395). CONCLUSIONS:NGS technologies and current bioinformatics tools can offer reliable results for detection of copy gain, loss, and LOH. However, when working with a hyper-diploid genome, some software tools can call excessive copy gain or loss due to inaccurate assessment of genome ploidy. With performance matrices on various experimental conditions, this study raises awareness within the cancer research community for the selection of sequencing platforms, sample preparation, sequencing coverage, and the choice of CNV detection tools.

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Abnormal immunological indicators associated with disease severity and mortality in patients with COVID-19 have been reported in several observational studies. However, there are marked heterogeneities in patient characteristics and research methodologies in these studies. We aimed to provide an updated synthesis of the association between immune-related indicators and COVID-19 prognosis. We conducted an electronic search of PubMed, Scopus, Ovid, Willey, Web of Science, Cochrane library, and CNKI for studies reporting immunological and/or immune-related parameters, including hematological, inflammatory, coagulation, and biochemical variables, tested on hospital admission of COVID-19 patients with different severities and outcomes. A total of 145 studies were included in the current meta-analysis, with 26 immunological, 11 hematological, 5 inflammatory, 4 coagulation, and 10 biochemical variables reported. Of them, levels of cytokines, including IL-1β, IL-1Ra, IL-2R, IL-4, IL-6, IL-8, IL-10, IL-18, TNF-α, IFN-γ, IgA, IgG, and CD4+ T/CD8+ T cell ratio, WBC, neutrophil, platelet, ESR, CRP, ferritin, SAA, D-dimer, FIB, and LDH were significantly increased in severely ill patients or non-survivors. Moreover, non-severely ill patients or survivors presented significantly higher counts of lymphocytes, monocytes, lymphocyte/monocyte ratio, eosinophils, CD3+ T,CD4+T and CD8+T cells, B cells, and NK cells. The currently updated meta-analysis primarily identified a hypercytokinemia profile with the severity and mortality of COVID-19 containing IL-1β, IL-1Ra, IL-2R, IL-4, IL-6, IL-8, IL-10, IL-18, TNF-α, and IFN-γ. Impaired innate and adaptive immune responses, reflected by decreased eosinophils, lymphocytes, monocytes, B cells, NK cells, T cells, and their subtype CD4+ and CD8+ T cells, and augmented inflammation, coagulation dysfunction, and nonpulmonary organ injury, were marked features of patients with poor prognosis. Therefore, parameters of immune response dysfunction combined with inflammatory, coagulated, or nonpulmonary organ injury indicators may be more sensitive to predict severe patients and those non-survivors.

BACKGROUND:Studies in comparison with allergic diseases and sensitization between rural and urban environments in westernized countries might be biased and not adequately reflect countries undergoing rapid transition.METHODS:A total of 5542 schoolchildren from urban area and 5139 from rural area were recruited for the EuroPrevall-INCO survey. A subsequent case-control sample with 196 children from urban area and 202 from rural area was recruited for a detailed face-to-face questionnaire and assessment of sensitization. Skin prick tests and serum-specific IgE measurements were used to assess sensitizations against food and aeroallergens. Logistic regression analysis was used to determine associations between risk/protective factors, food adverse reactions (FAR), allergic diseases, and sensitizations.RESULTS:Prevalence of self-reported allergic diseases, including asthma (6.6% vs.2.5%), rhinitis (23.2% vs.5.3%), and eczema (34.1% vs.25.9%), was higher in urban than in rural children. Urban children had a significantly higher prevalence of FAR and related allergic diseases, and lower food/inhalation allergen sensitization rate, than those of rural children. In urban children, frequent changing places of residency (odds ratio 2.85, 95% confidence interval: 1.45-5.81) and antibiotic usage (3.54, 1.77-7.32) in early life were risk factors for sensitization, while sensitization and family history of allergy were risk factors for allergic diseases. In rural children, exposure to rural environments in early life was protective against both allergen sensitizations (0.46, 0.21-0.96) and allergic diseases (0.03, 0.002-0.19).CONCLUSION:We observed a disparity in rates of allergic diseases and allergen sensitization between rural and urban children. In addition to family history, the development of allergic diseases and allergen sensitization were associated with specific urban/rural environmental exposures in early life.

There has been an important change in the clinical characteristics and immune profile of Coronavirus disease 2019 (COVID-19) patients during the pandemic thanks to the extensive vaccination programs. Here, we highlight recent studies on COVID-19, from the clinical and immunological characteristics to the protective and risk factors for severity and mortality of COVID-19. The efficacy of the COVID-19 vaccines and potential allergic reactions after administration are also discussed. The occurrence of new variants of concerns such as Omicron BA.2, BA.4, and BA.5 and the global administration of COVID-19 vaccines have changed the clinical scenario of COVID-19. Multisystem inflammatory syndrome in children (MIS-C) may cause severe and heterogeneous disease but with a lower mortality rate. Perturbations in immunity of T cells, B cells, and mast cells, as well as autoantibodies and metabolic reprogramming may contribute to the long-term symptoms of COVID-19. There is conflicting evidence about whether atopic diseases, such as allergic asthma and rhinitis, are associated with a lower susceptibility and better outcomes of COVID-19. At the beginning of pandemic, the European Academy of Allergy and Clinical Immunology (EAACI) developed guidelines that provided timely information for the management of allergic diseases and preventive measures to reduce transmission in the allergic clinics. The global distribution of COVID-19 vaccines and emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with reduced pathogenic potential dramatically decreased the morbidity, severity, and mortality of COVID-19. Nevertheless, breakthrough infection remains a challenge for disease control. Hypersensitivity reactions (HSR) to COVID-19 vaccines are low compared to other vaccines, and these were addressed in EAACI statements that provided indications for the management of allergic reactions, including anaphylaxis to COVID-19 vaccines. We have gained a depth knowledge and experience in the over 2 years since the start of the pandemic, and yet a full eradication of SARS-CoV-2 is not on the horizon. Novel strategies are warranted to prevent severe disease in high-risk groups, the development of MIS-C and long COVID-19.

Rural environments and microbiota are linked to a reduction in the prevalence of allergies. However, the mechanism underlying the reduced allergies modulated by rural residency is unclear. Here, we assessed gut bacterial composition and metagenomics in urban and rural children in the EuroPrevall-INCO cohort. Airborne dusts, including mattress and rural henhouse dusts, were profiled for bacterial and fungal composition by amplicon sequencing. Mice were repeatedly exposed to intranasal dust extracts and evaluated for their effects on ovalbumin (OVA)-induced allergic airway inflammation, and gut microbiota restoration was validated by fecal microbiota transplant (FMT) from dust-exposed donor mice. We found that rural children had fewer allergies and unique gut microbiota with fewer Bacteroides and more Prevotella. Indoor dusts in rural environments harbored higher endotoxin level and diversity of bacteria and fungi, whereas indoor urban dusts were enriched with Aspergillus and contained elevated pathogenic bacteria. Intranasal administration of rural dusts before OVA sensitization reduced respiratory eosinophils and blood IgE level in mice and also led to a recovery of gut bacterial diversity and Ruminiclostridium in the mouse model. FMT restored the protective effect by reducing OVA-induced lung eosinophils in recipient mice. Together, these results support a cause-effect relationship between exposure to dust microbiota and allergy susceptibility in children and mice. Specifically, rural environmental exposure modulated the gut microbiota, which was essential in reducing allergy in children from Southern China. Our findings support the notion that the modulation of gut microbiota by exposure to rural indoor dust may improve allergy prevention.

Background The cancer genome is commonly altered with thousands of structural rearrangements including insertions, deletions, translocation, inversions, duplications, and copy number variations. Thus, structural variant (SV) characterization plays a paramount role in cancer target identification, oncology diagnostics, and personalized medicine. As part of the SEQC2 Consortium effort, the present study established and evaluated a consensus SV call set using a breast cancer reference cell line and matched normal control derived from the same donor, which were used in our companion benchmarking studies as reference samples. Results We systematically investigated somatic SVs in the reference cancer cell line by comparing to a matched normal cell line using multiple NGS platforms including Illumina short-read, 10X Genomics linked reads, PacBio long reads, Oxford Nanopore long reads, and high-throughput chromosome conformation capture (Hi-C). We established a consensus SV call set of a total of 1788 SVs including 717 deletions, 230 duplications, 551 insertions, 133 inversions, 146 translocations, and 11 breakends for the reference cancer cell line. To independently evaluate and cross-validate the accuracy of our consensus SV call set, we used orthogonal methods including PCR-based validation, Affymetrix arrays, Bionano optical mapping, and identification of fusion genes detected from RNA-seq. We evaluated the strengths and weaknesses of each NGS technology for SV determination, and our findings provide an actionable guide to improve cancer genome SV detection sensitivity and accuracy. Conclusions A high-confidence consensus SV call set was established for the reference cancer cell line. A large subset of the variants identified was validated by multiple orthogonal methods.

Copy number variation (CNV) is a common type of mutation that often drives cancer progression. With advances in next-generation sequencing (NGS), CNVs can be detected in a detailed manner via newly developed computational tools but quality of such CNV calls has not been carefully evaluated. We analyzed CNV calls reported by 6 cutting-edge callers for 91 samples which were derived from the same cancer cell line, prepared and sequenced by varying the following factors: type of tissue sample (Fresh vs. Formalin Fixed Paraffin Embedded (FFPE)), library DNA amount, tumor purity, sequencing platform (Whole-Genome Sequencing (WGS) versus Whole-Exome Sequencing (WES)), and sequencing coverage. We found that callers greatly determined the pattern of CNV calls. Calling quality was drastically impaired by low purity (<50%) and became variable when WES, FFPE, and medium purity (50%-75%) were applied. Effects of low DNA amount and low coverage were relatively minor. Our analysis demonstrates the limitation of benchmarking somatic CNV callers when the real ground truth is not available. Our comprehensive analysis has further characterized each caller with respect to confounding factors and examined the consistency of CNV calls, thereby providing guidelines for conducting somatic CNV analysis. ### Competing Interest Statement The authors have declared no competing interest.