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Background: Abnormal epigenetic alterations influenced by external factors and affecting DNA expression contribute to the development of asthma. However, the role of nasal epithelium in airway inflammation remains unknown. Objective: The objective of this study is to identify novel DNA promoter hypermethylation, which completely suppresses mRNA expression in nasal epithelial of asthma. Methods: Microarray datasets were downloaded from the Gene Expression Omnibus [GEO] database. Gene expression and DNA promoter methylation sites in key correlated modules between asthma and normal were identified by weighted gene co-expression network analysis [WGCNA]. Gene Ontology and KEGG were conducted to analyse the function of genes. Further validation was performed in human BEAS-2B cells challenged by IL-4 or IL-13. Results: Lightcyan, lightgreen, midnightblue, cyan and tan modules in mRNA expression dataset showed a close relationship with asthma, in which genes were enriched in TNF, IL-17, ErbB, MAPK and Estrogen signalling pathways. Blue and turquoise modules in methylation profiling dataset were associated with asthma. 49 lowly expressed genes were identified to be correlated with aberrant DNA hypermethylation of promoters. Among these genes, the mRNA levels of BCL10, GADD45B, LSR and SQSTM1 were downregulated in BEAS-2B cells challenged with IL-4 or IL-13. Conclusion: Four potential genes in nasal epithelium, by hypermethylating their own DNA promoter, might mediate the inflammatory response in the pathogenesis of asthma. Analyzing epigenomic data by integrated bioinformatics helps to understand the role of DNA methylation in asthma, with the goal of providing new perspectives for diagnosis and therapy.

Introduction: circRNA played a role in a variety of diseases. This paper aimed to explore the differentially expressed circRNA in induced sputum cells of asthma patients, so as to provide new potential biomarkers and new ideas for the study of asthma. Methods: All subjects were from the First Affiliated Hospital of Sun Yat-sen University. Differentially expressed circRNAs of asthma patients were screened by high-throughput sequencing. qRT-PCR was used to verify the expression of differential circRNAs. The association between circRNA and asthma was explored by analyzing the correlation between circRNA and clinical data and some cytokines of asthma patients. The possible ceRNA network was analyzed and predicted by online software, and the expression of each molecule in the network was preliminary verified by qRT-PCR in induced sputum cells and 16HBE cell. Results: We screened a total of 49 circRNAs differentially expressed in asthma patients (including 12 circRNAs with elevated expression and 37 circRNAs with decreased expression), among which has_circSORT1 and has_circSERPINB1 were significantly elevated. Correlation analysis showed that has_circSORT1 was correlated with FeNO, EOS%, IL-17A, IFN-γ, and PC20, and has_circSERPINB1 was correlated with IL-6, IL-17A, IFN-γ, FEV1%, and FVC%. The possible existence of has_circSORT1/has-miR-185-3p/ZNFX1, a ceRNA regulatory network, in induced sputum cells of asthma patients was hypothesized by online software prediction and qRT-PCR in sputum cells and 16HBE. Conclusion: Differentially expressed circRNAs existed in induced sputum cells of asthma patients, among which has_circSORT1 and has_circSERPINB1 were significantly upregulated and may be involved in the process of asthma disease, which could be expected to be a potential biomarker for asthma diagnosis. In addition, a ceRNA regulatory network, has_circSORT1/has-miR-185-3p/ZNFX1, may exist in asthma.

Introduction: Asthma is a common chronic respiratory disease. This study aimed to explore the expression level of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in induced sputum supernatant, induced sputum cells, and serum of asthma patients. Methods: The protein levels of CEACAM5 in induced sputum supernatant and serum were detected by enzyme-linked immunosorbent assay. The expression of CEACAM5 in induced sputum cells was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We analyzed the correlations between CEACAM5 expression and the clinical characteristics (FeNO and IgE) of asthma. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of CEACAM5 in asthma. The expression level of CEACAM5 in 16HBE and BEAS-2B cells was detected by qRT-PCR. Results: The expression of CEACAM5 in induced sputum supernatant, induced sputum cells, and serum of asthma patients was significantly upregulated. Asthma patients with high CEACAM5 expression in induced sputum supernatant had higher levels of FeNO, IgE, and IL-13. The expression levels of CEACAM5 in induced sputum supernatant and induced sputum cells were positively correlated with FeNO and IgE. The ROC curve showed that CEACAM5 had a good diagnostic value in asthma. CEACAM5 expression was upregulated in BEAS-2B and 16HBE cells after IL-4 or IL-13 stimulation for 48 h. Conclusion: The expression levels of CEACAM5 in induced sputum supernatant, induced sputum cells, and serum of asthma patients were significantly increased. CEACAM5 may be involved in eosinophilic inflammation of asthma and may be used as a diagnostic biomarker and therapeutic target of asthma.

Macrophage migration inhibitory factor (MIF) inhibition can attenuate pulmonary fibrosis, but the antifibrotic mechanism is unclear. Here we investigated the antifibrotic effect of MIF knockdown in rats with bleomycin (BLM)-induced pulmonary fibrosis. The results showed that MIF inhibition attenuated lung injury and extracellular matrix deposition; significantly reduced the levels of cytokines including transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), hydroxyproline (hyp), fibroblast growth factor 23 (FGF23), and secreted phosphoprotein 1 (Spp1); and inhibited the expression of CD68, F4/80, and α-smooth muscle actin (α-SMA) protein. MIF inhibition is associated with reduction of proinflammatory mediators and macrophage infiltration in lungs. In addition, MIF knockdown in the day 14 group was significantly better than MIF knockdown in day 1 group in terms of the above mentioned cytokines TGF-β1, IL-17, TNF-α. MIF knockdown in day 14 group showed a better trend than MIF knockdown in day 1 group in inhibition of hyp and α-SMA formation. Furthermore, MIF inhibition downregulated the FGF23, Spp1, anti-integrin alpha 10 (Itga10), laminin subunit alpha 1 (Lama1), thrombospondin 2 (THBS2), and Serpin family B member 5 (SERPINB5) mRNA levels and the p-Smad2/3 protein level. MIF knockdown may inhibit fibrosis through the TGF-β1/Smads signaling pathway. In addition, MIF inhibition protects against vascular remodeling via Thbs2 and Serpinb5 signaling. In summary, our study showed that knockdown of MIF can significantly inhibit lung inflammation and fibrosis in rats with BLM-induced pulmonary fibrosis. The future development of inhibitors targeting MIF may contribute to the treatment of pulmonary fibrosis.

By implementing dynamic circulating tumor DNA (ctDNA) analysis, we explored the impact of TP53 mutations on tumor evolution and resistance mechanisms to ensartinib in patients with ALK-positive NSCLC.In a multicenter phase 2 trial, patients with ALK-positive NSCLC who progressed on crizotinib were treated with ensartinib. Blood samples for ctDNA analysis were collected at baseline, cycle 3 day 1, and progression disease (PD) and analyzed with a 212-gene panel.A total of 440 samples were collected from 168 patients. Baseline TP53 mutations (20.2%) significantly correlated with inferior progression-free survival (4.2 mo versus 11.7 mo, p0.0001). Patients with TP53 mutations had higher mutation load than those without TP53 mutations at baseline (13.79 ± 3.72 versus 4.67 ± 0.39, p0.001). Although there was no significant difference in mutation load between these groups at cycle 3 day 1 (5.89 ± 2.25 versus 3.72 ± 0.62, p = 0.425), patients with mutated TP53 developed more mutations at PD (7.07 ± 1.25 versus 3.20 ± 0.33, p = 0.003). Frequency and abundance of secondary ALK mutations G1269A, G1202R, and E1210K increased markedly at PD than baseline. In patients without secondary ALK mutations, we identified ALK-independent resistance mechanisms including bypass signaling activation, downstream effector protein reactivation, epithelial-mesenchymal transformation, and epigenetic dysregulation.Our study highlighted the advantage of ctDNA analysis for monitoring tumor evolution. TP53 mutations promoted genetic evolution and accelerated occurrence of resistance. We also unveiled ALK-dependent resistance mechanisms, mainly by G1269A, G1202R, and E1210K mutations, and ALK-independent resistance mechanisms to ensartinib.

There were gender differences in the prevalence and severity of allergic diseases. Group 2 innate lymphoid cells (ILC2s) were recently reported to play a critical role in allergic diseases. We investigated the sex-dependent differences in ILC2-dominant allergic airway inflammation model using T\B cell-deficient mice, and determined the gender differences of ILC2 levels in patients with asthma and allergic rhinitis. Female mice exhibited higher levels of inflammatory infiltration and large production of IL-5 and IL-13, especially for ILC2 levels compared to male mice with the induction of IL-33. However, no significant differences were found for the levels of circulating ILC2s between the genders of patients. The treatment of testosterone significantly decreased the intracellular type 2 cytokines in ILC2s and the proliferation of pure ILC2s in response to epithelial cytokines. Our study suggested the sex differences and the involvement of androgen on ILC2s in allergic diseases.

Introduction: CXCL14 involved in inflammatory processes was upregulated in the asthma expression profile datasets in our pilot study. However, the expression of CXCL14 in induced sputum and its potential clinical role in asthma were poorly reported. Objective: We sought to detect CXCL14 expression in airway epithelium and induced sputum cells of asthma and explore its potential clinical implications. Methods: The expression of CXCL14 in asthma was analyzed using R software based on multiple microarray datasets, including GSE43696, GSE63142, GSE67940, and GSE76262. Subsequent verification of the CXCL14 expression pattern in induced sputum and bronchial epithelium cells was performed by qRT-PCR and ELISA. Besides, the correlations between CXCL14 and eosinophilic inflammation indicators (FeNO, EOS#, and IgE), Th2 signature genes (SERPINB2, POSTN, and CLCA1), inflammatory cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, TSLP, IL-8, IL-17A, IFN-γ, and IL-2), and airway obstruction indicators (pulmonary function and mucin secretion) were further explored. Results: The expression of CXCL14 in epithelium and sputum cells was upregulated in asthma and positively correlated with clinical eosinophilic indicators. The protein levels of CXCL14 were positively associated with Th2 signature genes (SERPINB2, POSTN, and CLCA1) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, and TSLP). Increased expression of CXCL14 was also observed in BEAS-2B cells stimulated by the cytokine IL-4. Furthermore, the expression of CXCL14 was positively correlated with MUC5AC secretion and negatively associated with pulmonary function. Conclusions: Upregulated CXCL14 in asthma was positively correlated with inflammatory indicators and negatively correlated with pulmonary function, which indicated that upregulated CXCL14 might act as a pathogenic gene through involvement in Th2 inflammation in asthma.

Extensive-stage small-cell lung cancer (ES-SCLC) is associated with poor prognosis and treatment options are scarce. Immunotherapy has shown robust clinical activity in ES-SCLC in previous phase 3 trials. We aimed to assess the efficacy and safety of adebrelimab (SHR-1316), a novel anti-PD-L1 antibody, with standard chemotherapy as a first-line treatment for ES-SCLC.The CAPSTONE-1 study was a randomised, double-blind, placebo-controlled, phase 3 trial, done in 47 tertiary hospitals in China. Key inclusion criteria were patients aged 18-75 years, with previously untreated histologically or cytologically confirmed ES-SCLC and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. Eligible patients were randomly assigned (1:1) to receive four to six cycles of carboplatin (area under the curve of 5 mg/mL per min, day 1 of each cycle) and etoposide (100 mg/mBetween Dec 26, 2018, and Sept 4, 2020, 462 eligible patients were enrolled and randomly assigned: 230 (50%) patients received adebrelimab plus chemotherapy (adebrelimab group) and 232 (50%) patients received placebo plus chemotherapy (placebo group). At data cutoff (Oct 8, 2021), median follow-up was 13·5 months (IQR 8·9-20·1). Median overall survival was significantly improved in the adebrelimab group (median 15·3 months [95% CI 13·2-17·5]) compared with the placebo group (12·8 months [11·3-13·7]; hazard ratio 0·72 [95% CI 0·58-0·90]; one-sided p=0·0017). The most common treatment-related grade 3 or 4 adverse events were decreased neutrophil count (174 [76%] patients in the adebrelimab group and 175 [75%] patients in the placebo group), decreased white blood cell count (106 [46%] and 88 [38%]), decreased platelet count (88 [38%] and 78 [34%]), and anaemia (64 [28%] and 66 [28%]). Treatment-related serious adverse events occurred in 89 (39%) patients in the adebrelimab group and 66 (28%) patients in the placebo group. Four treatment-related deaths were reported: two each in the adebrelimab group (respiratory failure and interstitial lung disease and pneumonia) and placebo group (multiple organ dysfunction and unknown cause of death).Adding adebrelimab to chemotherapy significantly improved overall survival with an acceptable safety profile in patients with ES-SCLC, supporting this combination as a new first-line treatment option for this population.Jiangsu Hengrui Pharmaceuticals.

Background Baculoviral IAP repeat-containing 3 (BIRC3) which encodes a member of the IAP family of proteins upregulated in the asthma expression profile dataset. However, there was few research on studying the clinical implication of BIRC3 in asthma. Objective To validate BIRC3 expression and its clinical implications in induced sputum of asthma. Methods Based on the GSE76262 (118 asthma cases and 21 healthy controls) dataset, differentially expressed genes were screened using R software. Subsequently, BIRC3 mRNA and protein were clinically verified in induced sputum samples through quantitative real‐time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Besides, the correlations between BIRC3 expression and asthmatic eosinophilic/allergic inflammation indicators (FeNO, IgE, and EOS%), pulmonary function (FEV1, FEV1% pred, FVC% pred, and FEV1/FVC), and inflammatory cytokines (IL-4, IL-5, IL-13, IL-25, IL-10, IL-33, and TSLP) were analyzed. Finally, BIRC3 mRNA was detected in human primary bronchial epithelial cells stimulated by cytokines (IL-4 or IL-13). Results BIRC3 was screened as a candidate gene in the GSE76262, which was highly expressed in asthma. Highly expressed BIRC3 was positively correlated with eosinophilic and allergic indicators, including FeNO, blood eosinophil, and serum IgE. Moreover, BIRC3 protein was positively associated with inflammation cytokines, like IL-4, IL-5, IL-13, IL-25, IL-10, IL-33, and TSLP, while negatively correlated with FEV1, FEV1%pred, FVC% pred, and FEV1/FVC. Furthermore, the expression of BIRC3 could be induced in primary bronchial epithelial cells treated by cytokines IL-4 or IL-13. Conclusions BIRC3 significantly increased in induced sputum of asthma and positively correlated with airway eosinophilic and peripheral blood allergic inflammation, type 2 cytokines, and airway obstruction. Increased BIRC3 might be involved in the pathogenesis of asthma by affecting the eosinophilic and allergic inflammation.

Background Asthma is a common chronic airway disease in the world. The purpose of this study was to explore the expression of IL1-RL1 in sputum and its correlation with Th1 and Th2 cytokines in asthma. Methods We recruited 132 subjects, detected IL1-RL1 protein level in sputum supernatant by ELISA, and analyzed the correlation between the expression level of IL1-RL1 and fraction of exhaled nitric oxide (FeNO), IgE, peripheral blood eosinophil count (EOS#), and Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-33 and TSLP) and Th1 cytokines (IFN-γ, IL-2, IL-8). The diagnostic value of IL1-RL1 was evaluated by ROC curve. The expression of IL1-RL1 was further confirmed by BEAS-2B cell in vitro. Results Compared with the healthy control group, the expression of IL1-RL1 in sputum supernatant, sputum cells and serum of patients with asthma increased. The AUC of ROC curve of IL1-RL1 in sputum supernatant and serum were 0.6840 (p = 0.0034), and 0.7009 (p = 0.0233), respectively. IL1-RL1 was positively correlated with FeNO, IgE, EOS#, Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-33 and TSLP) and Th1 cytokines (IFN-γ, IL-2, IL-8) in induced sputum supernatant. Four weeks after inhaled glucocorticoids (ICS) treatment, the expression of IL1-RL1 in sputum supernatant and serum was increased. In vitro, the expression of IL1-RL1 in BEAS-2B was increased after stimulated by IL-4 or IL-13 for 24 h. Conclusion The expression of IL1-RL1 in sputum supernatant, sputum cells and serum of patients with asthma was increased, and was positively correlated with some inflammatory markers in patients with asthma. IL1-RL1 may be used as a potential biomarker for the diagnosis and treatment of asthma.

Background Aberrant expression of m6A-related proteins contributes to the occurrence and progression of non-small cell lung cancer (NSCLC). Current studies mainly focus on single m6A regulatory genes and their underlying mechanisms, and the expression of multiple m6A regulatory proteins in NSCLC remains unclear. Therefore, it is necessary to systematically examine these proteins, particularly in clinical specimens. Methods Bioinformatic analysis was used to determine the expression of m6A regulatory genes and their correlation with common gene mutations, such as TP53, EGFR, and KRAS, using The Cancer Genome Atlas (TCGA) and the AE-meta databases. Immunohistochemistry was employed to analyze the protein expression of m6A regulatory proteins in 61 benign lung tissues and 316 NSCLC tissues. Statistical analysis was performed to calculate the correlation between the expression of m6A regulatory proteins and clinicopathological features, survival, and common gene mutations in lung carcinoma patients. Results Analysis of the mRNA levels of 13 core m6A regulators, using information from TCGA and the AE-meta databases, revealed that YTHDF1 levels were upregulated in NSCLC compared with those in adjacent normal tissues. Immunohistochemical staining showed that the expression of METTL3, ALKBH5, YTHDC2, and YTHDF1 was significantly upregulated in NSCLC tissues. Further analyses demonstrated a positive correlation between differentially expressed m6A regulatory proteins, including METTL3, ALKBH5, YTHDC2, and YTHDF1, and the poor clinicopathological features and survival of NSCLC patients. According to the statistics of NSCLC patients enrolled in this study, the protein levels of METTL3 in patients with EGFR exon-19 mutation were higher than those in patients with wild-type EGFR. Conclusion Our results indicate that m6A regulators, including METTL3, ALKBH5, YTHDC2, and YTHDF1, could serve as predictive markers of NSCLC, which will facilitate early detection and diagnosis of NSCLC.

Clinical Practice Points • Osimertinib is widely used in patients with lung cancer with EGFR T790M after EGFR TKI treatment with high efficacy and approved in first-line treatment for all EGFR positive patients. However, the resistance profile to osimertinib is not well-elucidated. NTRK fusions are rare oncogenic drivers in lung cancer. • Here, we report a NOTCH2–NTRK1 rearranged fusion in lung adenocarcinoma with neuroendocrine differentiation after osimertinib treatment. The patient had significantly improved clinical symptoms and had stable disease for 2 months after larotrectinib and osimertinib treatment, with significant decrease in NOTCH2-NTRK1 fusion (AF from 24.85% to 0.63%). • The emergence of NTRK fusions after osimertinib treatment may suggest a therapeutic target. NTRK fusions screening is important after osimertinib resistance.

Summary Background Ensartinib is a potent new-generation ALK inhibitor with high activity against a broad range of known crizotinib-resistant ALK mutations and CNS metastases. We aimed to assess the efficacy and safety of ensartinib in ALK-positive patients with non-small-cell lung cancer (NSCLC), in whom crizotinib therapy was unsuccessful. The associations between ensartinib efficacy and crizotinib-resistant mutations were also explored. Methods We did a single-arm, open-label, phase 2 study at 27 centres in China. Patients were aged 18 years or older, had stage IIIb or stage IV ALK-positive NSCLC that had progressed while they were on crizotinib therapy, an Eastern Cooperative Oncology Group performance status of 2 or less, had measurable disease, and had received fewer than three previous treatments. Patients with CNS metastases were included if these metastases were asymptomatic and did not require steroid therapy. All patients received 225 mg ensartinib orally once daily on a continuous dosing schedule. The primary outcome was the proportion of patients with an objective response according to the Response Evaluation Criteria in Solid Tumors (version 1.1), as assessed by an independent review committee in all patients who received at least one dose of ensartinib with no major violations of the inclusion criteria (ie, the full analysis set). Safety was assessed in all enrolled patients who received at least one dose of ensartinib. This trial was registered with ClinicalTrials.gov , NCT03215693 . Findings Between Sept 28, 2017, and April 11, 2018, 160 patients were enrolled and had at least one dose of ensartinib (safety analysis set). Four patients had inclusion violations and were excluded from the efficacy analysis, which thus included 156 patients (full analysis set). 97 (62%) patients in the full analysis set had brain metastases. 76 (52% [95% CI 43–60]) of 147 patients in the full analysis set, with responses that could be assessed by the independent review committee, had an objective response. 28 (70% [53–83]) of 40 patients with measurable brain metastases as assessed by the independent review committee had an intracranial objective response. 145 (91%) of 160 patients had at least one treatment-related adverse event, which were mostly grade 1 or 2. The most common treatment-related adverse events were rash (89 [56%]), increased alanine aminotransferase concentrations (74 [46%]), and increased aspartate aminotransferase concentrations (65 [41%]). Interpretation Ensartinib has activity and is well tolerated in patients with crizotinib-refractory, ALK-positive NSCLC, including those with brain metastases. The role of ensartinib in patients in whom other second-generation ALK inhibitors have been unsuccessful warrants further studies. Funding Betta Pharmaceuticals.

Background Biotherapy for asthma may be useful in patients suffering from chronic obstructive pulmonary disease (COPD) with asthma characteristics. Therefore, the evaluation and close monitoring of asthma characteristics in severe and extremely severe COPD can guide treatment decisions to improve prognosis. Methods Stable patients suffering from COPD and having a forced expiratory volume in 1 s (FEV1%) of ≤50% (GOLD 3-4) in the First Affiliated Hospital of Sun Yat-Sen University from December 2014 to June 2018 were retrospectively enrolled in this study and evaluated in terms of their asthma characteristics (blood eosinophil counts, fractional exhaled NO [FeNO] values, and reversibility). Results A total of 178 patients with an average age of 65.62±9.28 years were enrolled in this study. A total of 85 patients had an improvement of ≥12% in FEV1%, and 61 of these patients had an absolute increase of >200 mL. Of 122 patients, 68 had blood eosinophil counts of ≥150 cells/μl, whereas 27 showed blood eosinophil counts ≥300 cells/μl. The blood eosinophil of ≥2% was found in 66/122 (54.10%) patients, whereas ≥3% was found in 51/122 (41.80%) patients. A total of 46 of 58 patients had an increased serum IgE level of ≥30 IU/mL, and 32 patients had an IgE of ≥100 IU/mL. The FeNO value of ≥25 ACO (ppb) was found in 51/155 (32.90%) patients. Furthermore, 43 patients had asthma-COPD overlap (ACO), and the FeNO values in the ACO group was 26.13±14.91 ppb, which was significantly higher than that in the COPD alone group (20.99±9.16 ppb; P=0.016). A total of 12 patients with ACO had a negative response after bronchodilation. In the COPD alone group, 34 patients had an absolute increase of >200 mL, whereas 55 of the 95 patients had blood eosinophil counts of ≥150 cells/μl. The blood eosinophilia of ≥2% was found in 54/95 (56.84%) patients. A total of 36 of 45 patients had an increased serum IgE level of ≥30 IU/mL. The FeNO value of 34/123 (27.64%) patients was ≥25 ppb. Conclusion The characteristics of asthma are common findings in patients with severe and extremely severe COPD. Biomarkers should be actively used to evaluate the characteristics of asthma in these patients. If the characteristics of asthma exist, then anti-IgE or anti-IL-5 therapy should be considered to reduce exacerbation.

Background Precise detection of anaplastic lymphoma kinase (ALK) rearrangement guides the application of ALK-targeted tyrosine kinase inhibitors (ALK-TKIs) in patients with non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS) has been widely used in clinics, but DNA-based NGS used to detect fusion genes has delivered false-negative results. However, fusion genes can be successfully detected at the transcription level and with higher sensitivity using RNA-based reverse transcription polymerase chain reaction (RT-PCR). Objective This study compared the performance of RT-PCR and NGS in the detection of echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion in Chinese patients with NSCLC. Methods Formalin-fixed paraffin-embedded tissues from 153 patients who were pathologically diagnosed as having NSCLC were collected from November 2017 to October 2019. Both DNA/RNA-based NGS and RNA-based RT-PCR were used to detect EML4-ALK fusion. For samples with discordant ALK status results, fluorescence in situ hybridization (FISH) or Sanger sequencing was used to further confirm the ALK status. Results In total, 124 samples were successfully analyzed using both NGS and RT-PCR. For 118 samples, results were consistent between NGS and RT-PCR, with 25 reported as ALK fusion positive and 93 as ALK fusion negative, achieving a concordance rate of 95.16%. Among the six samples with disconcordant results, five were positive using RT-PCR but negative using NGS, and one was positive using NGS but negative using RT-PCR. Four of six cases with disconcordant results (three RT-PCR positive and one NGS positive) were successfully validated using either FISH or Sanger sequencing. Conclusions Compared with NGS, RT-PCR appears to be a reliable method of detecting EML4-ALK fusion in patients with NSCLC. Supplementary Information The online version contains supplementary material available at 10.1007/s40291-021-00532-8.

BackgroundInhalation of fungal spores is a strong risk factor for severe asthma and experimentally leads to development of airway mycosis and asthma-like disease in mice. However, in addition to fungal spores, humans are simultaneously exposed to other inflammatory agents such as lipopolysaccharide (LPS), with uncertain relevance to disease expression. To determine how high dose inhalation of LPS influences the expression of allergic airway disease induced by the allergenic moldAspergillus niger(A. niger).MethodsC57BL/6J mice were intranasally challenged with the viable spores ofA. nigerwith and without 1 μg of LPS over two weeks. Changes in airway hyperreactivity, airway and lung inflammatory cell recruitment, antigen-specific immunoglobulins, and histopathology were determined.ResultsIn comparison to mice challenged only withA. niger, addition of LPS (1 μg) toA. nigerabrogated airway hyperresponsiveness and strongly attenuated airway eosinophilia, PAS+ goblet cells and TH2 responses while enhancing TH1 and TH17 cell recruitment to lung. Addition of LPS resulted in more severe, diffuse lung inflammation with scattered, loosely-formed parenchymal granulomas, but failed to alter fungus-induced IgE and IgG antibodies.ConclusionsIn contrast to the strongly allergic lung phenotype induced by fungal spores alone, addition of a relatively high dose of LPS abrogates asthma-like features, replacing them with a phenotype more consistent with acute hypersensitivity pneumonitis (HP). These findings extend the already established link between airway mycosis and asthma to HP and describe a robust model for further dissecting the pathophysiology of HP.

Summary Fungal airway infection (airway mycosis) is an important cause of allergic airway diseases such as asthma, but the mechanisms by which fungi trigger asthmatic reactions are poorly understood. Here, we leverage wild-type and mutant Candida albicans to determine how this common fungus elicits characteristic Th2 and Th17 cell-dependent allergic airway disease in mice. We demonstrate that rather than proteinases that are essential virulence factors for molds, C. albicans instead promoted allergic airway disease through the peptide toxin candidalysin. Candidalysin activated platelets through the Von Willebrand factor (VWF) receptor GP1bα to release the Wnt antagonist Dickkopf-1 (Dkk-1) to drive Th2 and Th17 cell responses that correlated with reduced lung fungal burdens. Platelets simultaneously precluded lethal pulmonary hemorrhage resulting from fungal lung invasion. Thus, in addition to hemostasis, platelets promoted protection against C. albicans airway mycosis through an antifungal pathway involving candidalysin, GP1bα, and Dkk-1 that promotes Th2 and Th17 responses.

Background Lung nodules are a diagnostic challenge. Current clinical management of lung nodule patients is inefficient and therefore causes patient misclassification, which increases healthcare expenses. However, a precise and robust lung nodule classifier to minimize discomfort for patients and healthcare costs is still lacking. The aim of the present protocol is to evaluate the effectiveness of using a liquid biopsy classifier to diagnose nodules compared to physician estimates and whether the classifier can reduce the number of unnecessary biopsies in benign cases. Methods A prospective cohort of 10,560 patients enrolled at 23 clinical centers in China with non-calcified pulmonary nodules, ranging from 0.5 to 3 cm in diameter, indicated by LDCT or CT will be included. After signed consent forms, the participants' pulmonary nodules will be assessed using three evaluation tools: (I) physician cancer probability estimates (II) validated lung nodule risk models, including Mayo Clinic and Veteran's Affairs models (III) ctDNA methylation classifier previously established. Each patient will undergo LDCT/CT follow-ups for 2 to 3 years and their information and one blood sample will be collected at baseline, 3, 6, 12, 24 and 36 months. The primary study outcomes will be the diagnostic accuracy of the methylation classifier in the cohort. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) will be used to compare the diagnostic value of each testing tool in differentiating benign and malignant pulmonary nodules. Discussion We are conducting an observational study to explore the accuracy of using a ctDNA methylation classifier for incidental lung nodules diagnosis. Trial registration Clinicaltrials.gov NCT03651986.

Bronchiectasis is an independent risk factor for cardiovascular disease（CVD）and cardiac dysfunction. Endothelial progenitor cells (EPCs) play a crucial role in maintaining endothelial function, and is inversely correlated with cardiovascular risk factors or cardiac dysfunction. However, the relationship between EPCs and bronchiectasis is unknown.Twenty-nine patients with stable bronchiectasis and 15 healthy controls were recruited. Fasting venous blood were collected for determining circulating EPC number and activity as well as systemic inflammatory cytokines.The number and migratory or proliferative activity of circulating EPCs in bronchiectasis patients were significantly reduced (p 0.001). In high E-FACED group, the number of circulating EPCs evaluated by cell culture assay and EPC proliferation were decreased (p 0.05). Similarly, the number and function of circulating EPCs were both reduced in low forced expiratory volume in 1 s (FEV1) or high mMRC group (p 0.05). There was a significant correlation between circulating EPCs and bronchiectasis disease severity, according to the E-FACED score (p 0.05), particularly to FEV1 (p 0.05) and mMRC dyspnea score (p 0.05). The count and activity of EPCs inversely correlated with hsCRP levels and IL-6 levels (p 0.01).Deficiencies in the number and function of circulating EPCs are present in patients with bronchiectasis. The changes are related to disease severity and may be partly attributed to systemic inflammation. The current findings may provide novel surrogate evaluation biomarkers and potential therapeutic target for bronchiectasis.

Background: The number of deaths caused by COVID-19 are on the rising worldwide. This study focused on severe and critically ill COVID-19, aim to explore independent risk factors associated with disease severity and to build a nomogram to predict patients’ prognosis.Methods: Patients with laboratory-confirmed COVID-19 admitted to the Union Hospital, Tongji Medical College and Hankou Hospital of Wuhan, China, from February 8th to April 6th, 2020. LASSO Regression and Multivariate Analysis were applied to screen independent factors. COX Nomogram was built to predict the 7-day, 14-day and 1-month survival probability.Results: A total of 115 severe [73 (63.5%)] and critically ill [42 (36.5%)] patients were included in this study, containing 93 (80.9%) survivors and 22 (19.1%) non-survivors. For disease severity, D-dimer [OR 6.33 (95%CI, 1.27-45.57], eosinophil percentage [OR 8.02 (95%CI, 1.82-45.04)], total bilirubin [OR 12.38 (95%CI, 1.24-223.65)] and lung involvement score [OR 1.22 (95%CI, 1.08-1.40)] were the independent factors associated with critical illness. Troponin [HR 9.02 (95%CI, 3.02, 26.97)] and total bilirubin [HR 3.16 (95%CI, 1.13, 8.85)] were the independent predictors for patients’ prognosis. Troponin≥26.2 ng/L and total bilirubin＞20 μmol/L were associated with poor prognosis. The nomogram based on the independent risk factors had a C-index of 0.92 (95%CI, 0.87, 0.98) for predicting survival probability. The survival nomogram validated in the critically ill patients had a C-index of 0.83 (95%CI: 0.75, 0.94).Conclusions: In conclusion, in severe and critically ill patients with COVID-19, D-dimer, eosinophil percentage, total bilirubin and lung involvement score were the independent risk factors associated with disease severity. The proposed survival nomogram accurately predicted prognosis. The survival analysis may suggest that early incidence of multiple organ dysfunction may be an important predictor of poor prognosis.

Background According to the proportion of glandular and squamous pathological components, adenosquamous carcinoma (ASC) could be divided into adenocarcinoma (AC) and squamous cell carcinoma (SCC) predominant subtypes. Due to its rarity, no study investigating the impact of different subtypes on the clinical features, radiologic findings and prognosis characteristics of ASC has been reported. Methods Sixty eight patients who underwent surgical resection for lung adenosquamous carcinoma in our institute between January 2006 and March 2017 were retrospectively reviewed. Data regarding the clinical features, radiologic findings and prognosis characteristics were collected. Results Thirty nine patients of the study cohort were with AC-predominant ASC and 29 with SCC-predominant ASC. There was no significant difference between the two subgroups in age, gender, smoking history, serum carcinoembryonic antigen (CEA) level and T,N classification. Air bronchogram was found more frequently in AC-predominant ASC than in SCC-predominant ASC (P = 0.046). Multivariate analysis identified pathological subtype (P = 0.022) and CT findings of peripheral location (P = 0.009) to be independent prognostic factors. Conclusions AC-predominant ASC were more commonly presented with air bronchogram, and were with a better prognosis than SCC-predominant ASC.

9071 Background: Investigational agent APG-2449 is a novel, orally active FAK inhibitor and a third-generation ALK/ROS1 TKI that has shown potent activity against a range of ALK-resistant mutations, including G1202R, L1196M, V1180L, E1210K, S1206F, G1269A, F1174L, I1171S, and C1156Y in preclinical NSCLC, mesothelioma, and other solid tumor models. Methods: This dose escalation and dose expansion study evaluated APG-2449 in patients with second-generation TKI-resistant ALK/ROS1+ NSCLC or mesothelioma. APG-2449 was administered orally once daily at the assigned doses on a 28-day cycle using a “3+3” dose escalation design under fasted/fed conditions. Study aims were to assess safety/tolerability, recommended phase 2 dose (RP2D), pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Results: As of the data cutoff date of December 30, 2021, 84 pts (median age 52 [range 21-78] years; 42% female) with NSCLC or mesothelioma enrolled were treated with APG-2449 at doses ranging from 150 to 1,500 mg. PK analyses indicated an approximately dose-proportional increase in plasma exposure under fed conditions across dose levels tested. Cerebrospinal fluid PK analyses showed that APG-2449 was brain-penetrant. Low-fat meals increased APG-2449 Cmax and AUC by approximately 40% to 80% compared to fasting conditions. Based on PK, biomarker, efficacy, and safety results, the RP2D was determined to be 1,200 mg. Four partial responses (PRs) were observed in 14 ALK+ pts resistant to second-generation TKIs treated at the RP2D. Another pt with the G1202R mutation following alectinib treatment had tumor shrinkage of 27.9%. Among 8 pts with brain metastases, 1 complete response and 3 PRs were observed intracranially. In 10 TKI-naïve pts, the overall response rate was 80% (ALK+, 6/8; ROS1+, 2/2) and the disease control rate (DCR) was 100%. Preliminary biomarker data showed decreased FAK phosphorylation in peripheral blood mononuclear cells and increased IFN-γ levels in serum after multiple doses of APG-2449. No dose-limiting toxicity was observed. A total of 66 (78.6%) pts experienced treatment-related adverse events (TRAEs). The most frequent TRAEs included elevated blood creatinine (33.3%), ALT (25.0%), and AST (19.0%) levels and gastrointestinal disorders: nausea (22.6%), vomiting (17.9%), and diarrhea (13.1%). Only 6 (7.1%) TRAEs were grade ≥ 3. Conclusions: APG-2449 has a favorable safety and PK profile and was well tolerated in 84 subjects. Preliminary efficacy was observed in pts whose disease was resistant to second-generation TKIs, especially among those with brain metastases, and in TKI-naïve pts. Biomarker data indicated potential target engagement on FAK and immunomodulatory effects of APG-2449. Clinical trial information: NCT03917043.

e21124 Background: The addition of bevacizumab to first-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) has shown promising efficacy in delaying TKI resistance and prolonging PFS in treating EGFR-mutated advanced lung cancer. However, data on the afatinib-bevacizumab combination therapy are limited. Methods: A total of 47 patients with EGFR-mutated advanced lung cancer were retrospectively analyzed, including 21 patients treated with afatinib combined with bevacizumab and 26 patients treated with afatinib monotherapy. Clinical data, survival outcomes and toxic effects were evaluated. Results: There was no significant difference in basic clinical features between the combination and monotherapy groups. The overall response rate (ORR), median progression-free survival (PFS) and overall survival (OS) in the combination group (afatinib 40 mg/day combined with bevacizumab 7.5 mg/kg every 3 weeks) were 42.9%, 16.5 months and 35.2 months, respectively, while the ORR, PFS and OS in the afatinib group was 53.8%, 13.8 months and 33.8 months, respectively. No significant difference was noted in ORR, PFS or OS between the two groups ( p＞0.05). However, among the subgroup with concurrent genetic mutations, combination therapy provided a significant PFS benefit (18.2 months, [95% CI 7.1-29.3] vs. 11.0 months [95%CI 7.5-14.5], p= 0.036). Although there was a longer trend in OS, no statistical difference was presented (35.2 months [95% CI 29.8-40.6] vs. 27.5 months [95% CI 13.1-41.9], p= 0.138). The safety of the combination therapy was acceptable and manageable. Conclusions: Afatinib-bevacizumab combination therapy could provide superior PFS benefit over afatinib alone in the subset of patients with concomitant genetic mutations in EGFR-mutated advanced NSCLC, and the safety of the combination therapy is acceptable.[Table: see text]

Asthma is a common chronic airway inflammatory disease worldwide. Studies on gene expression profiles in induced sputum may provide noninvasive diagnostic biomarkers and therapeutic targets for asthma.To investigate mRNA expression of MMP12 in induced sputum and its relationship with asthma airway eosinophilic inflammation.GSE76262 dataset was analyzed using R software, weighted gene coexpression network analysis (WGCNA), and protein-protein interaction (PPI) network construction. The top ten hub genes were screened with Cytoscape software (version 3.8.4). We then verified the mRNA expression of MMP12 in two other datasets (GSE137268 and GSE74075) via ROC curve estimates and our induced sputum samples using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Finally, we explored the correlation between MMP12 with asthmatic eosinophilic-related indicators.We obtained the top ten hub genes, namely, CCL17, CCL2, CSF1, CCL22, CCR3, CD69, FCGR2B, CD1C, CD1E, and MMP12 via expression profile screening and validation on the GSE76262 dataset. MMP12 was selected as the candidate gene through further validation on GSE137268 and GSE74075 datasets. Finally, we demonstrated that the mRNA expression of MMP12 is significantly upregulated in induced sputum of asthmatic patients (p 0.05) and significantly correlated with eosinophilic-related indicators (p 0.05). These findings indicated that MMP12 can act as a diagnostic biomarker for asthma.Our study successfully identified and demonstrated that MMP12 is a potential diagnostic biomarker for asthma due to its high expression and association with eosinophilic-related indicators. The results of this study can provide novel insights into asthmatic diagnosis and therapy in the future.

UNSTRUCTURED Dear Editor, Per your instruction, I have submitted a formal request to withdraw our manuscript 18108 on June 11th with an explanation for the withdraw and a scan of the signed letter. The same letter has been uploaded to your online system as well. I am sending this withdraw letter again due to the “request for a revised manuscript” email get to us again! Feel free to contact me should you have any question. But, please help DO NOT send any further junk mail to 40+ co-authors. It is really bad that we have submitted the withdraw petition several times with an explanation, but the system is still sending emails to all authorships. Best regards, Jian-Bing Fan (the corresponding author)

Background: Precisely detecting anaplastic lymphoma kinase (ALK) rearrangement ensures ideal efficacy of ALK targeted tyrosine kinase inhibitors (ALK-TKIs). Next generation sequencing (NGS) has been widely used in clinics, while, numbers of studies indicated NGS would yield false negative outcome, especially for fusion analysis. Instead, more fusions can be detected by RT-PCR at transcription level. This study aims to compare the capacity of RT-PCR and NGS approach to detect ALK rearrangement in Chinese non-small-cell lung cancer (NSCLC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissues from 153 patients pathologically diagnosed as NSCLC with eligible ALK status given by previous NGS testing (29 patients were ALK positive and 124 patients were ALK negative) were collected from November 2017 to October 2019. RT-PCR were applied to detect ALK rearrangements in both groups. For samples with discordant result of ALK status, fluorescence in situ hybridization (FISH) or Sanger's sequencing were used for validation. Results: One hundred and twenty-four samples (26 of 30 ALK positive samples; 98 of 124 ALK negative samples) were successfully subjected to RT-PCR. Five of 98 ALK negative samples given by NGS test were re-defined as ALK positive by RT-PCR. Meanwhile, in 26 ALK positive samples, RT- PCR yielded acceptable result with 96.15% of concordance rate. Only one NGS-defined ALK positive sample was uncovered by RT-PCR due to a rare ALK rearrangement. Four of 6 RT-PCR defined ALK positive cases (3 RT-PCR positive and 1 NGS positive) were validated by either FISH or sanger's sequencing, which resulted positive result. Conclusions: RT-PCR displays reliable and superior capacity to detect ALK fusion in NSCLC patients. Citation Format: Yukun Kuang, Peihang Xu, Jiyu Wang, Yifan Zheng, Xue Sun, Zimu Li, RunJing Gan, Huixia Li, Yubiao Guo, Guanshan Zhu, Changbin Zhu, Zunfu Ke, Kejing Tang. Detecting ALK rearrangement by RT-PCR: A superior approach to next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 467.

BACKGROUND: Epidermal growth factor receptor (EGFR) co-mutated with TP53 could reduce responsiveness to tyrosine kinase inhibitors (TKIs) and worsen patients’ prognosis compared to TP53 wild type patients in EGFR: mutated lung adenocarcinomas (LUAD). To identify this genetically unique subset prior to treatment through computed tomography (CT) images had not been reported yet. METHODS: Stage III and IV LUAD with known mutation status of EGFR and TP53 from The First Affiliated Hospital of Sun Yat-sen University (May 1, 2017 to June 1, 2020) were collected. Characteristics of pretreatment enhanced-CT images were analyzed. One-versus-one was used as the multiclass classification strategy to distinguish the three subtypes of co-mutations: EGFR(+) & TP53(+), EGFR(+) & TP53(−), EGFR(−). The clinical model, semantic model, radiomics model and integrated model were built. Area under the receiver-operating characteristic curves (AUCs) were used to evaluate the prediction efficacy. RESULTS: A total of 199 patients were enrolled, including 83 (42%) cases of EGFR(−), 55 (28%) cases of EGFR(+) & TP53(+), 61 (31%) cases of EGFR(+) & TP53(−). Among the four different models, the integrated model displayed the best performance for all the three subtypes of co-mutations: EGFR(−) (AUC, 0.857; accuracy, 0.817; sensitivity, 0.998; specificity, 0.663), EGFR(+) & TP53(+) (AUC, 0.791; accuracy, 0.758; sensitivity, 0.762; specificity, 0.783), EGFR(+) & TP53(−) (AUC, 0.761; accuracy, 0.813; sensitivity, 0.594; specificity, 0.977). The radiomics model was slightly inferior to the integrated model. The results for the clinical and the semantic models were dissatisfactory, with AUCs less than 0.700 for all the three subtypes. CONCLUSIONS: CT imaging based artificial intelligence (AI) is expected to distinguish co-mutation status involving TP53 and EGFR. The proposed integrated model may serve as an important alternative marker for preselecting patients who will be adaptable to and sensitive to TKIs.

Introduction: In the previous phase II clinical trial (NCT03215693), ensartinib, a next generation (NG) ALK TKI, showed comparable efficacy to other NG ALK TKIs in the post-crizotinib setting. In the preplanned biomarker analysis, we investigated the efficacy and the resistance mechanisms of ensartinib via longitudinal ctDNA analysis. Methods: Pts with stage IIIB/IV ALK+ NSCLC, and had progressive disease (PD) after crizotinib were eligible. Plasma samples from this trial were prospectively collected for ctDNA analysis at baseline (BL), cycle 3 day 1 (C3D1) and the end of treatment (EOT). Plasma DNA was analyzed using a 212-gene sequencing panel. ctDNA amount was defined as the sum of variant allele fraction (VAF) of ALK fusions and mutations. Cox model was applied for multivariate analysis. Results: Between 9/2017 and 7/2019, 182 pts were enrolled in the trial. In total, 178 were included in the full analysis set (FAS), with a median PFS (mPFS) of 9.8 months (95% CI, 7.5 to 11.7 months). 169 pts were evaluable by independent review committee (IRC), with an ORR of 57.7% (95% CI, 50.2% to 65.3%). A total of 431 ctDNA samples were analyzed, including 168 at BL, 165 at C3D1, and 106 at EOT (8 overlappings between C3D1 and EOT). 168 (99.4%) pts had evaluable ctDNA at BL. The median ctDNA amount was 0.00704. The higher ctDNA amount was associated with liver metastases, bone metastases, and TP53 mutations. Tumor burden, as measured by the sum of the target lesions' longest diameters, also positively correlated with ctDNA amount (Pearson correlation 0.261; p < 0.001). Multivariable Cox regression revealed that high level of ctDNA amount and TP53 mutations at BL was significantly associated with poor PFS independently (p =0.020 and p < 0.001) (Table). 106 (62.7%) pts had evaluable ctDNA at both BL and EOT. Compared to baseline, the frequency of certain ALK mutations significantly increased at EOT, such as G1269A (7.5% vs 21.7%), G1202R (3.8% vs 17.0%) and E1210K (0% vs 10.4%). TP53 statusctDNA amountTP53 Wildtype TP53 Mutations Low amountHigh amount(n = 34)(n = 134)(n = 46)(n = 47)mPFS, months (95% CI) 11.7 (9.8, 14.0) 4.2 (3.0, 5.6)9.3 (5.6, 11.7) 4.2 (2.8, 5.5)HR (95% CI); p value2.50 (1.65, 3.79) ; p < 0.0012.34 (1.52, 3.61) ; p < 0.001 Consistent with the change in frequency, the abundance of G1269A, G1202R, and E1210K also trended to increase at EOT, while the abundance of C1156Y and I1171X had a downward trend in most pts. Conclusions: Consistent with previous reports, ensartinib showed high clinical efficacy. Longitudinal ctDNA analysis could be a powerful tool in predicting treatment outcomes and revealing resistant mechanisms of ensartinib. We observed G1269A, G1202R and E1210K as the major resistant mutations to ensartinib. Additional analyses are ongoing and results will be updated and reported in the meeting. Citation Format: Yunpeng Yang, Jie Huang, Tao Wang, Jianya Zhou, Jing Zheng, Jifeng Feng, Wu Zhuang, Jianhua Chen, Jun Zhao, Wei Zhong, Yanqiu Zhao, Yiping Zhang, Yong Song, Yi Hu, Zhuang Yu, Youling Gong, Yuan Chen, Feng Ye, Shucai Zhang, Lejie Cao, Yun Fan, Gang Wu, Yubiao Guo, Chengzhi Zhou, Kewei Ma, Jian Fang, Weineng Feng, Yunpeng Liu, Zhendong Zheng, Gaofeng Li, Ning Wu, Wei Song, Xiaoqing Liu, Shijun Zhao, Lieming Ding, Li Mao, Giovanni Selvaggi, Larry Zhu, Shanshan Xiao, Li Zhang. Longitudinal circulating tumor DNA (ctDNA) analysis predicts response and reveals the resistance mechanisms of ensartinib in ALK+ NSCLC patients (pts) progressed on crizotinib: Updated analysis of a phase II clinical trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2997.

Background Programmed death ligand-1 (PD-L1) expression remains a crucial predictor in selecting patients for immunotherapy. The current study aimed to non-invasively predict PD-L1 expression based on chest computed tomography (CT) images in advanced lung adenocarcinomas (LUAD), thus help select optimal patients who can potentially benefit from immunotherapy. Methods A total of 127 patients with stage III and IV LUAD were enrolled into this study. Pretreatment enhanced thin-section CT images were available for all patients and were analyzed in terms of both morphologic characteristics by radiologists and deep learning (DL), so to further determine the association between CT features and PD-L1 expression status. Univariate analysis and multivariate logical regression analysis were applied to evaluate significant variables. For DL, the 3D DenseNet model was built and validated. The study cohort were grouped by PD-L1 Tumor Proportion Scores (TPS) cutoff value of 1% (positive/negative expression) and 50% respectively. Results Among 127 LUAD patients, 46 (36.2%) patients were PD-L1-positive and 38 (29.9%) patients expressed PD-L1-TPS ≥50%. For morphologic characteristics, univariate and multivariate analysis revealed that only lung metastasis was significantly associated with PD-L1 expression status despite of different PD-L1 TPS cutoff values, and its Area under the receiver operating characteristic curve (AUC) for predicting PD-L1 expression were less than 0.700. On the other hand, the predictive value of DL-3D DenseNet model was higher than that of the morphologic characteristics, with AUC more than 0.750. Conclusions The traditional morphologic CT characteristics analyzed by radiologists show limited prediction efficacy for PD-L1 expression. By contrast, CT-derived deep neural network improves the prediction efficacy, it may serve as an important alternative marker for clinical PD-L1 detection.

Objectives: The current study aimed to non-invasively predict PD-L1 expression based on chest computed tomography (CT) images in advanced lung adenocarcinomas (LUAD), thus help to select optimal patients who can potentially benefit from immunotherapy. Methods: A total of 127 patients with stage III and IV unresectable LUAD were enrolled into this study. Pretreatment enhanced thin-section CT images were available for all patients and were analyzed in terms of both morphologic characteristics and deep learning (DL), so to further determine the association between CT features and PD-L1 expression status. Univariate Analysis and Multivariate Logical Regression Analysis were applied to evaluate significant variables. For DL, the 3D DenseNet Model was built and validated. Fivefold cross validation was practiced to evaluate the prediction efficacy of the model. The study cohort were grouped by PD-L1 TPS cutoff value of 1% (positive/negative expression) and 50% (weakly/strongly positive expression) respectively. Results: Among 127 LUAD patients, 46 (36.2%) patients were PD-L1-positive and 38 (29.9%) patients were strongly positive. For morphologic characteristics, univariate analysis showed that vascular involvement and lung metastasis were associated with PD-L1-negative/positive expression status (P =0.022 and P=0.002, respectively) and they were more commonly presented in PD-L1-negative subgroup. Whilst, only lung metastasis was associated with PD-L1-weakly/strongly positive expression status (P=0.010), and it was more commonly presented in PD-L1-weakly positive expression subgroup. Multivariate Logistic Regression Analysis revealed that only lung metastasis was significantly associated with PD-L1 expression status despite of different PD-L1 TPS cutoff values, and its AUC for predicting PD-L1 expression were less than 0.700. On the other hand, the predictive value of DL-3D DenseNet Model was higher than that of the morphologic characteristics, with AUC more than 0.750 in LUAD. Conclusions: CT features were significantly associated with PD-L1 expression status. PD-L1-negative and weakly positive LUAD showed lung metastasis more frequently than that of PD-L1-positive and strongly positive ones. Compared to morphologic CT features, DL-3D DenseNet Model improves the prediction efficacy, it has promising application value in predicting PD-L1 expression status in advanced LUAD. Funding Statement: This work was supported by the National Natural Science Foundation of China (NO.81600065, NO.81771908 and NO.81571750). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This retrospective study was approved by the ethics committee of our institute and informed consents were waived.

Previous studies revealed that the concomitant prevalence of obstructive sleep apnea (OSA) and venous thromboembolism (VTE) was high, but the results were inconclusive due to various limitations. We aimed to systematically review the literature on the prevalence of OSA in patients with VTE. Relevant studies were identified on multiple electronic databases through July 2018. The DerSimonian-Laird random effects model was used to calculate the pooled prevalence of OSA, moderate-to-severe OSA, and severe OSA in VTE patients, respectively. Sensitivity analysis was performed based on diagnostic methods of OSA and races. A total of 11 studies comprising 895 patients were available for the meta-analysis, but one study was excluded because of the between-study heterogeneity in the following analysis. The pooled prevalence of OSA, moderate-to-severe OSA, and severe OSA in VTE patients were 70% (95% CI = 65%, 75%), 41% (95% CI = 29%, 54%), and 19% (95% CI = 15%, 23%), respectively. Sensitivity analysis indicated that the prevalence was similar in different diagnostic methods, but the contributions of races to OSA were complex. Although the lower prevalence of all OSA and moderate-to-severe OSA as compared with Western countries, Asian countries have similar or even a little bit higher prevalence of severe OSA. Findings from this meta-analysis supported that the prevalence of OSA in VTE patients was strikingly high. Screening for OSA in patients with VTE is necessary for developing effective treatment strategies.

The goal of the present study was to investigate the role of M1 macrophages in acute lung injury (ALI). To address this, we used lipopolysaccharide (LPS)-treated wild-type and CD11b-DTR mice, and examined their M1 macrophage levels, and the extent of their inflammation and pulmonary injuries. In addition, we evaluated pulmonary function by measuring the expressions of SP-A and SP-B in infiltrated M1 macrophages. Finally, we co-cultured the mouse type II-like alveolar epithelial cells (AT-II) and mouse pulmonary microvascular endothelial cells (PMECs) with M1 macrophages in the presence of TNF-α or H2O2 and assessed them for viability and apoptosis. After LPS treatment, we observed that the number of pulmonary M1/M2 macrophages and the serum levels of interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and reactive oxygen species (ROS) significantly increased. Furthermore, the increase in cytokines was accompanied with the initiation of lung injury indicated by the decreased levels of SP-A and SP-B. In macrophage-depleted CD11b-DTR mice, ALI was attenuated, serum levels of IL-1β, TNF-α and ROS were reduced, and lung levels of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) were decreased. After administering TNF-α and H2O2, the proapoptotic effect of M1 macrophages on AT-II or PMECs significantly increased, the cell viabilities significantly decreased, and apoptosis significantly increased. Our results suggest that M1 macrophages are recruited to the lungs where they significantly contribute to an increase in TNF-α and ROS production, thus initiating ALI.

PIWI interacting RNAs (piRNAs) are highly expressed in germline cells and are involved in maintaining genome integrity by silencing transposons. These are also involved in DNA/histone methylation and gene expression regulation in somatic cells of invertebrates. The functions of piRNAs in somatic cells of vertebrates, however, remain elusive. We found that snoRNA-derived and C (C')/D' (D)-box conserved piRNAs are abundant in human CD4 primary T-lymphocytes. piRNA (piR30840) significantly downregulated interleukin-4 (IL-4) via sequence complementarity binding to pre-mRNA intron, which subsequently inhibited the development of Th2 T-lymphocytes. Piwil4 and Ago4 are associated with this piRNA, and this complex further interacts with Trf4-Air2-Mtr4 Polyadenylation (TRAMP) complex, which leads to the decay of targeted pre-mRNA through nuclear exosomes. Taken together, we demonstrate a novel piRNA mechanism in regulating gene expression in highly differentiated somatic cells and a possible novel target for allergy therapeutics.