Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease.

Extracellular matrix (ECM) remodeling has been implicated in the irreversible obstruction of airways and destruction of alveolar tissue in chronic obstructive pulmonary disease (COPD). Studies investigating differences in the lung ECM in COPD have mainly focused on some collagens and elastin, leaving an array of ECM components unexplored. We investigated the differences in the ECM landscape comparing severe-early onset (SEO-) COPD and moderate COPD to control lung tissue for collagen type I α chain 1 (COL1A1), COL6A1, COL6A2, COL14A1, fibulin 2 and 5 (FBLN2, FBLN5), latent transforming growth factor-beta binding protein 4 (LTBP4), lumican (LUM), versican (VCAN), decorin (DCN), and elastin (ELN) using image analysis and statistical modelling. Percentage area and/or mean intensity of expression of LUM in the parenchyma, and COL1A1, FBLN2, LTBP4, DCN, and VCAN in the airway walls, was proportionally lower in COPD compared to controls. Lowered levels of most ECM proteins were associated with decreasing FEV1 measurements, indicating a relationship with disease severity. Furthermore, we identified six unique ECM signatures where LUM and COL6A1 in parenchyma and COL1A1, FBLN5, DCN, and VCAN in airway walls appear essential in reflecting the presence and severity of COPD. These signatures emphasize the need to examine groups of proteins to represent an overall difference in the ECM landscape in COPD, that are more likely to be related to functional effects, than individual proteins. Our study revealed differences in the lung ECM landscape between control and COPD and between SEO and moderate COPD signifying distinct pathological processes in the different subgroups.

Osteoprotegerin is an Early Marker of the Fibrotic Process and of Antifibrotic Treatment Responses in Ex Vivo Lung Fibrosis.

BACKGROUND: Lung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well. METHODS: Here we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker. RESULTS: OPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFß1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFß1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment. CONCLUSION: OPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.

IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

Genome-wide association meta-analysis identifies pleiotropic risk loci for aerodigestive squamous cell cancers.

Squamous cell carcinomas (SqCC) of the aerodigestive tract have similar etiological risk factors. Although genetic risk variants for individual cancers have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. To identify novel and pleotropic SqCC risk variants, we performed a meta-analysis of GWAS data on lung SqCC (LuSqCC), oro/pharyngeal SqCC (OSqCC), laryngeal SqCC (LaSqCC) and esophageal SqCC (ESqCC) cancers, totaling 13,887 cases and 61,961 controls of European ancestry. We identified one novel genome-wide significant (Pmeta<5x10-8) aerodigestive SqCC susceptibility loci in the 2q33.1 region (rs56321285, TMEM273). Additionally, three previously unknown loci reached suggestive significance (Pmeta<5x10-7): 1q32.1 (rs12133735, near MDM4), 5q31.2 (rs13181561, TMEM173) and 19p13.11 (rs61494113, ABHD8). Multiple previously identified loci for aerodigestive SqCC also showed evidence of pleiotropy in at least another SqCC site, these include: 4q23 (ADH1B), 6p21.33 (STK19), 6p21.32 (HLA-DQB1), 9p21.33 (CDKN2B-AS1) and 13q13.1(BRCA2). Gene-based association and gene set enrichment identified a set of 48 SqCC-related genes rel to DNA damage and epigenetic regulation pathways. Our study highlights the importance of cross-cancer analyses to identify pleiotropic risk loci of histology-related cancers arising at distinct anatomical sites.

Ex Vivo Optimization of Donor Lungs with Inhaled Sevoflurane during Normothermic Ex Vivo Lung Perfusion (VITALISE): A Pilot and Feasibility Study in Sheep.

Volatile anesthetics have been shown in different studies to reduce ischemia reperfusion injury (IRI). Ex vivo lung perfusion (EVLP) facilitates graft evaluation, extends preservation time and potentially enables injury repair and improvement of lung quality. We hypothesized that ventilating lungs with sevoflurane during EVLP would reduce lung injury and improve lung function. We performed a pilot study to test this hypothesis in a slaughterhouse sheep DCD model. Lungs were harvested, flushed and stored on ice for 3 h, after which EVLP was performed for 4 h. Lungs were ventilated with either an FiO2 of 0.4 (EVLP, n = 5) or FiO2 of 0.4 plus sevoflurane at a 2% end-tidal concentration (Cet) (S-EVLP, n = 5). Perfusate, tissue samples and functional measurements were collected and analyzed. A steady state of the target Cet sevoflurane was reached with measurable concentrations in perfusate. Lungs in the S-EVLP group showed significantly better dynamic lung compliance than those in the EVLP group (p = 0.003). Oxygenation capacity was not different in treated lungs for delta partial oxygen pressure (PO2; +3.8 (-4.9/11.1) vs. -11.7 (-12.0/-3.2) kPa, p = 0.151), but there was a trend of a better PO2/FiO2 ratio (p = 0.054). Perfusate ASAT levels in S-EVLP were significantly reduced compared to the control group (198.1 ± 93.66 vs. 223.9 ± 105.7 IU/L, p = 0.02). We conclude that ventilating lungs with sevoflurane during EVLP is feasible and could be useful to improve graft function.

A proteomics approach to identify COPD-related changes in lung fibroblasts.

Lung fibroblasts are implicated in abnormal tissue repair in chronic obstructive pulmonary disease (COPD). The exact mechanisms are unknown and comprehensive analysis comparing COPD- and control fibroblasts is lacking. The aim of this study is to gain insight into the role of lung fibroblasts in COPD pathology using unbiased proteomic and transcriptomic analysis. Protein and RNA were isolated from cultured parenchymal lung fibroblasts of 17 patients with stage IV COPD and 16 non-COPD controls. Proteins were analyzed using LC-MS/MS and RNA through RNA sequencing. Differential protein and gene expression in COPD was assessed via linear regression, followed by pathway enrichment, correlation analysis, and immunohistological staining in lung tissue. Proteomic and transcriptomic data were compared to investigate the overlap and correlation between both levels of data. We identified 40 differentially expressed (DE) proteins and zero DE genes between COPD and control fibroblasts. The most significant DE proteins were HNRNPA2B1 and FHL1. Thirteen of the 40 proteins were previously associated with COPD, including FHL1 and GSTP1. Six of the 40 proteins were related to telomere maintenance pathways, and were positively correlated with the senescence marker LMNB1. No significant correlation between gene and protein expression was observed for the 40 proteins. We hereby describe 40 DE proteins in COPD fibroblasts including previously described COPD proteins (FHL1, GSTP1) and new COPD research targets like HNRNPA2B1. Lack of overlap and correlation between gene and protein data supports the use of unbiased proteomics analysis and indicates that different types of information are generated with both methods.

Age-associated differences in the human lung extracellular matrix.

Extracellular matrix (ECM) remodeling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study, we aimed to identify and localize age-associated ECM differences in human lungs using comprehensive transcriptomic, proteomic, and immunohistochemical analyses. Our previously identified age-associated gene expression signature of the lung was re-analyzed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (false discovery rate, FDR < 0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from nine control patients (49-76 years) (FDR < 0.05). Extensive immunohistochemical analysis was used to localize and semi-quantify the age-associated ECM differences in lung tissues from 62 control patients (18-82 years). Comparative analysis of transcriptomic and proteomic data identified seven ECM proteins with higher expression with age at both gene and protein levels: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4, and LUM. With immunohistochemistry, we demonstrated higher protein levels with age for COL6A2 in whole tissue, parenchyma, airway wall, and blood vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in lung parenchyma. Our study revealed that higher age is associated with lung ECM remodeling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility to developing chronic lung diseases.NEW & NOTEWORTHY We identified seven age-associated extracellular matrix (ECM) proteins, i.e., COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4, and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for COL6A2 in whole tissue, parenchyma, airway wall, and vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in parenchyma. Our findings lay a new foundation for the investigation of ECM differences in age-associated chronic lung diseases.

Th2 high and mast cell gene signatures are associated with corticosteroid sensitivity in COPD.

RATIONALE: Severe asthma and chronic obstructive pulmonary disease (COPD) share common pathophysiological traits such as relative corticosteroid insensitivity. We recently published three transcriptome-associated clusters (TACs) using hierarchical analysis of the sputum transcriptome in asthmatics from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort comprising one Th2-high inflammatory signature (TAC1) and two Th2-low signatures (TAC2 and TAC3). OBJECTIVE: We examined whether gene expression signatures obtained in asthma can be used to identify the subgroup of patients with COPD with steroid sensitivity. METHODS: Using gene set variation analysis, we examined the distribution and enrichment scores (ES) of the 3 TACs in the transcriptome of bronchial biopsies from 46 patients who participated in the Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease COPD study that received 30 months of treatment with inhaled corticosteroids (ICS) with and without an added long-acting ß-agonist (LABA). The identified signatures were then associated with longitudinal clinical variables after treatment. Differential gene expression and cellular convolution were used to define key regulated genes and cell types. MEASUREMENTS AND MAIN RESULTS: Bronchial biopsies in patients with COPD at baseline showed a wide range of expression of the 3 TAC signatures. After ICS±LABA treatment, the ES of TAC1 was significantly reduced at 30 months, but those of TAC2 and TAC3 were unaffected. A corticosteroid-sensitive TAC1 signature was developed from the TAC1 ICS-responsive genes. This signature consisted of mast cell-specific genes identified by single-cell RNA-sequencing and positively correlated with bronchial biopsy mast cell numbers following ICS±LABA. Baseline levels of gene transcription correlated with the change in RV/TLC %predicted following 30-month ICS±LABA. CONCLUSION: Sputum-derived transcriptomic signatures from an asthma cohort can be recapitulated in bronchial biopsies of patients with COPD and identified a signature of airway mast cells as a predictor of corticosteroid responsiveness.

Smoking increases expression of the SARS-CoV-2 spike protein-binding long ACE2 isoform in bronchial epithelium.

After more than two years the COVID-19 pandemic, that is caused by infection with the respiratory SARS-CoV-2 virus, is still ongoing. The risk to develop severe COVID-19 upon SARS-CoV-2 infection is increased in individuals with a high age, high body mass index, and who are smoking. The SARS-CoV-2 virus infects cells of the upper respiratory tract by entering these cells upon binding to the Angiotensin-converting enzyme 2 (ACE2) receptor. ACE2 is expressed in various cell types in the lung but the expression is especially high in goblet and ciliated cells. Recently, it was shown that next to its full-length isoform, ACE2 also has a short isoform. The short isoform is unable to bind SARS-CoV-2 and does not facilitate viral entry. In the current study we investigated whether active cigarette smoking increases the expression of the long or the short ACE2 isoform. We showed that in active smokers the expression of the long, active isoform, but not the short isoform of ACE2 is higher compared to never smokers. Additionally, it was shown that the expression of especially the long, active isoform of ACE2 was associated with secretory, club and goblet epithelial cells. This study increases our understanding of why current smokers are more susceptible to SARS-CoV-2 infection, in addition to the already established increased risk to develop severe COVID-19.

Gene expression profiles in mesenchymal stromal cells from bone marrow, adipose tissue and lung tissue of COPD patients and controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage. Novel regenerative strategies are urgently awaited. Cultured mesenchymal stem/stromal cells (MSCs) have shown promising results in experimental models of COPD, but differences between sources may impact on their potential use in therapeutic strategies in patients. AIM: To assess the transcriptome of lung-derived MSCs (LMSCs), bone marrow-derived MSCs (BM-MSC) and adipose-derived MSCs (AD-MSCs) from COPD patients and non-COPD controls. METHODS: We studied differences in gene expression profiles between the MSC-subtypes, as well as between COPD and control using RNA sequencing (RNA-seq). RESULTS: We show that besides heterogeneity between donors, MSCs from different sources have strongly divergent gene signatures. The growth factors FGF10 and HGF were predominantly expressed in LMSCs. MSCs from all sources displayed altered expression profiles in COPD, with most pronounced significantly up- and downregulated genes in MSCs from adipose tissue. Pathway analysis revealed that the most differentially expressed genes in COPD-derived AD-MSCs are involved in extracellular matrix (ECM) binding and expression. In LMSCs, the gene that differed most strongly between COPD and control was CSGALNACT1, an ECM modulating gene. CONCLUSION: Autologous MSCs from COPD patients display abnormalities with respect to their transcriptome, which were surprisingly most profound in MSCs from extrapulmonary sources. LMSCs may be optimally equipped for lung tissue repair because of the expression of specific growth factor genes.

Differential roles for lysyl oxidase (like), family members in chronic obstructive pulmonary disease; from gene and protein expression to function.

Chronic obstructive pulmonary disease (COPD) is characterized by long-term airflow obstruction with cigarette smoke as a key risk factor. Extracellular matrix (ECM) alterations in COPD may lead to small airway wall fibrosis. Altered collagen cross-linking, potentially mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4), orchestrates disturbed ECM homeostasis. In this study, we investigated the effects of smoking status and presence and severity of COPD on LOs gene and protein expression in the airways and the impact of LOs inhibition on airway contraction in an ex vivo mouse model. We used gene expression data from bronchial brushings, airway smooth muscle (ASM) cells in vitro and immunohistochemistry in lung tissue to assess smoke- and COPD-associated differences in LOs gene and protein expression in the small airways. We found higher LOX expression in current- compared to ex-smokers and higher LOXL1 expression in COPD compared to non-COPD patients. LOX and LOXL2 expression were upregulated in COPD ASM cells treated with cigarette smoke extract. LOXL1 and LOXL2 protein levels were higher in small airways from current- compared to non-smokers. In COPD patients, higher LOXL1 and lower LOX protein levels were observed, but no differences for LOXL2, LOXL3, and LOXL4 protein were detected in small airways. Inhibiting LOs activity increased airway contraction in murine lung slices. COPD-associated changes in LOs, in particular LOX and LOXL1, may be related to smoking and contribute to impaired airway function, providing potential novel targets for preventing or treating small airways changes in COPD.

Smoking induces shifts in cellular composition and transcriptome within the bronchial mucus barrier.

BACKGROUND AND OBJECTIVE: Smoking disturbs the bronchial-mucus-barrier. This study assesses the cellular composition and gene expression shifts of the bronchial-mucus-barrier with smoking to understand the mechanism of mucosal damage by cigarette smoke exposure. We explore whether single-cell-RNA-sequencing (scRNA-seq) based cellular deconvolution (CD) can predict cell-type composition in RNA-seq data. METHODS: RNA-seq data of bronchial biopsies from three cohorts were analysed using CD. The cohorts included 56 participants with chronic obstructive pulmonary disease [COPD] (38 smokers; 18 ex-smokers), 77 participants without COPD (40 never-smokers; 37 smokers) and 16 participants who stopped smoking for 1 year (11 COPD and 5 non-COPD-smokers). Differential gene expression was used to investigate gene expression shifts. The CD-derived goblet cell ratios were validated by correlating with staining-derived goblet cell ratios from the COPD cohort. Statistics were done in the R software (false discovery rate p-value < 0.05). RESULTS: Both CD methods indicate a shift in bronchial-mucus-barrier cell composition towards goblet cells in COPD and non-COPD-smokers compared to ex- and never-smokers. It shows that the effect was reversible within a year of smoking cessation. A reduction of ciliated and basal cells was observed with current smoking, which resolved following smoking cessation. The expression of mucin and sodium channel (ENaC) genes, but not chloride channel genes, were altered in COPD and current smokers compared to never smokers or ex-smokers. The goblet cell-derived staining scores correlate with CD-derived goblet cell ratios. CONCLUSION: Smoking alters bronchial-mucus-barrier cell composition, transcriptome and increases mucus production. This effect is partly reversible within a year of smoking cessation. CD methodology can predict goblet-cell percentages from RNA-seq.

In-depth molecular analysis of combined and co-primary pulmonary large cell neuroendocrine carcinoma and adenocarcinoma.

Up to 14% of large cell neuroendocrine carcinomas (LCNECs) are diagnosed in continuity with nonsmall cell lung carcinoma. In addition to these combined lesions, 1% to 7% of lung tumors present as co-primary tumors with multiple synchronous lesions. We evaluated molecular and clinicopathological characteristics of combined and co-primary LCNEC-adenocarcinoma (ADC) tumors. Ten patients with LCNEC-ADC (combined) and five patients with multiple synchronous ipsilateral LCNEC and ADC tumors (co-primary) were included. DNA was isolated from distinct tumor parts, and 65 cancer genes were analyzed by next generation sequencing. Immunohistochemistry was performed including neuroendocrine markers, pRb, Ascl1 and Rest. Pure ADC (N = 37) and LCNEC (N = 17) cases were used for reference. At least 1 shared mutation, indicating tumor clonality, was found in LCNEC- and ADC-parts of 10/10 combined tumors but only in 1/5 co-primary tumors. A range of identical mutations was observed in both parts of combined tumors: 8/10 contained ADC-related (EGFR/KRAS/STK11 and/or KEAP1), 4/10 RB1 and 9/10 TP53 mutations. Loss of pRb IHC was observed in 6/10 LCNEC- and 4/10 ADC-parts. The number and intensity of expression of Ascl1 and neuroendocrine markers increased from pure ADC (low) to combined ADC (intermediate) and combined and pure LCNEC (high). The opposite was true for Rest expression. In conclusion, all combined LCNEC-ADC tumors were clonally related indicating a common origin. A relatively high frequency of pRb inactivation was observed in both LCNEC- and ADC-parts, suggesting an underlying role in LCNEC-ADC development. Furthermore, neuroendocrine differentiation might be modulated by Ascl1(+) and Rest(-) expression.

Leukapheresis increases circulating tumour cell yield in non-small cell lung cancer, counts related to tumour response and survival.

BACKGROUND: Circulating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes. METHODS: Patients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 108 leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs. RESULTS: Fifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p < 0.01). CTCs per 7.5 mL DLA product were median 9.2 times (interquartile range = 5.6-24.0) higher than CTCs in 7.5 mL blood. RosetteSEP did not significantly improve CTC detection (pretreatment: 34/55, 62%, post treatment: 16/34, 47%) and CTCs per mL even decreased compared to DLA (p = 0.04).. Patients with advanced-stage disease with DLA-CTC after treatment showed fewer tumour responses and shorter progression-free survival (PFS) than those without DLA-CTC (median PFS, 2.0 vs 12.0 months, p < 0.01). DLA-CTC persistence after treatment was independent of clinical factors associated with shorter PFS (hazard ratio (HR) = 5.8, 95% confidence interval (CI), 1.4-35.5, p = 0.02). All evaluable CTCs showed aneuploidy. CONCLUSIONS: DLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS. TRIAL REGISTRATION: The study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).

Bronchial gene expression signature associated with rate of subsequent FEV1 decline in individuals with and at risk of COPD.

BACKGROUND: COPD is characterised by progressive lung function decline. Leveraging prior work demonstrating bronchial airway COPD-associated gene expression alterations, we sought to determine if there are alterations associated with differences in the rate of FEV1 decline. METHODS: We examined gene expression among ever smokers with and without COPD who at baseline had bronchial brushings profiled by Affymetrix microarrays and had longitudinal lung function measurements (n=134; mean follow-up=6.38±2.48 years). Gene expression profiles associated with the rate of FEV1 decline were identified by linear modelling. RESULTS: Expression differences in 171 genes were associated with rate of FEV1 decline (false discovery rate <0.05). The FEV1 decline signature was replicated in an independent dataset of bronchial biopsies from patients with COPD (n=46; p=0.018; mean follow-up=6.76±1.32 years). Genes elevated in individuals with more rapid FEV1 decline are significantly enriched among the genes altered by modulation of XBP1 in two independent datasets (Gene Set Enrichment Analysis (GSEA) p<0.05) and are enriched in mucin-related genes (GSEA p<0.05). CONCLUSION: We have identified and replicated an airway gene expression signature associated with the rate of FEV1 decline. Aspects of this signature are related to increased expression of XBP1-regulated genes, a transcription factor involved in the unfolded protein response, and genes related to mucin production. Collectively, these data suggest that molecular processes related to the rate of FEV1 decline can be detected in airway epithelium, identify a possible indicator of FEV1 decline and make it possible to detect, in an early phase, ever smokers with and without COPD most at risk of rapid FEV1 decline.

Identification of asthma-associated microRNAs in bronchial biopsies.

BACKGROUND: Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. METHODS: Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. RESULTS: In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. CONCLUSION: Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

The discovAIR project: a roadmap towards the Human Lung Cell Atlas.

The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The Lung Biological Network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and cell-cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework programme. discovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Human Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Human Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions.

Elastin in pulmonary pathology: relevance in tumours with a lepidic or papillary appearance. A comprehensive understanding from a morphological viewpoint.

Elastin and collagen are the main components of the lung connective tissue network, and together provide the lung with elasticity and tensile strength. In pulmonary pathology, elastin staining is used to variable extents in different countries. These uses include evaluation of the pleura in staging, and the distinction of invasion from collapse of alveoli after surgery (iatrogenic collapse). In the latter, elastin staining is used to highlight distorted but pre-existing alveolar architecture from true invasion. In addition to variable levels of use and experience, the interpretation of elastin staining in some adenocarcinomas leads to interpretative differences between collapsed lepidic patterns and true papillary patterns. This review aims to summarise the existing data on the use of elastin staining in pulmonary pathology, on the basis of literature data and morphological characteristics. The effect of iatrogenic collapse and the interpretation of elastin staining in pulmonary adenocarcinomas is discussed in detail, especially for the distinction between lepidic patterns and papillary carcinoma.