Cancer diagnostics and therapeutics have widely changed, and in advanced non-small cell lung cancer (aNSCLC), molecular analysis is crucial to identify actionable biomarkers. Targeted therapies specifically interfere with molecular mechanisms involved in tumor growth and proliferation to improve clinical outcomes and quality of life compared to conventional chemotherapy. However, suboptimal testing practices, including errors in neoplastic cell percentage (NCP) assessment, limit reliable downstream analyses, and therefore, hinder accurate targeted therapy decisions for NSCLC patients. This study conducted two assessment rounds in which participants evaluated cytology or biopsy cases to examine NCP assessment accuracy and molecular testing decisions among pathologists and non-pathologists. No evidence was found for changes in NCP assessment performance across rounds (OR = 1.996, 95

Background Chronic lung allograft dysfunction (CLAD) is the leading cause of late mortality after lung transplantation. Bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) are the main underlying clinical entities. Their molecular and cellular signatures are unclear and, therefore, we aimed to identify molecular programmes associated with morphological disease severity in CLAD. Methods We performed high-resolution imaging-based gene expression profiling of 128 lung samples from explanted CLAD and donor lungs, using weighted gene co-expression network analysis, cellular and pathway enrichment, and hub gene identification. Findings were validated across four datasets, including a murine transplant model, human BOS lungs, transbronchial biopsies, and bronchoalveolar lavage fluid of lung transplant recipients. Results Unsupervised clustering revealed two transcriptomic CLAD endotypes aligning with mild-fibrotic (BOS, mild RAS) and advanced-fibrotic disease (moderate/severe RAS). Samples from the same patient often diverged molecularly, underscoring intra-patient heterogeneity and limitations of current phenotypical classification. Five molecular programmes emerged: (1) epithelial stress and innate immunity in early-fibrotic CLAD, (2) progressive adaptive immunity and cytotoxicity in advanced CLAD, (3) transient extracellular matrix remodelling, (4) progressive endothelial loss/dysfunction, and (5) progressive loss of homeostasis, wherein multiple potential druggable targets were detected. Finally, we identified a 26 CLAD hub gene-panel, that showed robust diagnostic performance to discriminate CLAD. Conclusion CLAD is a spatially heterogeneous, yet molecularly continuous disease process, wherein BOS and RAS represent variable stages of a shared immunopathological continuum. Our findings support the development of lung-specific molecular classifiers to guide diagnostics and reveal novel targets for personalised therapies in transplantation.

Background:Immune checkpoint blockade (ICB) is widely used to treat patients with non-small cell lung cancer (NSCLC). However, intratumor (immune) heterogeneity may significantly impact the therapeutic efficacy of ICBs. It remains unknown whether larger core biopsies better represent the intratumor heterogeneity. This study aimed to explore the intra- and interpatient heterogeneity of selected immune-related parameters on varying simulated biopsy core sizes. Methods:In this exploratory prospective cohort study, patients with stage I-III NSCLC who had undergone curative-intent surgery, with or without neoadjuvant chemotherapy or chemoradiation, were enrolled. Intratumor immune heterogeneity was assessed using quadruple immunofluorescence stainings (CD31, Ki67, CD4, CD8, CD68, FOXP3, PD-L1, Pan-keratin, and DAPI) on resected tumor specimens. Digital images were used to simulate biopsy cores ranging from 1- to 4-mm in diameter. Heterogeneity was quantified using the quartile coefficient of dispersion (QCD) across 250 randomly selected subregions per core size. Results:Tumor specimens from 29 patients were analyzed, including 10 patients who had received neoadjuvant therapy. Intratumor QCD values decreased significantly with increasing simulated biopsy core diameter for all markers. Nonetheless, substantial spatial heterogeneity persisted for programmed death-ligand 1 (PD-L1), with 58.8% of patients showing heterogeneous distribution, even in 4-mm simulated biopsy cores. Conclusions:Larger biopsy core sizes were associated with reduced intratumor heterogeneity for most immune markers, suggesting a better reflection of the tumor immune landscape of the whole tumor. However, PD-L1 expression remained spatially heterogeneous even in larger cores. Further validation in larger prospective cohort studies will be essential to determine the translational relevance of heterogeneity metrics and to better inform biopsy sampling strategies in routine clinical practice.

OBJECTIVE:Formalin-fixed, paraffin-embedded (FFPE) cell blocks (CBs) are widely used for processing cytology specimens, but preparation methods remain variable and non-standardised. Low cellularity is a common limitation that may compromise diagnostic utility. This study aimed to evaluate the cellularity of routinely prepared CBs across different preparation methods, laboratories and sample types. METHODS:Each laboratory in this multicentre observational study retrospectively assessed 50 consecutive CBs prepared using a single method. Cellularity of Haematoxylin and Eosin (H&E)-stained sections was semi-quantitatively evaluated by two independent reviewers per laboratory in four categories: acellular, low (≤ 100 cells), medium (100-500 cells) and high cellular (> 500 cells). The proportion of poorly cellular CBs (acellular + low cellular) was compared across methods, laboratories and sample types using non-parametric tests. RESULTS:Cellularity was assessed for 1817 CBs prepared using Agar (22%), HistoGel (19%), Plasma-Thrombin (19%), Cellient (17%), In-house (15%) and Shandon/Epredia (8%) methods. The proportion of poorly cellular CBs ranged from 4% to 80% across laboratories (mean 27%, median 25%), with no consistent clustering by method. Median proportions by method ranged from 12% (Cellient) to 35% (Agar), but inter-method differences were not statistically significant (p > 0.05). Substantial variability was observed both across and within sample types and preparation methods. CONCLUSIONS:The study revealed marked variability in the proportion of poorly cellular CBs among preparation methods and laboratories, highlighting the need for improved processing and monitoring of CB adequacy.

BACKGROUND:Post-infectious bronchiolitis obliterans (PIBO) may occur following childhood infections. Subsequent dysanaptic lung growth, with differential development of the alveolar compartment over the airways, remains elusive. We performed a morphological characterization of the whole airway tree in PIBO compared to bronchiolitis obliterans syndrome (BOS) after lung transplantation. METHODS:Lungs from matched PIBO (n=5), BOS (n=5), and non-diseased donors (n=5) were evaluated using ex vivo high-resolution computed tomography (CT) scans followed by three-dimensional (3D)-airway segmentation. Matched lung tissue samples (n=4 locations/lung) were scanned with micro-CT (resolution: 5-10 µm) for 3D terminal bronchiole assessment and histology. RESULTS:No significant difference in the number of airways per generation (until generation 11) was observed between groups (p=0.86). Airway diameters in PIBO (generations 6-11) and BOS (generations 7-10) were increased compared to controls (overall p=0.018), without significant differences between PIBO and BOS. More obstructed airways were present in BOS versus PIBO (p=0.016), but airway obstructions were larger in PIBO versus BOS (p=0.032). There were significantly fewer terminal bronchioles in PIBO compared to BOS and controls (PIBO: median 2934 terminal bronchioles/lung (IQR:2247-4115), BOS 8424 terminal bronchioles/lung (IQR:6207-10480), controls 10 903 terminal bronchioles/lung (IQR:7583-12 820), p=0.0009), but terminal bronchiole diameters were not significantly different (p=0.37). Obstruction of pre-terminal bronchioles was segmental (i.e., focal with normal distal terminal bronchiole) in BOS, but partly non-focal (non-reopening bronchiole) in PIBO. CONCLUSIONS:PIBO lungs display an almost threefold decrease in terminal bronchioles compared to BOS, with morphological differences in the type and location of airway obstructions, providing structural evidence supporting dysanaptic lung growth.

Although the SARS-CoV-2 pandemic is now largely controlled through widespread vaccination and available therapeutics, emerging variants could undermine their overall effectiveness. 4 '-Thiouridine (TU) and its 5 '-isobutyryl prodrug (iBu TU) were identified as potent inhibitors of SARS-CoV-2 replication across various cell models, including advanced primary human respiratory models. Both compounds exhibited no significant toxicity across a range of cell culture systems, with no signs of mitochondrial damage or elevated lactic acid production. Furthermore, TU and iBu TU tested negative for mutagenicity, aneugenic and clastogenic effects, alleviating concerns about genotoxicity. They also showed no interaction with a broad spectrum of ion channels, GPCRs, and enzymes, further confirming their favorable safety profile. TU is metabolized intracellularly to its active triphosphate form (TU-TP), which effectively disrupts viral RNA replication by acting as a delayed chain terminator. iBu TU is rapidly converted to TU in plasma and displays superior oral bioavailability compared to TU (F = 90% vs 42% in monkeys). In a Syrian hamster model infected with SARS-CoV-2, oral iBu TU significantly reduced viral loads (1.7 to 4.5 log10) and improved lung health without adverse effects. These findings highlight iBu TU as a promising oral antiviral candidate for treating SARS-CoV-2 infection.

Pregnant women are at higher risk of severe COVID-19, with vaccine access and hesitancy remaining a challenge. Here, we use a pregnant female hamster model of COVID-19 to explore the effects of maternal infection on pregnancy, revealing a significant increase in intrauterine growth restriction (IUGR) due to placental inflammation. Viral infection causes bronchopneumonia and weight loss in infected dams, but no vertical transmission occurs. IUGR is instead linked to placental damage, characterized by fibrin deposition, thrombosis, and elevated placental expression of IP10, IL6, and IL10, irrespective of fetal sex. Enoxaparin treatment reduces placental damage and improves fetal outcomes, while vaccination enhances viral clearance, protects the placenta, and reduces the risk of IUGR. These findings underscore placentitis as a key driver of fetal complications upon SARS-CoV-2 infection and highlight the potential of vaccination and anticoagulant therapy to protect both mother and child.

Although the SARS-CoV-2 pandemic is now largely controlled through widespread vaccination and available therapeutics, emerging variants could undermine their overall effectiveness. 4'-Thiouridine (TU) and its 5'-isobutyryl prodrug (iBu TU) were identified as potent inhibitors of SARS-CoV-2 replication across various cell models, including advanced primary human respiratory models. Both compounds exhibited no significant toxicity across a range of cell culture systems, with no signs of mitochondrial damage or elevated lactic acid production. Furthermore, TU and iBu TU tested negative for mutagenicity, aneugenic and clastogenic effects, alleviating concerns about genotoxicity. They also showed no interaction with a broad spectrum of ion channels, GPCRs, and enzymes, further confirming their favorable safety profile. TU is metabolized intracellularly to its active triphosphate form (TU-TP), which effectively disrupts viral RNA replication by acting as a delayed chain terminator. iBu TU is rapidly converted to TU in plasma and displays superior oral bioavailability compared to TU (F = 90% vs 42% in monkeys). In a Syrian hamster model infected with SARS-CoV-2, oral iBu TU significantly reduced viral loads (1.7 to 4.5 log10) and improved lung health without adverse effects. These findings highlight iBu TU as a promising oral antiviral candidate for treating SARS-CoV-2 infection.

Intravascular large B-cell lymphoma forms a rare entity that may mimic disease symptoms according to the affected sites. It is characterized by the presence of large atypical lymphoid B cells restricted to intravascular spaces, especially capillaries. We present a 65-year-old man who presented with a concurrent intravascular large B-cell lymphoma and Waldenström macroglobulinemia with prominent pulmonary symptoms masquerading as a hypersensitivity pneumonitis. A subsequent surgical lung biopsy illustrated peribronchial and interstitial nodular aggregates which consisted of numerous capillary structures filled with neoplastic B lymphocytes. This report hints toward potential induction of prominent neo-angiogenesis by intravascular large B-cell lymphoma, with development of intrapulmonary nodular aggregates mimicking interstitial lung disease.

Rationale: Cystic fibrosis (CF) is characterized by bronchiectasis on imaging, while functionally evolving toward obstructive impairment. Despite its assumed importance in CF, small airway remodeling and its relation to bronchiectasis remains poorly understood. Objectives: The aim of our study was to explore both large and small airway disease morphometrically, by using detailed imaging techniques, such as ex vivo high-resolution computed tomography (HRCT) and micro-computed tomography (μCT), and histological analysis in advanced CF. Methods: On HRCT (600 μm; CF, n = 21; control, n = 6) and μCT (150 μm; CF, n = 3; control, n = 1) scans of inflated explanted lungs, the ratio of visible airway volume to total lung volume (AV%) was calculated as a marker of bronchiectasis, while airway segmentation was used for generation analysis. Clinical data were retrospectively collected. On μCT (8.5 μm) images of lung cores (±2.8 cm3), extracted randomly from each lobe (three per lobe), distal airway (DA) diameter, number of airway collapses, and number of open terminal bronchioles per milliliter were analyzed. Morphometric analysis was supplemented with histological analysis of DA collapse. Results: AV% on HRCT was heterogeneous among CF lungs (0.7-4.6%), overlapping with controls (0.4-1.2%). However, the pattern of airway loss on μCT was homogeneous among CF lungs and most pronounced from generations 9-16. AV% did not correlate with the number of open terminal bronchioles per milliliter or percentage predicted forced expiratory volume in 1 second, which correlated with each other. Open DAs in CF lungs were narrowed compared with DA in controls. On the other hand, collapsed DAs in CF lungs showed varying degrees of proximal dilation, with DA diameter correlating with AV%. On histology, collapsed CF DAs showed constrictive bronchiolitis. Conclusions: Airway remodeling in end-stage CF is heterogeneous, ranging from minimal bronchiectasis, overlapping with control lungs, to extensive bronchiectasis with small airway dilation. However, the degree of bronchiectasis is unrelated to functional impairment or the amount of small airway loss, underscoring the importance of small airway disease.

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory diseases, particularly in young children, the elderly and immunocompromised. There are no approved antiviral drugs against this virus. We report that the combination of ribavirin with either remdesivir or its parent nucleoside GS-441524 results in a pronounced antiviral effect against HPIV-3 in LLC-MK2 cells and in human airway epithelial cells grown at the air–liquid interface. In AG129 mice intranasally inoculated with HPIV-3, the combined treatment with ribavirin and GS-441524 decreased infectious viral lung titers by >2.5 log10 to undetectable levels in 4 out of 11 mice and by 1.6 log10 in the remaining 7 mice as compared with the vehicle. The lungs of all mice that received the combined treatment appeared histologically normal or virtually normal, whereas 8 of 11 vehicle-treated mice presented with bronchopneumonia. By contrast, ribavirin alone did not result in a reduction in infectious viral lung titers; GS-441524 alone reduced infectious viral lung titers by 1.2 log10. Moreover, several mice in the single-treatment groups exhibited severe lung pathology. These findings may warrant exploring this combination in patients with severe HPIV-3 infections and possibly also against infections with other viruses that are susceptible in vitro to these two drugs.

To evaluate the diagnostic accuracy of PET/CT, cranial MRI, ultrasound and temporal artery biopsy (TAB) in patients with suspected giant cell arteritis (GCA) in a direct comparison. Consecutive patients with a suspicion of GCA and at least 2 diagnostic tests ≤ 7 days after initiation of glucocorticoids between June 2021 and June 2024, were included retrospectively. The gold standard for the diagnosis of GCA was the judgment of experienced clinicians after a follow-up of ≥ 6 months. Examinations were compared within subgroups undergoing the same tests. Sixty-one GCA patients and 50 patients with an alternative diagnosis were included. Combined cranial and large vessel PET/CT had the highest sensitivity (89

PURPOSE:CD276 (B7-H3) is an immunoregulatory protein that plays an important role in the inhibition of T-cell function. CD276 is overexpressed on a variety of human solid cancer cells with limited expression in normal tissues, making it an appealing target for innovative cancer immunotherapy approaches. Pleural mesothelioma (PM) is a highly aggressive disease with a need for new treatment options. Our objective was to investigate the expression of CD276 in the multicenter PM cohort of the European Thoracic Oncology Platform Mesoscape project and correlate the results with annotated clinical data. MATERIALS AND METHODS:Using tissue microarrays (TMAs), the expression of CD276, assessed using a semiquantitative aggregate H-score method on the membrane (and secondarily in the cytoplasm), was correlated with clinicopathologic characteristics and survival outcome. RESULTS:CD276 immunohistochemistry results were available for 353 patients, with mostly epithelioid histology (71%). Membranous CD276 expression was present in 86%. High membranous CD276 expression (H-score ≥the median H-score of 120) was significantly more common in females (P = .0029; 71% v 47%) and in epithelioid histology (P < .001; 59% v 29%), whereas no significant association in clinical outcome (overall survival [OS]/progression-free survival) was found. Cross-validation of the TMA method using whole sections revealed a moderate agreement for membranous assessment (Cohen's kappa = 0.47) and a lower agreement for cytoplasm assessment (Cohen's kappa = 0.37). In an exploratory analysis, high cytoplasmic CD276 expression was associated with worse prognosis (OS, log-rank P = .043), but was not significant when adjusting for other clinical variables. CONCLUSION:Although no prognostic value of CD276 expression was found, its high membranous expression (86%) in the PM samples of the study supports further research of its potential as a therapeutic target for this disease.

Introduction:Invasive mucinous adenocarcinoma (IMA) of the lung has a less aggressive behavior than other adenocarcinoma subtypes. Overall, the propensity for nodal and distant metastases is low, but spread throughout the lungs is frequent. The radiographic "ground glass" presentation makes differentiation between infectious and inflammatory consolidations challenging, and the diagnosis of malignancy is often unexpected. Case Presentation:A 50-year-old patient underwent double lung transplantation (LTx) in July 2018 for progressive fibrosing interstitial lung disease (ILD). IMA was unexpectedly found in the explant lungs. Pre-transplant PET-CT scans suggested inflammatory ILD without malignancy. Endobronchial ultrasound-guided transbronchial fine needle aspiration of the enlarged mediastinal lymph nodes demonstrated no evidence of malignancy. Post-transplant pathology confirmed stage IVA IMA with a KRAS G12D mutation. After 3 years, recurrent IMA was detected. The asymptomatic patient remains under close surveillance with stable lung function, and tailored treatment will be considered if progression occurs. Conclusion:IMA is currently rarely considered an indication for LTx. The risk of recurrence after transplantation is substantial, and recurrence negatively impacts long-term post-transplant prognosis. Incidental adenocarcinoma in explant lungs will remain a complication of imperfect transplant recipient selection. A high index of suspicion of disease recurrence in the donor lungs should be maintained in these patients. Further research is required to understand the optimal screening, treatment, and follow-up of these patients.

Postmortem microbiological tests can help confirm infectious causes of death and identify the responsible microorganism (MO). However, challenges arise due to the lack of consensus and regulatory guidelines. This prospective single-center study conducted at University Hospitals Leuven from 2013 to 2016, aimed to assess the relevance as well as site- and time-related aspects of postmortem microbiology. A total of 200 clinical and forensic autopsies were included in the study, with 1321 samples collected. Approximately one-fifth of cases revealed an infectious cause of death, predominantly pneumonia. Histopathological classification into infectious and non-infectious causes of death served as the gold standard against which microbiological results were compared. The group of MO (i.e., group 1 and group 2 pathogens as well as a small number of specific pathogens), the number of positive sites, specific sampling sites, and the unique presence of a pathogen at a given site were all significantly related to infectious deaths. We propose that the concept of polymicrobial overgrowth as contamination primarily pertains to non-pathogenic MOs. We recommend the sampling of internal or external peripheral blood, heart blood, left lung, and spleen as part of a standardized protocol. Although the postmortem interval (PMI) was shown to affect microbiological results, its clinical relevance warrants further investigation.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fibrosing interstitial lung disease with poor prognosis. Previously, we have shown that Claudin-1 (CLDN1) expression is increased in IPF lungs, upon becoming exposed outside tight junctions. In mild disease, the presence of exposed CLDN1 was reported in the epithelium lining fibroblastic foci, areas of bronchiolization and squamous metaplasia. Here, we aimed to identify the populations expressing CLDN1 and investigate its regulatory network in relation to disease progression. METHODS: Spatial transcriptomics was performed using the 10X Xenium platform to evaluate 389 genes across 8 samples, including 6 IPF (2 of each disease stage: mild, moderate, severe) and 2 control lungs. A consensus approach based on manual annotation and multiple reference datasets was used for cell type mapping. Regions of interest were manually annotated by expert histopathologists. Differential gene expression and gene regulatory network (GRN) analyses were conducted to investigate the role of CLDN1 in IPF lungs via key regulatory interactions and pathways. Simultaneously, the therapeutic effectiveness of an anti-CLDN1 monoclonal antibody was tested in a bleomycin-induced lung fibrosis mouse model. Efficacy was evaluated using the Ashcroft Score, flexiVent® for lung function, and bulk RNA-seq. RESULTS: In control lungs, CLDN1 is predominantly expressed in airways and minimally in alveolar cells (Fig1A). In IPF, CLDN1 was detected in aberrant basaloid cells adjacent to fibroblastic foci, and in bronchial and alveolar cells in honeycombing cysts and fibrotic alveoli (Fig1A). High levels of CLDN1 were also observed in mesothelial cells in IPF lungs (Fig1B), consistent with previously published single cell data (Adams et al., Mayr et al., Sikkema et al.). GRN analysis revealed that CLDN1 is associated with extracellular matrix (ECM) organization (Fig1C). In bleomycin-induced mice, anti-CLDN1 mAb treatment significantly improved lung fibrosis (p < 0.001) and lung function (p < 0.05, Fig1D). Additionally, bulk transcriptomic analysis indicated that anti-CLDN1 mAb treatment reversed the human IPF-related gene signature, associated with ECM and inflammation (Fig1D). CONCLUSIONS: Our findings suggest a functional role for CLDN1 in IPF progression, as CLDN1 is prominently localized within aberrant epithelial populations in regions of active matrix deposition and tissue remodeling. GRN analysis and in vivo studies further support a link between CLDN1 and the regulation of ECM organization and inflammatory pathways. Finally, anti-CLDN1 mAb treatment significantly improved lung fibrosis and function in a bleomycin-induced mouse model, providing a strong preclinical proof-of-concept for Lixudebart as a novel therapy for IPF.

Rationale: The precise nature of small airway obstructions in chronic obstructive pulmonary disease (COPD) remains poorly understood, especially at early disease stages. Objectives: This study aimed to characterize small airway obstructions and numbers up to the terminal bronchioles (TBs) in smokers with limited emphysema and end-stage COPD. We hypothesized that obstruction subtypes would differ in morphology, nature, and number from early to end-stage COPD. Methods: Whole lungs were inflated and processed from seven control donors (control: declined for extrapulmonary reasons); from eight donors with a history of smoking, of whom three had <5% emphysema (smokers with no emphysema) and five had >5% emphysema (smokers with emphysema); and from eight patients with end-stage COPD. Micro-computed tomography of tissue was used to assess number of TBs, aerated TBs, and number and type of obstructions and was cross-correlated with histopathology. Measurements and Main Results: Obstructions were mainly present in smokers with emphysema and patients with COPD, resulting in less aerated TBs. On the basis of emphysema extent, more nonaerated TBs were present in regions with no emphysema than in regions with mild emphysema; however, destruction was more prominent in mild emphysema. Multiple types of obstructions were identified, comprising occlusions, webs, and collapses. In smokers with emphysema, obstructions primarily comprised webs and occlusions, whereas all obstruction types were present in COPD. On histopathology, obstructions were identified as mucus plugs. Conclusions: Multiple types of obstruction characterized as mucus plugs were identified in smokers with emphysema and patients with end-stage COPD. Their morphology, nature, and number evolved from smokers with emphysema to end-stage COPD. A shift from obstruction-dominant dysfunction to destruction-dominant pathology was found in smokers on the basis of emphysema presence.