Pulmonology: What You May Have Missed in 2023.

The field of pulmonology saw significant advances in 2023. The publications highlighted in this article address advances and changes in practice related to asthma, chronic obstructive pulmonary disease (COPD), interstitial lung disease, pleural disorders, and sleep-disordered breathing. One article reviews data examining the efficacy of vaccination against respiratory syncytial virus, a respiratory viral illness that has had devastating effects globally. Four studies evaluate the role of various therapies in COPD, including dupilumab, ensifentrine, pulmonary rehabilitation programs, and lung volume reduction versus endobronchial valves. Another study explores the effect on vascular events of positive-pressure ventilation in patients with sleep-disordered breathing and recent stroke. The use of combination therapy with rituximab and mycophenolate mofetil on progression-free survival in patients with nonspecific interstitial pneumonia is the topic of another study. We also highlight an update of clinical recommendations for the evaluation of patients with pleural disorders and a systematic review analyzing the effectiveness of inhaled corticosteroids as a supplement to dual therapy for COPD.

Respiratory Health Effects of In Vivo Sub-Chronic Diesel and Biodiesel Exhaust Exposure.

Biodiesel, which can be made from a variety of natural oils, is currently promoted as a sustainable, healthier replacement for commercial mineral diesel despite little experimental data supporting this. The aim of our research was to investigate the health impacts of exposure to exhaust generated by the combustion of diesel and two different biodiesels. Male BALB/c mice (n = 24 per group) were exposed for 2 h/day for 8 days to diluted exhaust from a diesel engine running on ultra-low sulfur diesel (ULSD) or Tallow or Canola biodiesel, with room air exposures used as control. A variety of respiratory-related end-point measurements were assessed, including lung function, responsiveness to methacholine, airway inflammation and cytokine response, and airway morphometry. Exposure to Tallow biodiesel exhaust resulted in the most significant health impacts compared to Air controls, including increased airway hyperresponsiveness and airway inflammation. In contrast, exposure to Canola biodiesel exhaust resulted in fewer negative health effects. Exposure to ULSD resulted in health impacts between those of the two biodiesels. The health effects of biodiesel exhaust exposure vary depending on the feedstock used to make the fuel.

Clonal hematopoiesis detection in patients with cancer using cell-free DNA sequencing.

In the context of cancer, clonal hematopoiesis of indeterminate potential (CHIP) is associated with the development of therapy-related myeloid neoplasms and shorter overall survival. Cell-free DNA (cfDNA) sequencing is becoming widely adopted for genomic screening of patients with cancer but has not been used extensively to determine CHIP status because of a requirement for matched blood and tumor sequencing. We present an accurate classification approach to determine the CH status from cfDNA sequencing alone, applying our model to 4324 oncology clinical cfDNA samples. Using this method, we determined that 30.3% of patients in this cohort have evidence of CH, and the incidence of CH varies by tumor type. Matched RNA sequencing data show evidence of increased inflammation, especially neutrophil activation, within the tumors and tumor microenvironments of patients with CH. In addition, patients with CH had evidence of neutrophil activation systemically, pointing to a potential mechanism of action for the worse outcomes associated with CH status. Neutrophil activation may be one of many mechanisms, however, because patients with estrogen receptor-positive breast cancer harboring TET2 frameshift mutations had worse outcomes but similar neutrophil frequencies to patients without CH. Together, these data show the feasibility of detecting CH through cfDNA sequencing alone and an application of this method, demonstrating increased inflammation in patients with CH both systemically and in the tumor microenvironment.

Combined PD-1, BRAF and MEK inhibition in BRAFV600E colorectal cancer: a phase 2 trial.

While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and lack durability. Preclinical data suggest that BRAF/MAPK pathway inhibition may augment the tumor immune response. We performed a proof-of-concept single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC. The primary end point was overall response rate, and the secondary end points were progression-free survival, disease control rate, duration of response and overall survival. The study met its primary end point with a confirmed response rate (24.3% in all patients; 25% in microsatellite stable patients) and durability that were favorable relative to historical controls of BRAF-targeted combinations alone. Single-cell RNA sequencing of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed greater induction of tumor cell-intrinsic immune programs and more complete MAPK inhibition in patients with better clinical outcome. Immune program induction in matched patient-derived organoids correlated with the degree of MAPK inhibition. These data suggest a potential tumor cell-intrinsic mechanism of cooperativity between MAPK inhibition and immune response, warranting further clinical evaluation of optimized targeted and immune combinations in CRC. ClinicalTrials.gov registration: NCT03668431.

Biodiesel feedstock determines exhaust toxicity in 20% biodiesel: 80% mineral diesel blends.

To address climate change concerns, and reduce the carbon footprint caused by fossil fuel use, it is likely that blend ratios of renewable biodiesel with commercial mineral diesel fuel will steadily increase, resulting in biodiesel use becoming more widespread. Exhaust toxicity of unblended biodiesels changes depending on feedstock type, however the effect of feedstock on blended fuels is less well known. The aim of this study was to assess the impact of biodiesel feedstock on exhaust toxicity of 20% blended biodiesel fuels (B20). Primary human airway epithelial cells were exposed to exhaust diluted 1/15 with air from an engine running on conventional ultra-low sulfur diesel (ULSD) or 20% blends of soy, canola, waste cooking oil (WCO), tallow, palm or cottonseed biodiesel in diesel. Physico-chemical exhaust properties were compared between fuels and the post-exposure effect of exhaust on cellular viability and media release was assessed 24 h later. Exhaust properties changed significantly between all fuels with cottonseed B20 being the most different to both ULSD and its respective unblended biodiesel. Exposure to palm B20 resulted in significantly decreased cellular viability (96.3 ± 1.7%; p < 0.01) whereas exposure to soy B20 generated the greatest number of changes in mediator release (including IL-6, IL-8 and TNF-α, p < 0.05) when compared to air exposed controls, with palm B20 and tallow B20 closely following. In contrast, canola B20 and WCO B20 were the least toxic with only mediators G-CSF and TNF-α being significantly increased. Therefore, exposure to palm B20, soy B20 and tallow B20 were found to be the most toxic and exposure to canola B20 and WCO B20 the least. The top three most toxic and the bottom three least toxic B20 fuels are consistent with their unblended counterparts, suggesting that feedstock type greatly impacts exhaust toxicity, even when biodiesel only comprises 20% of the fuel.

Understanding paediatric data standards challenges through academia-industry partnerships: A conect4children (c4c) qualitative study.

INTRODUCTION: The conect4children (c4c) consortium was setup to facilitate the development of new drugs and therapies for paediatric populations and address key challenges associated with paediatric clinical trials. Two of the major adopting principles for c4c were academia-industry partnership and data harmonisation and interoperability through common eCRF definitions. To understand the challenges arising out of these principles, the c4c team at Newcastle University conducted semi-structured interviews with four c4c industry partners. METHODS: Each partner was asked 10 questions about the data standards used in their company, management and maintenance of data dictionaries, how they dealt with paediatric-specific issues, major knowledge gaps and how academia could aid in bridging these gaps. Thematic analysis was performed to identify patterns in their answers. RESULTS: All companies use the Clinical Data Interchange Standards Consortium (CDISC) standards but face problems when certain terminology is not included in CDISC (e.g., paediatric-specific terminologies). All companies were committed to interoperability and had strict policies about how additional terminology could be added to their dictionaries. Three of the four companies maintained a single dictionary but also had lighter versions for specific usage. The two major knowledge gaps identified from the interviews were handling of non-CDISC terminology and maintenance of normal lab ranges in dictionaries. DISCUSSION: To address these gaps, c4c has been working on a four-point plan including the development of a cross-cutting paediatric dictionary and a paediatric user guide in collaboration with CDISC.

Use of expert elicitation in the field of occupational hygiene: Comparison of expert and observed data distributions.

The concept of professional judgement underpins the way in which an occupational hygienist assesses an exposure problem. Despite the importance placed on professional judgement in the discipline, a method of assessment to characterise accuracy has not been available. In this paper, we assess the professional judgement of four occupational hygienists ('experts') when completing exposure assessments on a range of airborne contaminants across a number of job roles within a surface mining environment in the Pilbara region of Western Australia. The job roles assessed were project driller, mobile equipment operator, fixed plant maintainer, and drill and blast operator. The contaminants of interest were respirable crystalline silica, respirable dust, and inhalable dust. The novel approach of eliciting exposure estimates focusing on contaminant concentration and attribution of an exposure standard estimate was used. The majority of the elicited values were highly skewed; therefore, a scaled Beta distribution were fitted. These elicited fitted distributions were then compared to measured data distributions, the results of which had been collected as part of an occupational hygiene program assessing full-shift exposures to the same contaminants and job roles assessed by the experts. Our findings suggest that the participating experts within this study tended to overestimate exposures. In addition, the participating experts were more accurate at estimating percentage of an exposure standard than contaminant concentration. We demonstrate that this elicitation approach and the encoding methodology contained within can be applied to assess accuracy of exposure judgements which will impact on worker protection and occupational health outcomes.

Toxicity of different biodiesel exhausts in primary human airway epithelial cells grown at air-liquid interface.

Biodiesel is created through the transesterification of fats/oils and its usage is increasing worldwide as global warming concerns increase. Biodiesel fuel properties change depending on the feedstock used to create it. The aim of this study was to assess the different toxicological properties of biodiesel exhausts created from different feedstocks using a complex 3D air-liquid interface (ALI) model that mimics the human airway. Primary human airway epithelial cells were grown at ALI until full differentiation was achieved. Cells were then exposed to 1/20 diluted exhaust from an engine running on Diesel (ULSD), pure or 20% blended Canola biodiesel and pure or 20% blended Tallow biodiesel, or Air for control. Exhaust was analysed for various physio-chemical properties and 24-h after exposure, ALI cultures were assessed for permeability, protein release and mediator response. All measured exhaust components were within industry safety standards. ULSD contained the highest concentrations of various combustion gases. We found no differences in terms of particle characteristics for any of the tested exhausts, likely due to the high dilution used. Exposure to Tallow B100 and B20 induced increased permeability in the ALI culture and the greatest increase in mediator response in both the apical and basal compartments. In contrast, Canola B100 and B20 did not impact permeability and induced the smallest mediator response. All exhausts but Canola B20 induced increased protein release, indicating epithelial damage. Despite the concentrations of all exhausts used in this study meeting industry safety regulations, we found significant toxic effects. Tallow biodiesel was found to be the most toxic of the tested fuels and Canola the least, both for blended and pure biodiesel fuels. This suggests that the feedstock biodiesel is made from is crucial for the resulting health effects of exhaust exposure, even when not comprising the majority of fuel composition.

In Vitro primary human airway epithelial whole exhaust exposure.

The method outlined in this article is a customization of the whole exhaust exposure method generated by Mullins et al. (2016) using reprogrammed primary human airway epithelial cells as described by Martinovich et al. (2017). It has been used successfully to generate recently published data (Landwehr et al. 2021). The goal was to generate an exhaust exposure model where exhaust is collected from a modern engine, real-world exhaust concentrations are used and relevant tissues exposed to assess the effects of multiple biodiesel exposures. Exhaust was generated, gently vacuumed into a dilution chamber where it was diluted 1/15 with air and then vacuumed into an incubator containing the primary cell cultures for exposure. Exhaust physico-chemical properties including combustion gas concentrations and particle spectra were then analyzed using a combustion gas analyzer and a Universal Scanning Mobility Particle Sizer. 24 h after exposure, cellular viability and mediator release were measured using Annexin-V/PI staining and meditator multiplexing kits respectively. This method was generated to test biodiesel exhaust exposures but can be easily adapted for any type of engine exhaust exposure or even potentially other respirable environmental exposures such as woodsmoke. The main customization points for this method are:•Exhaust generated by a diesel engine equipped with EURO VI exhaust after treatment devices including diesel particulate filter and diesel oxidation catalyst.•The generated exhaust was diluted 1/15 with air to replicate real world exposure concentrations.•Used primary human airway epithelial cells obtained from bronchoscope brushings from multiple volunteers and reprogrammed to allow multiple, comparative exposures from the same individual.

Fuel feedstock determines biodiesel exhaust toxicity in a human airway epithelial cell exposure model.

BACKGROUND: Biodiesel is promoted as a sustainable replacement for commercial diesel. Biodiesel fuel and exhaust properties change depending on the base feedstock oil/fat used during creation. The aims of this study were, for the first time, to compare the exhaust exposure health impacts of a wide range of biodiesels made from different feedstocks and relate these effects with the corresponding exhaust characteristics. METHOD: Primary airway epithelial cells were exposed to diluted exhaust from an engine running on conventional diesel and biodiesel made from Soy, Canola, Waste Cooking Oil, Tallow, Palm and Cottonseed. Exhaust properties and cellular viability and mediator release were analysed post exposure. RESULTS: The exhaust physico-chemistry of Tallow biodiesel was the most different to diesel as well as the most toxic, with exposure resulting in significantly decreased cellular viability (95.8 ± 6.5%) and increased release of several immune mediators including IL-6 (+223.11 ± 368.83 pg/mL) and IL-8 (+1516.17 ± 2908.79 pg/mL) above Air controls. In contrast Canola biodiesel was the least toxic with exposure only increasing TNF-α (4.91 ± 8.61). CONCLUSION: This study, which investigated the toxic effects for the largest range of biodiesels, shows that exposure to different exhausts results in a spectrum of toxic effects in vitro when combusted under identical conditions.

Cell-free DNA captures tumor heterogeneity and driver alterations in rapid autopsies with pre-treated metastatic cancer.

In patients with metastatic cancer, spatial heterogeneity of somatic alterations may lead to incomplete assessment of a cancer's mutational profile when analyzing a single tumor biopsy. In this study, we perform sequencing of cell-free DNA (cfDNA) and distinct metastatic tissue samples from ten rapid autopsy cases with pre-treated metastatic cancer. We show that levels of heterogeneity in genetic biomarkers vary between patients but that gene expression signatures representative of the tumor microenvironment are more consistent. Across nine patients with plasma samples available, we are able to detect 62/62 truncal and 47/121 non-truncal point mutations in cfDNA. We observe that mutation clonality in cfDNA is correlated with the number of metastatic lesions in which the mutation is detected and use this result to derive a clonality threshold to classify truncal and non-truncal driver alterations with reasonable specificity. In contrast, mutation truncality is more often incorrectly assigned when studying single tissue samples. Our results demonstrate the utility of a single cfDNA sample relative to that of single tissue samples when treating patients with metastatic cancer.

The conect4children (c4c) Consortium: Potential for Improving European Clinical Research into Medicines for Children.

The need for information about new and existing drugs used in children was recognized in the European Union (EU) with the implementation of the Paediatric Regulation in 2007. In 2017, the 10-year review of the Paediatric Regulation identified barriers to the conduct of clinical trials, including delays in setting up and completing paediatric trials. Across Europe, the difficulties with clinical research are compounded by variation within countries and between countries. Ethics and regulatory review have national specificities. This paper describes the Collaborative Network for European Clinical Trials for Children (conect4children, c4c), which addresses selected difficulties in the design and conduct of paediatric clinical trials. c4c is a time-limited public-private consortium funded by the Innovative Medicines Initiative (IMI2). The elements of c4c are as follows: expert advice providing input on study design and/or paediatric development programmes (including patient involvement activities); a network of sites following harmonised procedures coordinated by National Hubs and a single point of contact for Europe; a facility for education and training for sites and trial teams; and support for managing data used by the network and a common paediatric data dictionary. c4c does not sponsor trials. c4c is taking a phased approach with careful piloting through industry and non-industry studies intended to demonstrate the viability of the network (proof-of-viability studies). c4c uses a co-design approach involving industry and academics within a clearly defined scope. A sustainable, successor organization open to all potential service users will be open for business before the end of IMI2 funding in 2024.

Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based ctDNA Assays: A Joint Consensus Recommendation of the BloodPAC's Analytical Variables Working Group.

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has demonstrated considerable promise for numerous clinical intended uses. Successful validation and commercialization of novel ctDNA tests have the potential to improve the outcomes of patients with cancer. The goal of the Blood Profiling Atlas Consortium (BloodPAC) is to accelerate the development and validation of liquid biopsy assays that will be introduced into the clinic. To accomplish this goal, the BloodPAC conducts research in the following areas: Data Collection and Analysis within the BloodPAC Data Commons; Preanalytical Variables; Analytical Variables; Patient Context Variables; and Reimbursement. In this document, the BloodPAC's Analytical Variables Working Group (AV WG) attempts to define a set of generic analytical validation protocols tailored for ctDNA-based Next-Generation Sequencing (NGS) assays. Analytical validation of ctDNA assays poses several unique challenges that primarily arise from the fact that very few tumor-derived DNA molecules may be present in circulation relative to the amount of nontumor-derived cell-free DNA (cfDNA). These challenges include the exquisite level of sensitivity and specificity needed to detect ctDNA, the potential for false negatives in detecting these rare molecules, and the increased reliance on contrived samples to attain sufficient ctDNA for analytical validation. By addressing these unique challenges, the BloodPAC hopes to expedite sponsors' presubmission discussions with the Food and Drug Administration (FDA) with the protocols presented herein. By sharing best practices with the broader community, this work may also save the time and capacity of FDA reviewers through increased efficiency.

The Effect of Caffeine and Trifluralin on Chromosome Doubling in Wheat Anther Culture.

Challenges for wheat doubled haploid (DH) production using anther culture include genotype variability in green plant regeneration and spontaneous chromosome doubling. The frequency of chromosome doubling in our program can vary from 14% to 80%. Caffeine or trifluralin was applied at the start of the induction phase to improve early genome doubling. Caffeine treatment at 0.5 mM for 24 h significantly improved green plant production in two of the six spring wheat crosses but had no effect on the other crosses. The improvements were observed in Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Caffeine had no significant effect on chromosome doubling, despite a higher frequency of doubling in several caffeine treatments in the first experiment (67-68%) compared to the control (56%). In contrast, trifluralin significantly improved doubling following a 48 h treatment, from 38% in the control to 51% and 53% in the 1 µM and 3 µM trifluralin treatments, respectively. However, trifluralin had a significant negative effect on green plant regeneration, declining from 31.8 green plants per 20 anthers (control) to 9-25 green plants per 20 anthers in the trifluralin treatments. Further work is required to identify a treatment regime with caffeine and/or anti-mitotic herbicides that consistently increases chromosome doubling in wheat without reducing green plant regeneration.

A community-based lung cancer rapid tissue donation protocol provides high-quality drug-resistant specimens for proteogenomic analyses.

BACKGROUND: For the advancement of cancer research, the collection of tissue specimens from drug-resistant tumors after targeted therapy is crucial. Although patients with lung cancer are often provided targeted therapy, post-therapy specimens are not routinely collected due to the risks of collection, limiting the study of targeted therapy resistance mechanisms. Posthumous rapid tissue donation (RTD) is an expedient collection process that provides an opportunity to understand treatment-resistant lung cancers. METHODS: Consent to participate in the thoracic RTD protocol was obtained during patient care. When death occurred, tumor and paired non-tumor, cytology, and blood specimens were collected within 48 hours and preserved as formalin-fixed and frozen specimens. Tissue sections were evaluated with hematoxylin and eosin staining and immunohistochemistry (IHC) against multiple biomarkers, including various programmed death ligand 1 (PD-L1) clones. Next-generation sequencing was performed on 13 specimens from 5 patients. RESULTS: Postmortem specimens (N = 180) were well preserved from 9 patients with lung cancer. PD-L1 IHC revealed heterogeneity within and between tumors. An AGK-BRAF fusion was newly identified in tumor from a donor with a known echinoderm microtubule-associated protein-like 4 to anaplastic lymphoma kinase (EML4-ALK) fusion and history of anaplastic lymphoma kinase (ALK) inhibitor therapy. RNA expression analysis revealed a clonal genetic origin of metastatic cancer cells. CONCLUSIONS: Post-therapy specimens demonstrated PD-L1 heterogeneity and an acyl glycerol kinase to B-rapidly accelerated fibrosarcoma (AGK-BRAF) fusion in a patient with an EML4-ALK-positive lung adenocarcinoma as a potential resistance mechanism to ALK inhibitor therapy. Rapid tissue donation collection of postmortem tissue from lung cancer patients is a novel approach to cancer research that enables studies of molecular evolution and drug resistance.

Soy Biodiesel Exhaust is More Toxic than Mineral Diesel Exhaust in Primary Human Airway Epithelial Cells.

As global biodiesel production increases, there are concerns over the potential health impact of exposure to the exhaust, particularly in regard to young children who are at high risk because of their continuing lung development. Using human airway epithelial cells obtained from young children, we compared the effects of exposure to exhaust generated by a diesel engine with Euro V/VI emission controls running on conventional diesel (ultra-low-sulfur mineral diesel, ULSD), soy biodiesel (B100), or a 20% blend of soy biodiesel with diesel (B20). The exhaust output of biodiesel was found to contain significantly more respiratory irritants, including NOx, CO, and CO2, and a larger overall particle mass. Exposure to biodiesel exhaust resulted in significantly greater cell death and a greater release of immune mediators compared to both air controls and ULSD exhaust. These results have concerning implications for potential global health impacts, particularly for the pediatric population.

Author Correction: Characterization of Nigerian breast cancer reveals prevalent homologous recombination deficiency and aggressive molecular features.

The original version of this Article contained an error in the author affiliations. The affiliation of Kevin P. White with Tempus Labs, Inc. Chicago, IL, USA was inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the Article.

Characterization of Nigerian breast cancer reveals prevalent homologous recombination deficiency and aggressive molecular features.

Racial/ethnic disparities in breast cancer mortality continue to widen but genomic studies rarely interrogate breast cancer in diverse populations. Through genome, exome, and RNA sequencing, we examined the molecular features of breast cancers using 194 patients from Nigeria and 1037 patients from The Cancer Genome Atlas (TCGA). Relative to Black and White cohorts in TCGA, Nigerian HR + /HER2 - tumors are characterized by increased homologous recombination deficiency signature, pervasive TP53 mutations, and greater structural variation-indicating aggressive biology. GATA3 mutations are also more frequent in Nigerians regardless of subtype. Higher proportions of APOBEC-mediated substitutions strongly associate with PIK3CA and CDH1 mutations, which are underrepresented in Nigerians and Blacks. PLK2, KDM6A, and B2M are also identified as previously unreported significantly mutated genes in breast cancer. This dataset provides novel insights into potential molecular mechanisms underlying outcome disparities and lay a foundation for deployment of precision therapeutics in underserved populations.

Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia.

We identified genetic mutations in CD19 and loss of heterozygosity at the time of CD19- relapse to chimeric antigen receptor (CAR) therapy. The mutations are present in the vast majority of resistant tumor cells and are predicted to lead to a truncated protein with a nonfunctional or absent transmembrane domain and consequently to a loss of surface antigen. This irreversible loss of CD19 advocates for an alternative targeting or combination CAR approach.