During the past decade, volatile organic compounds (VOCs) in exhaled breath have emerged as promising biomarkers for malignant pleural mesothelioma (MPM). However, as these biomarkers lack external validation, no breath test for MPM has been implemented in clinical practice. To address this issue, we performed the first external validation of a VOC-based prediction model for MPM. The external validation cohort was prospectively recruited, consisting of 47 MPM patients and 76 asbestos-exposed (AEx) controls. The predictive performance of the previously developed model was assessed by determining the degree of agreement between the predicted and actual outcome of the participants (patient/control). Additionally, to optimise the performance, the model was updated by refitting it to the validation cohort. External validation revealed a poor performance of the original model as the accuracy was estimated at only 41%, indicating poor generalisability. However, subsequent updating of the model improved the differentiation between MPM patients and AEx controls significantly (73% accuracy, 92% sensitivity, and 92% negative predictive value), substantiating the validity of the original predictors. This updated model will be more generalisable to the target population and exhibits key characteristics of a potential screening test for MPM, which could significantly impact MPM management.

Pleural mesothelioma (PM) is an aggressive cancer of the serosal lining of the thoracic cavity, predominantly caused by asbestos exposure. Due to nonspecific symptoms, PM is characterized by an advanced-stage diagnosis, resulting in a dismal prognosis. However, early diagnosis improves patient outcome. Currently, no diagnostic biomarkers or screening tools are available. Therefore, exhaled breath was explored as this can easily be obtained and contains volatile organic compounds, which are considered biomarkers for multiple (patho)physiological processes. A breath test, which differentiates asbestos-exposed (AEx) individuals from PM patients with 87% accuracy, was developed. However, before being implemented as a screening tool, the clinical utility of the test must be determined. Occupational AEx individuals underwent annual breath tests using multicapillary column/ion mobility spectrometry. A baseline breath test was taken and their individual risk of PM was estimated. PM patients were included as controls. In total, 112 AEx individuals and six PM patients were included in the first of four screening rounds. All six PM patients were correctly classified as having mesothelioma (100% sensitivity) and out of 112 AEx individuals 78 were classified by the breath-based model as PM patients (30% specificity). Given the large false positive outcome, the breath test will be repeated annually for three more consecutive years to adhere to the ‘test, re-test’ principle and improve the false positivity rate. A low-dose computed tomography scan in those with two consecutive positive tests will correlate test positives with radiological findings and the possible growth of a pleural tumor. Finally, the evaluation of the clinical value of a breath-based prediction model may lead to the initiation of a screening program for early detection of PM in Aex individuals, which is currently lacking. This clinical study received approval from the Antwerp University Hospital Ethics Committee (B300201837007).

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.

Background: Volatile organic compounds (VOCs) present in breath are promising biomarkers for the detection of thoracic malignancies. To gain insight into the biological origin of these VOCs and their link to disease pathogenesis, it is important to investigate VOC production on a cellular level. Aim: We aimed to determine the headspace VOC profile of malignant pleural mesothelioma (MPM) and lung cancer (LC) cell lines in order to identify specific and discriminatory VOCs for each cell type. Methods: Six MPM cell lines (H2795, H2818, H2731, H-MESO-1, NKI04, MSTO-211H), representing different histological subtypes of MPM (epithelioid, sarcomatoid and biphasic), and two non-small cell LC cell lines (H2228, H1975) were cultured under appropriate conditions (37°C and 5% CO2). The VOCs present in their headspace air were analysed with gas chromatography – mass spectrometry. As controls, headspace air was sampled from flasks only containing culture medium. The data were pre-processed and medium-corrected before being subjected to unsupervised (principle component analysis, hierarchical clustering) and supervised (Lasso regression) data analysis. Results: Conclusion: Discrimination between almost all cell types was possible with high accuracy, highlighting the potential of VOC analysis for the (differential) diagnosis of thoracic malignancies and even subtyping of MPM.

Objectives: Breath analysis based upon volatile organic compounds (VOCs) has shown to be promising for detecting malignant pleural mesothelioma (MPM) (Lamote et al, 2017, Eur Respir J). However, the biochemical origin of these VOCs remains unclear, limiting its clinical application. To investigate which VOCs arise from the tumour cells, we analysed VOCs in the headspace of different MPM cell lines by gas chromatography-mass spectrometry (GC-MS). Methods: Epithelioid (H2795), sarcomatoid (H2731) & biphasic (NKI04) MPM cell lines were cultured in triplicate under appropriate conditions. From each flask, 1600mL air was sampled over a Tenax®GR adsorption column which was subsequently analysed by GC-MS. As controls, 1600mL air was sampled in triplicate from empty culture flasks (blank) and flasks only containing culture medium, under the same conditions. After GC-MS analysis, VOCs were characterised and subjected to unsupervised clustering algorithms and principle component analysis (PCA) to differentiate between the conditions and seek for the associated VOCs. Results: Conclusion: The different cell types show a distinct smellprint, even compared to controls. This in vitro approach pinpoints MPM subtype-specific VOCs, which can optimize the breath test for patients. These MPM-specific VOCs hold promise to diagnose and subtype MPM in a non-invasive way, improving the management of asbestos-exposed individuals and MPM patients.