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Pancreatic ductal adenocarcinoma (PDAC) is reported to be amongst the cancers with the lowest survival rate at 5 years. In the present study we aimed to validate a targeted next-generation sequencing (tNGS) panel to use in clinical routine, investigating genes important for PDAC diagnostic, prognostic and potential theragnostic aspect. In this NGS panel we also designed target regions to inquire about loss of heterozygosity (LOH) of chromosome 18 that has been described to be possibly linked to a worse disease progression. Copy number alteration has also been explored for a subset of genes. The last two methods are not commonly used for routine diagnostic with tNGS panels and we investigated their possible contribution to better characterize PDAC. A series of 140 formalin-fixed paraffin-embedded (FFPE) PDAC samples from 140 patients was characterized using this panel. Ninety-two % of patients showed alterations in at least one of the investigated genes (most frequent KRAS, TP53, SMAD4, CDKN2A and RNF43). Regarding LOH evaluation, we were able to detect chr18 LOH starting at 20% cell tumor percentage. The presence of LOH on chr18 is associated with a worse disease- and metastasis-free survival, in uni- and multivariate analyses. The present study validates the use of a tNGS panel for PDAC characterization, also evaluating chr18 LOH status for prognostic stratification., Competing Interests: Declaration of competing interest N.D. declares that she participated to advisory board and/or has given consultancy/presentations for: Pfizer, Astra Zeneca, Biocartis, MSD, Lilly, Roche, Bayer, BMS, Boerhinger, Amgen, Janssen, Stemline Therapeutics. The other authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Pulmonary hypertension and right ventricular (RV) dysfunction are major prognostic determinants in patients with heart failure with preserved ejection fraction (HFpEF). The underlying pathomechanisms remain unknown. In this context, we sought to study the pathogenesis of pulmonary hypertension and RV dysfunction in a rat model of obesity-associated HFpEF., Methods and Results: HFpEF was induced in obesity-prone rats fed a high-fat diet (n=13) and compared with obesity-resistant rats fed with standard chow (n=9). After 12 months, the animals underwent echocardiographic and hemodynamic evaluation followed by tissue sampling for pathobiological assessment. HFpEF rats presented mild RV pressure overload (with increased RV systolic pressure and pulmonary vascular resistance). No changes in pulmonary artery medial thickness and ex vivo vasoreactivity (to acetylcholine and endothelin-1) were observed and RNA sequencing analysis failed to identify gene clustering in HFpEF lungs. However, released nitric oxide levels were decreased in HFpEF pulmonary artery, while lung expression of preproendothelin-1 was increased. In HFpEF rats, RV structure and function were altered, with RV enlargement, decreased RV fractional area change and free wall longitudinal fractional shortening, together with altered right ventricle-pulmonary artery coupling (estimated by tricuspid annular plane systolic excursion/systolic pulmonary artery pressure). Hypertrophy and apoptosis (evaluated by transferase biotin- dUTP nick-end labeling staining) were increased in right and left ventricles of HFpEF rats. There was an inverse correlation between tricuspid annular plane systolic excursion/systolic pulmonary artery pressure and RV apoptotic rate. Plasma levels of soluble suppression of tumorigenicity-2, interleukin-1β, -6 and -17A were increased in HFpEF rats., Conclusions: Obesity-associated HFpEF in rats spontaneously evolves to pulmonary hypertension-HFpEF associated with impaired right ventricle-pulmonary artery coupling that appears disproportionate to a slight increase in RV afterload.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. We evaluated the impact of CDK4/6 inhibition by palbociclib in 28 MPM cell lines including 19 patient-derived ones, using various approaches including RNA-sequencing. Palbociclib strongly and durably inhibited the proliferation of 23 cell lines, indicating a unique sensitivity of MPM to CDK4/6 inhibition. When observed, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4. This was associated with high p16 INK4A (CDKN2A) levels that accompany RB1 defects or inactivation, or (unexpectedly) CCNE1 overexpression in the presence of wild-type RB1. Prolonged palbociclib treatment irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype including various potentially immunogenic components was irreversibly induced. Phosphorylated CDK4 was detected in 80% of 47 MPMs indicating their sensitivity to CDK4/6 inhibitors. Its absence in some highly proliferative MPMs was linked to very high p16 (CDKN2A) expression, which was also observed in public datasets in tumours from short-survival patients. Our study supports the evaluation of CDK4/6 inhibitors for MPM treatment, in monotherapy or combination therapy., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Extracellular histones have been associated with severity and outcome in sepsis. The aim of the present study was to assess the effects of sodium-β-O-Methyl cellobioside sulfate (mCBS), a histone-neutralizing polyanion, on the severity and outcome of sepsis in an experimental model., Methods: This randomized placebo-controlled experimental study was performed in 24 mechanically ventilated female sheep. Sepsis was induced by fecal peritonitis. Animals were randomized to three groups: control, early treatment, and late treatment (n = 8 each). mCBS was given as a bolus (1 mg/kg) followed by a continuous infusion (1 mg/kg/h) just after sepsis induction in the early treatment group, and 4 h later in the late treatment group. Fluid administration and antimicrobial therapy were initiated 4 h T4 after feces injection, peritoneal lavage performed, and a norepinephrine infusion titrated to maintain mean arterial pressure (MAP) between 65-75 mmHg. The experiment was blinded and lasted maximum 24 h., Results: During the first 4 h, MAP remained > 65 mmHg in the early treatment group but decreased significantly in the others (p < 0.01 for interaction, median value at T4: (79 [70-90] mmHg for early treatment, 57 [70-90] mmHg for late treatment, and 55 [49-60] mmHg for the control group). mCBS-treated animals required significantly less norepinephrine to maintain MAP than controls (p < 0.01 for interaction) and had lower creatinine (p < 0.01), lactate (p < 0.01), and interleukin-6 (p < 0.01) levels, associated with reduced changes in H3.1 nucleosome levels (p = 0.02). Early treatment was associated with lower norepinephrine requirements than later treatment. Two control animals died; all the mCBS-treated animals survived., Conclusions: Neutralization of extracellular histones with mCBS was associated with reduced norepinephrine requirements, improved tissue perfusion, less renal dysfunction, and lower circulating IL-6 in experimental septic shock and may represent a new therapeutic approach to be tested in clinical trials., (© 2023. The Author(s).)

Background: Right ventricular (RV) dysfunction remains a major problem after heart transplantation and may be associated with brain death (BD) in a donor. A calcineurin inhibitor tacrolimus was recently found to have beneficial effects on heart function. Here, we examined whether tacrolimus might prevent BD-induced RV dysfunction and the associated pathobiological changes., Methods: After randomized tacrolimus ( n = 8; 0.05 mg·kg -1 ·day -1 ) or placebo ( n = 9) pretreatment, pigs were assigned to a BD procedure and hemodynamically investigated 1, 3, 5, and 7 h after the Cushing reflex. After euthanasia, myocardial tissue was sampled for pathobiological evaluation. Seven pigs were used as controls., Results: Calcineurin inhibition prevented increases in pulmonary vascular resistance and RV-arterial decoupling induced by BD. BD was associated with an increased RV pro-apoptotic Bax-to-Bcl2 ratio and RV and LV apoptotic rates, which were prevented by tacrolimus. BD induced increased expression of the pro-inflammatory IL-6-to-IL-10 ratio, their related receptors, and vascular cell adhesion molecule-1 in both the RV and LV. These changes were prevented by tacrolimus. RV and LV neutrophil infiltration induced by BD was partly prevented by tacrolimus. BD was associated with decreased RV expression of the β-1 adrenergic receptor and sarcomere (myosin heavy chain [MYH]7-to-MYH6 ratio) components, while β-3 adrenergic receptor, nitric oxide-synthase 3, and glucose transporter 1 expression increased. These changes were prevented by tacrolimus., Conclusions: Brain death was associated with isolated RV dysfunction. Tacrolimus prevented RV dysfunction induced by BD through the inhibition of apoptosis and inflammation activation.

Background: With the increasing use of low dose CT scans, numerous pulmonary nodules are detected. As majority of them are benign, development of efficient non-surgical diagnostic intervention is mandatory. Electromagnetic navigation bronchoscopy (ENB) has been developed to reach difficult to access lesions. The aim of the present study was to compare the diagnostic yield of ENB procedures performed in a classical endoscopy suite or in a hybrid room equipped by a cone beam CT (CBCT)., Methods: A monocentric randomized study was performed in the Erasme Hospital between January 2020 and December 2021. Lung nodules of maximum 30 mm of diameter were eligible. In both arms (endoscopy or CBCT suites), ENB, fluoroscopic guidance and a radial endobronchial ultrasound were used to reach the lesion. Then six trans-bronchial biopsies (TBB) and one trans-bronchial lung cryobiopsy (TBLC) were performed. Primary outcomes were the diagnostic yield and diagnostic accuracy of the procedure., Results: Forty-nine patients were randomized (24 in the endoscopy and 25 in the CBCT arms). The lesion size was 15,9 ± 4,6 mm and 16,6 ± 6,0 mm respectively (mean ± SD, p = NS). The diagnostic yield of ENB performed under CBCT guidance was 80% compared to 42% when performed in the endoscopy suite under standard fluoroscopic guidance (p < 0,05). Similarly, the diagnostic accuracy in the CBCT group was 87% compared to 54% for the endoscopy group (p < 0,05). Duration of the procedure in the CBCT and endoscopy arms was 80 ± 23 and 61 ± 13 min respectively (mean ± SD, p < 0,01). Performing TBLC in addition to TBB increased the diagnostic yield by 14% (17 and 12,5% in CBCT and endoscopy suites respectively, p = NS)., Conclusion: This study highlighted the additional value to perform ENB procedure under CBCT guidance for small size (less than 2 cm of diameter) pulmonary nodules., Trial Registration: Clinical trial registration number: NCT05257382., (© 2023. The Author(s).)

Introduction: COVID-19-infected patients harbour neurological symptoms such as stroke and anosmia, leading to the hypothesis that there is direct invasion of the central nervous system (CNS) by SARS-CoV-2. Several studies have reported the neuropathological examination of brain samples from patients who died from COVID-19. However, there is still sparse evidence of virus replication in the human brain, suggesting that neurologic symptoms could be related to mechanisms other than CNS infection by the virus. Our objective was to provide an extensive review of the literature on the neuropathological findings of postmortem brain samples from patients who died from COVID-19 and to report our own experience with 18 postmortem brain samples., Material and Methods: We used microscopic examination, immunohistochemistry (using two different antibodies) and PCR-based techniques to describe the neuropathological findings and the presence of SARS-CoV-2 virus in postmortem brain samples. For comparison, similar techniques (IHC and PCR) were applied to the lung tissue samples for each patient from our cohort. The systematic literature review was conducted from the beginning of the pandemic in 2019 until June 1st, 2022., Results: In our cohort, the most common neuropathological findings were perivascular haemosiderin-laden macrophages and hypoxic-ischaemic changes in neurons, which were found in all cases (n = 18). Only one brain tissue sample harboured SARS-CoV-2 viral spike and nucleocapsid protein expression, while all brain cases harboured SARS-CoV-2 RNA positivity by PCR. A colocalization immunohistochemistry study revealed that SARS-CoV-2 antigens could be located in brain perivascular macrophages. The literature review highlighted that the most frequent neuropathological findings were ischaemic and haemorrhagic lesions, including hypoxic/ischaemic alterations. However, few studies have confirmed the presence of SARS-CoV-2 antigens in brain tissue samples., Conclusion: This study highlighted the lack of specific neuropathological alterations in COVID-19-infected patients. There is still no evidence of neurotropism for SARS-CoV-2 in our cohort or in the literature., (© 2023. The Author(s).)

A drug combination, vancomycin (VAN) plus tetrahydrolipstatin (THL), has demonstrated an effective synergistic action in vitro against Mycobacterium tuberculosis (Mtb). The poor oral bioavailability of VAN and THL and the predominant tropism of Mtb infection to the lungs make their pulmonary administration very attractive. To evaluate their local tolerability, bronchial cells, alveolar cells and monocytes were exposed to concentrations around and above their minimal inhibitory concentration (MIC). The VAN had no inhibitory activity on the tested human cell lines, even at a concentration 125 times higher than its MIC, whereas the THL, alone or in combination with VAN, presented a cytostatic action. Monolayer epithelium showed no significant irreversible damage at concentrations up to 100 times the combination MIC. BALB/cAnNRj mice exposed to concentration of 50 times the combination MIC delivered endotracheally 3 times a week for 3 weeks showed no clinical signs or significant weight loss. The increase of proinflammatory biomarkers (i.e., IL-1, IL-6, TNF-α and proportion of inflammatory cells) and cytotoxicity in bronchoalveolar lavage fluid (BALF) were non-significant. Lung histopathology did not show significant tissue damage. The VAN/THL combination at doses up to 50 times the combination MIC is found to be thus well tolerated by pulmonary route. This study is a promising result and encouraging further investigations of pulmonary administration of VAN/THL combination as dry powder for anti-tuberculosis treatment., (© 2022 John Wiley & Sons Ltd.)

Background & Aims: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs., Methods: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling., Results: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies., Conclusion: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy., Lay Summary: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer., Competing Interests: Conflict of interest JC consults for Crosscope, KB is Crosscope Chief Technology Officer, JS is Crosscope Chief Executive Officer. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Rationale: Donor brain death-induced lung injury may compromise graft function after transplantation. Establishing strategies to attenuate lung damage remains a challenge because the underlying mechanisms remain uncertain. Objectives: The effects of tacrolimus pretreatment were evaluated in an experimental model of brain death-induced lung injury. Methods: Brain death was induced by slow intracranial infusion of blood in anesthetized pigs after randomization to tacrolimus (orally administered at 0.25 mg ⋅ kg -1 twice daily the day before the experiment and intravenously at 0.05 mg ⋅ kg -1 1 h before the experiment; n  = 8) or placebo ( n  = 9) pretreatment. Hemodynamic measurements were performed 1, 3, 5, and 7 hours after brain death. After euthanasia of the animals, lung tissue was sampled for pathobiological and histological analysis, including lung injury score (LIS). Measurements and Main Results: Tacrolimus pretreatment prevented increases in pulmonary arterial pressure, pulmonary vascular resistance, and pulmonary capillary pressure and decreases in systemic arterial pressure and thermodilution cardiac output associated with brain death. After brain death, the ratio of Pa O 2 to Fi O 2 decreased, which was prevented by tacrolimus. Tacrolimus pretreatment prevented increases in the ratio of IL-6 to IL-10, VCAM1 (vascular cell adhesion molecule 1), circulating concentrations of IL-1β, and glycocalyx-derived molecules. Tacrolimus partially decreased apoptosis ( Bax [ Bcl2 -associated X apoptosis regulator]-to- Bcl2 [B-cell lymphoma-2] ratio [ P  = 0.07] and number of apoptotic cells in the lungs [ P  < 0.05]) but failed to improve LIS. Conclusions: Immunomodulation through tacrolimus pretreatment prevented pulmonary capillary hypertension as well as the activation of inflammatory and apoptotic processes in the lungs after brain death; however, LIS did not improve.

Background: Chemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value., Methods: Blood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels - Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC)., Results: 21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. Plasma chemerin concentration were significantly higher in ICU patients than in HC at all time-points analyzed (p<0.0001). Moreover, they were higher in deceased patients compared to survivors (p<0.05). Logistic univariate regression and multivariate analysis demonstrated that chemerin level at day 14 of admission was an independent risk factor for death. Accordingly, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein and tumor necrosis factor α. Finally, IHC analysis revealed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts in advanced acute respiratory distress syndrome (ARDS)., Discussion: Increased plasma chemerin levels are a marker of severity and may predict death of COVID-19 patients. However, multicentric studies are needed, before chemerin can be considered as a biomarker of severity and death used in daily clinical practice. Further studies are also necessary to identify the precise mechanisms of the chemerin/ChemR23 system in ARDS secondary to viral pneumonia., Competing Interests: BB received a financial support from Amgen and Boehringer Ingelheim. These funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article and the decision to submit for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lavis, Morra, Orte Cano, Albayrak, Corbière, Olislagers, Dauby, Del Marmol, Marchant, Decaestecker, Mascart, De Vos, Van de Borne, Salmon, Remmelink, Parmentier, Cardozo and Bondue.)

Background: Many postmortem studies address the cardiovascular effects of COVID-19 and provide valuable information, but are limited by their small sample size., Objectives: The aim of this systematic review is to better understand the various aspects of the cardiovascular complications of COVID-19 by pooling data from a large number of autopsy studies., Data Sources: We searched the online databases Ovid EBM Reviews, Ovid Embase, Ovid Medline, Scopus, and Web of Science for concepts of autopsy or histopathology combined with COVID-19, published between database inception and February 2021. We also searched for unpublished manuscripts using the medRxiv services operated by Cold Spring Harbor Laboratory., Study Eligibility Criteria: Articles were considered eligible for inclusion if they reported human postmortem cardiovascular findings among individuals with a confirmed SARS coronavirus type 2 (CoV-2) infection., Participants: Confirmed COVID-19 patients with post-mortem cardiovascular findings., Interventions: None., Methods: Studies were individually assessed for risk of selection, detection, and reporting biases. The median prevalence of different autopsy findings with associated interquartile ranges (IQRs)., Results: This review cohort contained 50 studies including 548 hearts. The median age of the deceased was 69 years. The most prevalent acute cardiovascular findings were myocardial necrosis (median: 100.0%; IQR, 20%-100%; number of studies = 9; number of patients = 64) and myocardial oedema (median: 55.5%; IQR, 19.5%-92.5%; number of studies = 4; number of patients = 46). The median reported prevalence of extensive, focal active, and multifocal myocarditis were all 0.0%. The most prevalent chronic changes were myocyte hypertrophy (median: 69.0%; IQR, 46.8%-92.1%) and fibrosis (median: 35.0%; IQR, 35.0%-90.5%). SARS-CoV-2 was detected in the myocardium with median prevalence of 60.8% (IQR 40.4-95.6%)., Conclusions: Our systematic review confirmed the high prevalence of acute and chronic cardiac pathologies in COVID-19 and SARS-CoV-2 cardiac tropism, as well as the low prevalence of myocarditis in COVID-19., (Copyright © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Our aims were to explore the feasibility, safety, and efficacy of peroperative transbronchial lung cryobiopsy (TBLC) guided by electromagnetic navigation bronchoscopy (ENB) and ENB-guided methylene blue marking of presumably non-palpable pulmonary nodules, and to assess its impact on video-assisted thoracoscopic surgery (VATS) and postoperative lung function., Methods: This approach was applied to 16 consecutive patients (Group A, mean age 64 years) who were compared retrospectively to a historical group of 49 patients (Group B, mean age 62 years) with similar nodules resected without guidance. The usefulness of dye marking was graded. The success rates of both ENB-guided TBLC and nodule localization through dye marking were computed. The type of resection, volume of resected parenchyma, duration of procedures, and postoperative lung function were compared between groups. Unpaired t-test, chi-square test, unpaired Wilcoxon test, and exact Fisher test were used when appropriate., Results: Malignancy was pathologically proven in all patients. TBLC revealed malignancy in 9 patients in Group A. The success rate of ENB-guided dye marking was 94%. Lobectomy was less frequently performed in Group A than in Group B (p = 0.022). Forced expiratory volume in 1 s and total lung capacity were significantly less reduced in Group A than in Group B (p = 0.006 and p = 0.019, respectively). Combined procedure was longer than surgery alone (p<0.001), but its surgical part was shorter than VATS without guidance (p < 0.001)., Conclusion: Peroperative ENB-guided TBLC with methylene blue marking of non-palpable lung nodules is feasible. A sparing lung parenchyma procedure could be achieved thanks to the ENB-guided dye marking before VATS., Competing Interests: Declarations of Competing Interest None., (Copyright © 2022 SPLF and Elsevier Masson SAS. All rights reserved.)