Predominance of M2 macrophages in organized thrombi in chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.

Thymic epithelial tumours: histopathological classification and differential diagnosis.

The epithelial and lymphoid compartments of the thymus can give rise to a wide variety of tumours, including thymomas, thymic carcinomas, lymphoreticular proliferations, germ cell tumours, and sarcomas. While some of these have close similarity to their counterparts in other organs, both in terms of histology and immunohistochemistry, as well as molecular features, others are unique to the thymus. The epithelial tumours, which can develop in the thymus, will be discussed in this review, with a particular emphasis on resolving differential diagnosis by means of morphology, immunohistochemical profiles, and molecular diagnostics.

Increase in venous thromboembolism in SARS-CoV-2 infected lung tissue: proteome analysis of lung parenchyma, isolated endothelium, and thrombi.

AIMS: COVID-19 pneumonia is characterized by an increased rate of deep venous thrombosis and pulmonary embolism. To better understand the pathophysiology behind thrombosis in COVID-19, we performed proteomics analysis on SARS-CoV-2 infected lung tissue. METHODS: Liquid chromatography mass spectrometry was performed on SARS-CoV-2 infected postmortem lung tissue samples. Five protein profiling analyses were performed: whole slide lung parenchyma analysis, followed by analysis of isolated thrombi and endothelium, both stratified by disease (COVID-19 versus influenza) and thrombus morphology (embolism versus in situ). Influenza autopsy cases with pulmonary thrombi were used as controls. RESULTS: Compared to influenza controls, both analyses of COVID-19 whole-tissue and isolated endothelium showed upregulation of proteins and pathways related to liver metabolism including urea cycle activation, with arginase being among the top upregulated proteins in COVID-19 lung tissue. Analysis of isolated COVID-19 thrombi showed significant downregulation of pathways related to platelet activation compared to influenza thrombi. Analysis of isolated thrombi based on histomorphology shows that in situ thrombi have significant upregulation of coronavirus pathogenesis proteins. CONCLUSIONS: The decrease in platelet activation pathways in severe COVID-19 thrombi suggests a relative increase in venous thromboembolism, as thrombi from venous origin tend to contain fewer platelets than arterial thrombi. Based on histomorphology, in situ thrombi show upregulation of various proteins related to SARS-CoV-2 pathogenesis compared to thromboemboli, which may indicate increased in situ pulmonary thrombosis in COVID-19. Therefore, this study supports the increase of venous thromboembolism without undercutting the involvement of in situ thrombosis in severe COVID-19.

Prediction of molecular subclasses of uveal melanoma by deep learning using routine haematoxylin-eosin-stained tissue slides.

AIMS: Uveal melanoma has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations, and these can only be obtained after genetic testing. In this study we evaluated the efficacy of patient outcome prediction using deep learning on haematoxylin and eosin (HE)-stained primary uveal melanoma slides in comparison to molecular testing. METHODS: In this retrospective study of patients with uveal melanoma, 113 patients from the Erasmus Medical Centre who underwent enucleation had tumour tissue analysed for molecular classification between 1993 and 2020. Routine HE-stained slides were scanned to obtain whole-slide images (WSI). After annotation of regions of interest, tiles of 1024 × 1024 pixels were extracted at a magnification of 40×. An ablation study to select the best-performing deep-learning model was carried out using three state-of-the-art deep-learning models (EfficientNet, Vision Transformer, and Swin Transformer). RESULTS: Deep-learning models were subjected to a training cohort (n = 40), followed by a validation cohort (n = 20), and finally underwent a test cohort (n = 48). A k-fold cross-validation (k = 3) of validation and test cohorts (n = 113 of three classes: BAP1, SF3B1, EIF1AX) demonstrated Swin Transformer as the best-performing deep-learning model to predict molecular subclasses based on HE stains. The model achieved an accuracy of 0.83 ± 0.09 on the validation cohort and 0.75 ± 0.04 on the test cohort. Within the subclasses, this model correctly predicted 70% BAP1-mutated, 61% SF3B1-mutated and 80% EIF1AX-mutated UM in the test set. CONCLUSIONS: This study showcases the potential of the deep-learning methodology for predicting molecular subclasses in a multiclass manner using HE-stained WSI. This development holds promise for advanced prognostication of UM patients without the need of molecular or immunohistochemical testing. Additionally, this study suggests there are distinct histopathological features per subclass; mainly utilizing epithelioid cellular morphology for BAP1-classification, but an unknown feature distinguishes EIF1AX and SF3B1.

Clinical Relevance of Rapid FOXF1-Targeted Sequencing in Patients Suspected of Alveolar Capillary Dysplasia With Misalignment of Pulmonary Veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.

Systematic review and meta-analysis of the prognostic impact of lymph node micrometastasis and isolated tumour cells in patients with stage I-IIIA non-small cell lung cancer.

Lymph node micrometastases could be one of the reasons for the high recurrence rate after complete surgical resection in stage I-IIIA non-small cell lung cancer (NSCLC). The standard evaluation of a single haematoxylin and eosin (H&E) slide of a paraffin-embedded section of a lymph node is insufficient for the detection of micrometastases, and there is a need for additional histopathological evaluation. The association of lymph node micrometastases with survival remains as yet unresolved. The aim of this systematic review and meta-analysis is to investigate if lymph node micrometastases and isolated tumour cells in patients with stage I-IIIA NSCLC, detected with multiple sectioning and/or immunohistochemistry (IHC) and/or reverse transcriptase polymerase chain reaction (RT-PCR), are associated with overall survival (OS) and disease-free survival (DFS) after surgical resection. We performed a meta-analysis of time-to-event outcomes based on 15 articles using ancillary techniques to detect micrometastases. We extracted the OS and DFS every 3-6 months after surgery, for patients with and without occult lymph node micrometastasis, from the survival curves published in each article. These data were used to reconstruct OS and DFS for 'micrometastasis' and 'no micrometastasis' groups. Based on all included studies that used IHC, serial sectioning, or RT-PCR, we found a 5-year OS of 55% (micrometastasis) vs. 75% (no micrometastasis), and a 5-year DFS of 53% (micrometastasis) vs. 75% (no micrometastasis). Patients with stage I-IIIA NSCLC with lymph node micrometastases detected by ancillary histopathological and molecular techniques have a significantly poorer OS and DFS compared to patients without lymph node micrometastases.

Increase of mast cells in COVID-19 pneumonia may contribute to pulmonary fibrosis and thrombosis.

AIMS: Lung tissue from COVID-19 patients shares similar histomorphological features with chronic lung allograft disease, also suggesting activation of autoimmune-related pathways in COVID-19. To more clearly understand the underlying spectrum of pathophysiology in COVID-19 pneumonia, we analysed mRNA expression of autoimmune-related genes in post-mortem lung tissue from COVID-19 patients. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded lung tissue samples of 18 COVID-19 patients and eight influenza patients were used for targeted gene expression profiling using NanoString technology. Multiplex immunofluorescence for tryptase and chymase was applied for validation. Genes related to mast cells were significantly increased in COVID-19. This finding was strengthened by multiplex immunofluorescence also showing a significant increase of tryptase- and chymase-positive cells in COVID-19. Furthermore, receptors for advanced glycation end-products (RAGE) and pro-platelet basic protein (PPBP) were up-regulated in COVID-19 compared to influenza. Genes associated with Type I interferon signalling showed a significant correlation to detected SARS-CoV2 pathway-related genes. The comparison of lung tissue samples from both groups based on the presence of histomorphological features indicative of acute respiratory distress syndrome did not result in finding any specific gene or pathways. CONCLUSION: Two separate means of measuring show a significant increase of mast cells in SARS-CoV-2-infected lung tissue compared to influenza. Additionally, several genes involved in fibrosis and thrombosis, among which are RAGE and PPBP, are up-regulated in COVID-19. As mast cells are able to induce thrombosis and fibrosis, they may play an important role in the pathogenesis of COVID-19.

Epicardial fat volume is related to the degree of cardiac allograft vasculopathy.

OBJECTIVES: Increasing evidence suggests a role for epicardial fat in the development of coronary artery disease in the general population. Heart transplantation patients are at increased risk of developing a specific form of coronary artery disease, cardiac allograft vasculopathy (CAV), which has far-reaching consequences in terms of morbidity and mortality. Until now, the role of epicardial fat volume (EFV) in the development of CAV remains unknown. Hence, we investigated the relationship between EFV and CAV as well as the influence of donor/recipient sex on EFV. METHODS: Adult heart transplant patients who underwent coronary computed tomography angiography (CCTA) for CAV screening who were four or more years post-HT were included. Using the CT examinations, we quantified the EFV and the degree of CAV. Ordinal and linear regression models were used to assess the association of EFV with CAV. RESULTS: In total, 149 (median age 44.5 years, 36% women) patients were included. The median time between HT and the CT scan was 11.0 (7.3-16.1) years. CAV grade 0, 1, 2 and 3 were seen in 85 (57%), 32 (22%), 14 (9%), and 18 (12%) patients, respectively. The median EFV was 208.4 (128.9-276.0) mL. Larger EFV were related to higher degrees of CAV (median of 164.7 to 290.6 mL for CAV grade 0 and 3, respectively, OR 5.23 (2.47-11.06), p < 0.001). Male recipients had significantly more EFV than female recipients irrespective of the donor sex (232.7 mL vs. 147.2 mL respectively, p < 0.001). Determinants for EFV were recipient sex, number of rejections, donor age, time between HT and CT scan, recipient BMI, and diabetes mellitus. CONCLUSIONS: EFV was associated with higher degrees of CAV. The recipient sex influenced the EFV more than the donor sex. KEY POINTS: • Patients after heart transplantation have a high amount of epicardial fat while larger amounts of epicardial fat are related to higher grades of cardiac allograft vasculopathy. • Determinants of higher epicardial fat volume included recipient sex, number of rejections, donor age, time between HT and CT scan, recipient BMI, and diabetes mellitus. • Longitudinal studies are needed to further disentangle the role of epicardial fat in the development and progression of cardiac allograft vasculopathy. Demonstration of four patients (from CAV grade 0 to CAV grade 3) in whom epicardial fat volume was determined. In red, the voxels identified as epicardial fat.

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.

Personalised selection of experimental treatment in patients with advanced solid cancer is feasible using whole-genome sequencing.

BACKGROUND: Biomarker-guided therapy in an experimental setting has been suggested to improve patient outcomes. However, trial-specific pre-screening tests are time and tissue consuming and complicate the personalised treatment of patients eligible for early-phase clinical trials. In this study the feasibility of whole-genome sequencing (WGS) as a one-test-for-all for guided inclusion in early-phase trials was investigated. METHODS: Phase I Molecular Tumor Board (MTB) at the Erasmus MC Cancer Institute reviewed patients with advanced cancer without standard-of-care treatment (SOC) options for a 'fresh-frozen' (FF) tumour biopsy for WGS based on clinical-pathological features. Clinical grade WGS was performed by Hartwig Medical Foundation. MTB matched the patient with a trial, if available. RESULTS: From September 2019-March 2021, 31 patients with highly diverse tumour types underwent a tumour biopsy for WGS. The median turnaround time (TAT) was 15 days [10-42 days]. At least one actionable event was found in 84% of the patients (26/31). One-third of the patients (11/31) received matched experimental treatment. CONCLUSIONS: WGS on fresh FF biopsies is a feasible tool for the selection of personalised experimental therapy in patients with advanced cancer without SOC options. WGS is now possible in an acceptable TAT and thus could fulfil the role of a universal genomic pre-screening test.

Incidence, treatment and survival of malignant pleural and peritoneal mesothelioma: a population-based study.

INTRODUCTION: Malignant mesothelioma (MM) is an aggressive cancer that primarily arises from the pleura (MPM) or peritoneum (MPeM), mostly due to asbestos exposure. This study reviewed the Dutch population-based incidence, treatment and survival since the national ban on asbestos in 1993. MATERIALS AND METHODS: Patients with MPM or MPeM diagnosed from 1993 to 2018 were selected from the Dutch cancer registry. Annual percentage change (APC) was calculated for (age-specific and sex-specific) revised European standardised incidence rates (RESR). Treatment pattern and Kaplan-Meier overall survival analyses were performed. RESULTS: In total, 12 168 patients were included in the study. For male patients younger than 80 years, the MM incidence significantly decreased in the last decade (APC ranging between -9.4% and -1.8%, p<0.01). Among both male and female patients aged over 80 years, the incidence significantly increased during the entire study period (APC 3.3% and 4.6%, respectively, p<0.01). From 2003 onwards, the use of systemic chemotherapy increased especially for MPM (from 9.3% to 39.4%). Overall, 62.2% of patients received no antitumour treatment. The most common reasons for not undergoing antitumour treatment were patient preference (42%) and performance status (25.6%). The median overall survival improved from 7.3 (1993-2003) to 8.9 (2004-2011) and 9.3 months from 2012 to 2018 (p<0.001). CONCLUSION: The peak of MM incidence was reached around 2010 in the Netherlands, and currently the incidence is declining in most age groups. The use of systemic chemotherapy increased from 2003, which likely resulted in improved overall survival over time. The majority of patients do not receive treatment though and prognosis is still poor.

Expression of phosphorylated ribosomal protein S6 in mesothelioma patients - correlation with clinico-pathological characteristics and outcome: results from the European Thoracic Oncology Platform (ETOP) Mesoscape project.

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p < 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies.

Development and validation of a supervised deep learning algorithm for automated whole-slide programmed death-ligand 1 tumour proportion score assessment in non-small cell lung cancer.

AIMS: Immunohistochemical programmed death-ligand 1 (PD-L1) staining to predict responsiveness to immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) has several drawbacks: a robust gold standard is lacking, and there is substantial interobserver and intraobserver variance, with up to 20% discordance around cutoff points. The aim of this study was to develop a new deep learning-based PD-L1 tumour proportion score (TPS) algorithm, trained and validated on a routine diagnostic dataset of digitised PD-L1 (22C3, laboratory-developed test)-stained samples. METHODS AND RESULTS: We designed a fully supervised deep learning algorithm for whole-slide PD-L1 assessment, consisting of four sequential convolutional neural networks (CNNs), using aiforia create software. We included 199 whole slide images (WSIs) of 'routine diagnostic' histology samples from stage IV NSCLC patients, and trained the algorithm by using a training set of 60 representative cases. We validated the algorithm by comparing the algorithm TPS with the reference score in a held-out validation set. The algorithm had similar concordance with the reference score (79%) as the pathologists had with one another (75%). The intraclass coefficient was 0.96 and Cohen's κ coefficient was 0.69 for the algorithm. Around the 1% and 50% cutoff points, concordance was also similar between pathologists and the algorithm. CONCLUSIONS: We designed a new, deep learning-based PD-L1 TPS algorithm that is similarly able to assess PD-L1 expression in daily routine diagnostic cases as pathologists. Successful validation on routine diagnostic WSIs and detailed visual feedback show that this algorithm meets the requirements for functioning as a 'scoring assistant'.

Molecular pathology testing for non-small cell lung cancer: an observational study of elements currently present in request forms and result reports and the opinion of different stakeholders.

BACKGROUND: For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. METHODS: This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. RESULTS: Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. CONCLUSIONS: Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: The Impact of the Timing of the Liquid Biopsy.

Background: In heart transplant recipients, donor-derived cell-free DNA (ddcfDNA) is a potential biomarker for acute rejection (AR), in that increased values may indicate rejection. For the assessment of ddcfDNA as new biomarker for rejection, blood plasma sampling around the endomyocardial biopsy (EMB) seems a practical approach. To evaluate the effect of the EMB procedure on ddcfDNA values, ddcfDNA values before the EMB were pairwise compared to ddcfDNA values after the EMB. We aimed at evaluating whether it matters whether the ddcfDNA sampling is done before or after the EMB-procedure. Methods: Plasma samples from heart transplant recipients were obtained pre-EMB and post-EMB. A droplet digital PCR method was used for measuring ddcfDNA, making use of single-nucleotide polymorphisms that allowed both relative quantification, as well as absolute quantification of ddcfDNA. Results: Pairwise comparison of ddcfDNA values pre-EMB with post-EMB samples (n = 113) showed significantly increased ddcfDNA concentrations and ddcfDNA% in post-EMB samples: an average 1.28-fold increase in ddcfDNA concentrations and a 1.31-fold increase in ddcfDNA% was observed (p = 0.007 and p = 0.03, respectively). Conclusion: The EMB procedure causes iatrogenic injury to the allograft that results in an increase in ddcfDNA% and ddcfDNA concentrations. For the assessment of ddcfDNA as marker for AR, collection of plasma samples before the EMB procedure is therefore essential.

High Levels of Neutrophil Extracellular Traps Persist in the Lower Respiratory Tract of Critically Ill Patients With Coronavirus Disease 2019.

Lower respiratory tract (LRT) disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can deteriorate to acute respiratory distress syndrome (ARDS). Because the release of neutrophil extracellular traps (NETs) is implicated in ARDS pathogenesis, we investigated the presence of NETs and correlates of pathogenesis in blood and LRT samples of critically ill patients with COVID-19. Plasma NET levels peaked early after intensive care unit admission and were correlated with the SARS-CoV-2 RNA load in sputum and levels of neutrophil-recruiting chemokines and inflammatory markers in plasma samples. The baseline plasma NET quantity was correlated with disease severity but was not associated with soluble markers of thrombosis or with development of thrombosis. High NET levels were present in LRT samples and persisted during the course of COVID-19, consistent with the detection of NETs in bronchi and alveolar spaces in lung tissue from deceased patient with COVID-19. Thus, NETs are produced and retained in the LRT of critically ill patients with COVID-19 and could contribute to SARS-CoV-2-induced ARDS disease.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Mucinous adenocarcinoma arising in congenital pulmonary airway malformation: clinicopathological analysis of 37 cases.

AIMS: Mucinous adenocarcinoma arising in congenital pulmonary airway malformation (CPAM) is a rare complication, with little being known about its natural course. The aims of this article are to describe a series of mucinous adenocarcinomas arising from CPAMs, and present their clinicopathological features, genetics, and clinical outcome. METHODS AND RESULTS: Thirty-seven cases were collected within a 34-year period, and the subtype of adenocarcinoma and CPAM, tumour location, stage, growth patterns, molecular data and follow-up were recorded. The cohort comprised CPAM type 1 (n = 33) and CPAM type 2 (n = 4). Morphologically, 34 cases were mucinous adenocarcinomas (21 in situ; 13 invasive), and three were mixed mucinous and non-mucinous adenocarcinoma. Seventeen cases showed purely extracystic (intra-alveolar) adenocarcinoma, 15 were mixed intracystic and extracystic, and five showed purely intracystic proliferation. Genetically, nine of 10 cases tested positive for KRAS mutations, four with exon 2 G12V mutation and five with exon 2 G12D mutation. Residual disease on completion lobectomy was observed in two cases, and three cases recurred 7, 15 and 32 years after the original diagnosis. Two patients died of metastatic invasive mucinous adenocarcinoma. CONCLUSIONS: Most adenocarcinoma that arise in type 1 CPAMs, are purely mucinous, and are early-stage disease. Intracystic proliferation is associated with lepidic growth, an absence of invasion, and indolent behaviour, whereas extracystic proliferation may be associated with more aggressive behaviour and advanced stage. Most cases are cured by lobectomy, and recurrence/residual disease seems to be associated with limited surgery. Long-term follow-up is needed, as recurrence can occur decades later.

Fast detection of FOXF1 variants in patients with alveolar capillary dysplasia with misalignment of pulmonary veins using targeted sequencing.

BACKGROUND: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with heterozygous variants in the FOXF1 gene or its regulatory region. Patients with ACD/MPV unnecessarily undergo invasive and expensive treatments while awaiting a diagnosis. The aim of this study was to reduce the time to diagnose ACD/MPV by developing a targeted next-generation sequencing (NGS) panel that detects FOXF1 variants. METHODS: A FOXF1-targeted NGS panel was developed for detection of mutations and large genomic alterations and used for retrospective testing of ACD/MPV patients and controls. Results were confirmed with Sanger sequencing and SNP array analysis. RESULTS: Each amplicon of the FOXF1-targeted NGS panel was efficiently sequenced using DNA isolated from blood or cell lines of 15 ACD/MPV patients and 8 controls. Moreover, testing of ACD/MPV patients revealed six novel and six previously described pathogenic or likely pathogenic FOXF1 alterations. CONCLUSION: We successfully designed a fast and reliable targeted genetic test to detect variants in the FOXF1 gene and its regulatory region in one run. This relatively noninvasive test potentially prevents unnecessary suffering for patients and reduces the use of futile and expensive treatments like extra-corporeal membrane oxygenation. IMPACT: FOXF1-targeted NGS potentially prevents ACD/MPV patients from unnecessary suffering and expensive treatments. FOXF1-targeted NGS potentially reduces the number of misdiagnosis in ACD/MPV patients. Retrospective testing of ACD/MPV patients using FOXF1-targeted NGS revealed six novel pathogenic or likely pathogenic variants.

Central Role of Dendritic Cells in Pulmonary Arterial Hypertension in Human and Mice.

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old Tnfaip3DNGR1-KO mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 (Tnfaip3) gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of Tnfaip3DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3DNGR1-KO mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in Tnfaip3DNGR1-KO mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (Bmpr2), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the Bmpr2 gene.