From Morphology to Molecules: Advances in the Distinction of Multiple Primary Lung Cancers From Intrapulmonary Metastases in Non-Small Cell Lung Cancer.

The increasing incidence of multiple lung nodules underscores the need for precise differentiation between multiple primary lung cancers (MPLCs) and intrapulmonary metastases (IPMs). This distinction impacts patient prognosis and treatment strategies. The prevalence of multiple lung nodules, ranging from 19.7% to 55.5%, highlights the clinical significance of this challenge. Historically, the role of histopathology, particularly comprehensive histology assessment (CHA), has been pivotal in differentiating MPLCs and IPMs. However, CHA has significant limitations, resulting in a constant search for a better way to distinguish those lesions. The best strategy for delineating MPLCs from IPMs is a multidisciplinary approach combining clinical data, radiology, histology, and molecular methods. Histology provides architectural and cellular characteristics, radiology contributes anatomic context and lesion characterization, and molecular methods reveal molecular features critical for accurate differentiation. Incorporating clinical data further enhances diagnostic precision. This review presents current knowledge and current approaches to multiple lung tumors. It is clear that even with a combination of pathology, radiology, and molecular data, definitive classification of multifocal lung tumors is not always possible.

Developments in predictive biomarker testing and targeted therapy in advanced stage non-small cell lung cancer and their application across European countries.

In the past two decades, the treatment of metastatic non-small cell lung cancer (NSCLC), has undergone significant changes due to the introduction of targeted therapies and immunotherapy. These advancements have led to the need for predictive molecular tests to identify patients eligible for targeted therapy. This review provides an overview of the development and current application of targeted therapies and predictive biomarker testing in European patients with advanced stage NSCLC. Using data from eleven European countries, we conclude that recommendations for predictive testing are incorporated in national guidelines across Europe, although there are differences in their comprehensiveness. Moreover, the availability of recently EMA-approved targeted therapies varies between European countries. Unfortunately, routine assessment of national/regional molecular testing rates is limited. As a result, it remains uncertain which proportion of patients with metastatic NSCLC in Europe receive adequate predictive biomarker testing. Lastly, Molecular Tumor Boards (MTBs) for discussion of molecular test results are widely implemented, but national guidelines for their composition and functioning are lacking. The establishment of MTB guidelines can provide a framework for interpreting rare or complex mutations, facilitating appropriate treatment decision-making, and ensuring quality control.

Future perspective for the application of predictive biomarker testing in advanced stage non-small cell lung cancer.

For patients with advanced stage non-small cell lung cancer (NSCLC), treatment strategies have changed significantly due to the introduction of targeted therapies and immunotherapy. In the last few years, we have seen an explosive growth of newly introduced targeted therapies in oncology and this development is expected to continue in the future. Besides primary targetable aberrations, emerging diagnostic biomarkers also include relevant co-occurring mutations and resistance mechanisms involved in disease progression, that have impact on optimal treatment management. To accommodate testing of pending biomarkers, it is necessary to establish routine large-panel next-generation sequencing (NGS) for all patients with advanced stage NSCLC. For cost-effectiveness and accessibility, it is recommended to implement predictive molecular testing using large-panel NGS in a dedicated, centralized expert laboratory within a regional oncology network. The central molecular testing center should host a regional Molecular Tumor Board and function as a hub for interpretation of rare and complex testing results and clinical decision-making.

Immunophenotypes of anti-SARS-CoV-2 responses associated with fatal COVID-19.

Background: The relationship between anti-SARS-CoV-2 humoral immune response, pathogenic inflammation, lymphocytes and fatal COVID-19 is poorly understood. Methods: A longitudinal prospective cohort of hospitalised patients with COVID-19 (n=254) was followed up to 35 days after admission (median, 8 days). We measured early anti-SARS-CoV-2 S1 antibody IgG levels and dynamic (698 samples) of quantitative circulating T-, B- and natural killer lymphocyte subsets and serum interleukin-6 (IL-6) response. We used machine learning to identify patterns of the immune response and related these patterns to the primary outcome of 28-day mortality in analyses adjusted for clinical severity factors. Results: Overall, 45 (18%) patients died within 28 days after hospitalisation. We identified six clusters representing discrete anti-SARS-CoV-2 immunophenotypes. Clusters differed considerably in COVID-19 survival. Two clusters, the anti-S1-IgGlowestTlowestBlowestNKmodIL-6mod, and the anti-S1-IgGhighTlowBmodNKmodIL-6highest had a high risk of fatal COVID-19 (HR 3.36-21.69; 95% CI 1.51-163.61 and HR 8.39-10.79; 95% CI 1.20-82.67; p≤0.03, respectively). The anti-S1-IgGhighestTlowestBmodNKmodIL-6mod and anti-S1-IgGlowThighestBhighestNKhighestIL-6low cluster were associated with moderate risk of mortality. In contrast, two clusters the anti-S1-IgGhighThighBmodNKmodIL-6low and anti-S1-IgGhighestThighestBhighNKhighIL-6lowest clusters were characterised by a very low risk of mortality. Conclusions: By employing unsupervised machine learning we identified multiple anti-SARS-CoV-2 immune response clusters and observed major differences in COVID-19 mortality between these clusters. Two discrete immune pathways may lead to fatal COVID-19. One is driven by impaired or delayed antiviral humoral immunity, independently of hyper-inflammation, and the other may arise through excessive IL-6-mediated host inflammation response, independently of the protective humoral response. Those observations could be explored further for application in clinical practice.

Severe and Rare Case of Human Dirofilaria repens Infection with Pleural and Subcutaneous Manifestations, Slovenia.

We report a case of human Dirofilaria repens infection in a woman in Slovenia who had concomitant pleural and subcutaneous manifestations of the infection. This case report illustrates the clinical course of a severe symptomatic parasitic infection that had multisystemic manifestations.

Is hypermethylation of SOX1 gene an independent prognostic marker in surgically resected non-small cell lung cancer?

Background: Promoter hypermethylation of tumor suppressor genes presents promising markers for lung cancer diagnosis and prognosis. The purpose of this study was to determine the association between the promoter hypermethylation of multiple genes and 5-year survival rate in patients with Non-small cell lung cancer (NSCLC). Materials and Methods: Primary tumor samples (n = 65), corresponding nonmalignant lung tissues (n = 65), and circulating blood were obtained from NSCLC patients who underwent curative surgery. Promoter methylation status in seven genes (RASSF1A, CDH13, MGMT, ESR1, DAPK, SOX1, and HOXA9) was quantified by using bisulfite pyrosequencing. Five-year survival data were obtained by a clinician. Cox proportional hazards models were used to analyze the associations between gene methylation status and overall patient survival. Results: The 5-year survival of the patients with SOX1 aberrant tumor methylation was found to be statistically significantly shorter than for those patients without aberrant tumor methylation (P = 0.01). This effect was independent of TNM stage. No significant survival differences were associated with aberrant methylation in other genes tested in either of the two tissue types. Conclusion: Our study shows that SOX1 promoter hypermethylation in NSCLC tumors is significantly associated with inferior survival, showing promise as a useful prognostic biomarker in patients with NSCLC.

Real-life long-term outcomes of upfront surgery in patients with resectable stage I-IIIA non-small cell lung cancer.

BACKGROUND: Treatment of early-stage non-small cell lung cancer (NSCLC) is rapidly evolving. When introducing novelties, real-life data on effectiveness of currently used treatment strategies are needed. The present study evaluated outcomes of stage I-IIIA NSCLC patients treated with upfront radical surgery in everyday clinical practice, between 2010-2017. PATIENTS AND METHODS: Data of 539 consecutive patients were retrieved from a prospective hospital-based registry. All diagnostic, treatment and follow-up procedures were performed at the same thoracic oncology centre according to the valid guidelines. The primary outcome was overall survival (OS) analysed by clinical(c) and pathological(p) TNM (tumour, node, metastases) stage. The impact of clinicopathological characteristics on OS was evaluated using univariable (UVA) and multivariable regression analysis (MVA). RESULTS: With a median follow-up of 53.9 months, median OS and 5-year OS rate in the overall population were 90.4 months and 64.4%. Five-year OS rates by pTNM stage I, II and IIIA were 70.2%, 60.21%, and 49.9%, respectively. Both cTNM and pTNM stages were associated with OS; but only pTNM retained its independent prognostic value (p = 0.003) in MVA. Agreement between cTNM and pTNM was 69.0%. Next to pTNM, age (p = 0.001) and gender (p = 0.004) retained their independent prognostic value for OS. CONCLUSIONS: The study showed favourable outcomes of resectable stage I-IIIA NSCLC treated with upfront surgery in real-life. Relatively low agreement between cTNM and pTNM stages and independent prognostic value of only pTNM, observed in real-life data, suggest that surgery remains the most accurate provider of the anatomical stage of disease and important upfront therapy.

Impairment of the NKT-STAT1-CXCL9 Axis Contributes to Vessel Fibrosis in Pulmonary Hypertension Caused by Lung Fibrosis.

Rationale: Pulmonary hypertension (PH) is a common, severe comorbidity in interstitial lung diseases such as pulmonary fibrosis (PF), and it has limited treatment options. Excessive vascular fibrosis and inflammation are often present in PH, but the underlying mechanisms are still not well understood. Objectives: To identify a novel functional link between natural killer T (NKT) cell activation and vascular fibrosis in PF-PH. Methods: Multicolor flow cytometry, secretome, and immunohistological analyses were complemented by pharmacological NKT cell activation in vivo, in vitro, and ex vivo. Measurements and Main Results: In pulmonary vessels of patients with PF-PH, increased collagen deposition was linked to a local NKT cell deficiency and decreased IL-15 concentrations. In a mouse model of PH caused by lung fibrosis, pharmacological NKT cell activation using a synthetic α-galactosylceramide analog (KRN7000) restored local NKT cell numbers and ameliorated vascular remodeling and right ventricular systolic pressure. Supplementation with activated NKT cells reduced collagen deposition in isolated human pulmonary arterial smooth muscle cells (hPASMCs) and in ex vivo precision-cut lung slices of patients with end-stage PF-PH. Coculture with activated NKT cells induced STAT1 signaling in hPASMCs. Secretome analysis of peripheral blood mononuclear cells identified CXCL9 and CXCL10 as indicators of NKT cell activation. Pharmacologically, CXCL9, but not CXCL10, potently inhibited collagen deposition in hPASMCs via the chemokine receptor CXCR3. Conclusions: Our results indicate that the absence of NKT cells impairs the STAT1-CXCL9-CXCR3 axis in PF-PH and that restoration of this axis by NKT cell activation may unravel a novel therapeutic strategy to target vascular fibrosis in interstitial lung disease.

Case report: Congenital extrahepatic portocaval shunt presenting as pulmonary arterial hypertension in a pregnant patient.

Congenital extrahepatic portocaval shunt (CEPS) is a rare condition in which a rare congenital vascular anomaly of the portal system is present. CEPS may manifest as pulmonary arterial hypertension (PAH). When diagnosed and treated early, PAH can be reversible. We report a case of a previously asymptomatic woman, who manifested with severe pulmonary hypertension during pregnancy and was consequently diagnosed with CEPS. After unsuccessful medical treatment, urgent lung transplantation was done.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Utility of Telomerase Gene Mutation Testing in Patients with Idiopathic Pulmonary Fibrosis in Routine Practice.

Recent studies have suggested that causative variants in telomerase complex genes (TCGs) are present in around 10% of individuals with idiopathic pulmonary fibrosis (IPF) regardless of family history of the disease. However, the studies used a case-control rare variant enrichment study design which is not directly translatable to routine practice. To validate the prevalence results and to establish the individual level, routine clinical practice, and utility of those results we performed next generation sequencing of TCGs on a cohort of well-characterized consecutive individuals with IPF (diagnosis established according to ATS/ERS/JRS/ALAT guidelines). Of 27 IPF patients, three had a family history of idiopathic interstitial pneumonia (familial IPF) and 24 did not (sporadic IPF). Pathogenic/likely-pathogenic variants (according to American College of Medical Genetics criteria) in TCG were found in three individuals (11.1%) of the whole cohort; specifically, they were present in 2 out of 24 (8.3%) of the sporadic and in 1 out of 3 (33.3%) of the patients with familial IPF. Our results, which were established on an individual-patient level study design and in routine clinical practice (as opposed to the case-control study design), are roughly in line with the around 10% prevalence of causative TCG variants in patients with IPF.

COVID-19 Pulmonary Pathology: The Experience of European Pulmonary Pathologists throughout the First Two Waves of the Pandemic.

Autoptic studies of patients who died from COVID-19 constitute an important step forward in improving our knowledge in the pathophysiology of SARS-CoV-2 infection. Systematic analyses of lung tissue, the organ primarily targeted by the disease, were mostly performed during the first wave of the pandemic. Analyses of pathological lesions at different times offer a good opportunity to better understand the disease and how its evolution has been influenced mostly by new SARS-CoV-2 variants or the different therapeutic approaches. In this short report we summarize responses collected from a questionnaire survey that investigated important pathological data during the first two pandemic waves (spring-summer 2020; autumn-winter 2020-2021). The survey was submitted to expert lung pathologists from nine European countries involved in autoptic procedures in both pandemic waves. The frequency of each lung lesion was quite heterogeneous among the participants. However, a higher frequency of pulmonary superinfections, both bacterial and especially fungal, was observed in the second wave compared to the first. Obtaining a deeper knowledge of the pathological lesions at the basis of this complex and severe disease, which change over time, is crucial for correct patient management and treatment. Autoptic examination is a useful tool to achieve this goal.

COVID-19 pandemic impact on cytopathology practice in the post-lockdown period: An international, multicenter study.

BACKGROUND: In a previous worldwide survey, the authors showed a drastic reduction in the number of cytological specimens processed during the coronavirus disease 2019 "lockdown" period along with an increase in malignancy rates. To assess the continued impact of the pandemic on cytological practices around the world, they undertook a second follow-up worldwide survey collecting data from the post-lockdown period (2020). METHODS: Participants were asked to provide data regarding their cytopathology activity during the first 12 weeks of their respective national post-lockdown period (2020), which ranged from April 4 to October 31. Differences between the post-lockdown period and the corresponding 2019 period were evaluated, and the authors specifically focused on rates of malignant diagnoses. RESULTS: A total of 29 respondents from 17 countries worldwide joined the survey. Overall, a lower number of cytological specimens (n = 236,352) were processed in comparison with the same period in 2019 (n = 321,466) for a relative reduction of 26.5%. The overall malignancy rate showed a statistically significant increase (12,442 [5.26%] vs 12,882 [4.01%]; P < .001) during the same time period. Similar results were obtained if both malignancy and suspicious for malignancy rates were considered together (15,759 [6.58%] vs 16,011 [4.98%]; P < .001). CONCLUSIONS: The data showed a persistent reduction in the cytological specimen volume during the post-lockdown period (2020). However, the relative increase in the cytological workload in the late part of the post-lockdown is a promising finding of a slow return to normality.

Heterogeneous Response of Airway Eosinophilia to Anti-IL-5 Biologics in Severe Asthma Patients.

Many questions concerning responders (R) and nonresponders (NR) in severe eosinophilic asthma (SEA) after blocking the IL-5 (interleukin 5) pathway are still not clear, especially regarding the early parameters of response to biologics in personalized treatment strategies. We evaluated 17 SEA patients treated with anti-IL-5 biologics (16 patients mepolizumab, one patient benralizumab) before the introduction of biologics, and at a week 16 follow-up. Clinical, cellular and immunological parameters in peripheral blood were measured in R and NR. Sputum induction with the measurement of cellular and immunological parameters was performed at 16 weeks only. There were 12 R and 5 NR to biologics. After 16 weeks, there was a significant improvement in percentages of FEV1 (p = 0.001), and asthma control test (ACT) (p = 0.001) in the R group, but not in NR. After 16 weeks, the eosinophils in induced sputum were 27.0% in NR and 4.5% in R (p = 0.05), with no difference in IL-5 concentrations (p = 0.743). Peripheral eosinophilia decreased significantly in NR (p = 0.032) and R (p = 0.002). In patients with SEA on anti-IL-5 therapy, there was a marked difference in airway eosinophilic inflammation between R and NR already at 16 weeks, after anti-IL-5 introduction.

Automated Analysis of Proliferating Cells Spatial Organisation Predicts Prognosis in Lung Neuroendocrine Neoplasms.

Lung neuroendocrine neoplasms (lung NENs) are categorised by morphology, defining a classification sometimes unable to reflect ultimate clinical outcome. Subjectivity and poor reproducibility characterise diagnosis and prognosis assessment of all NENs. Here, we propose a machine learning framework for tumour prognosis assessment based on a quantitative, automated and repeatable evaluation of the spatial distribution of cells immunohistochemically positive for the proliferation marker Ki-67, performed on the entire extent of high-resolution whole slide images. Combining features from the fields of graph theory, fractality analysis, stochastic geometry and information theory, we describe the topology of replicating cells and predict prognosis in a histology-independent way. We demonstrate how our approach outperforms the well-recognised prognostic role of Ki-67 Labelling Index on a multi-centre dataset comprising the most controversial lung NENs. Moreover, we show that our system identifies arrangement patterns in the cells positive for Ki-67 that appear independently of tumour subtyping. Strikingly, the subset of these features whose presence is also independent of the value of the Labelling Index and the density of Ki-67-positive cells prove to be especially relevant in discerning prognostic classes. These findings disclose a possible path for the future of grading and classification of NENs.

Gene Expression Levels of the Prolyl Hydroxylase Domain Proteins PHD1 and PHD2 but Not PHD3 Are Decreased in Primary Tumours and Correlate with Poor Prognosis of Patients with Surgically Resected Non-Small-Cell Lung Cancer.

BACKGROUND: Hypoxia correlates with poor prognosis in several cancer types, including lung cancer. Prolyl hydroxylase domain proteins (PHDs) play a role in cell oxygen sensing, negatively regulating the hypoxia-inducible factor (HIF) pathway. Our study aim was to evaluate PHD1, PHD2 and PHD3 mRNA expression levels in primary tumours and normal lungs of non-small-cell lung cancer (NSCLC) patients and to correlate it with selected regulators of HIF signalling, with clinicopathological characteristics and overall survival (OS). METHODS: Tumour tissue samples were obtained from 60 patients with surgically resected NSCLC who were treated with radical surgery. In 22 out of 60 cases, matching morphologically normal lung tissue was obtained. PHD1, PHD2 and PHD3 mRNA expressions were measured using RT-qPCR. RESULTS: The PHD1 and PHD2 mRNA levels in primary tumours were significantly decreased compared to those in normal lungs (both p < 0.0001). PHD1 and PHD2 expression in tumours was positively correlated (rs = 0.82; p < 0.0001) and correlated well with HIF pathway downstream genes HIF1A, PKM2 and PDK1. Decreased PHD1 and PHD2 were associated with larger tumour size, higher tumour stage (PHD1 only) and squamous cell carcinoma. Patients with low PHD1 and patients with low PHD2 expression had shorter OS than patients with high PHD1 (p = 0.02) and PHD2 expression (p = 0.01). PHD1 showed borderline independent prognostic values in multivariate analysis (p = 0.06). In contrast, we found no associations between PHD3 expression and any of the observed parameters. CONCLUSIONS: Our results show that reduced expression of PHD1 and PHD2 is associated with the development and progression of NSCLC. PHD1 could be further assessed as a prognostic marker in NSCLC.

Prognostic impact of PD-1 and PD-L1 expression in malignant pleural mesothelioma: an international multicenter study.

BACKGROUND: Programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) immune-checkpoint blockade is a promising new therapeutic strategy in cancer. However, expression patterns and prognostic significance of PD-L1 and PD-1 are still controversial in human malignant pleural mesothelioma (MPM). METHODS: Formalin-fixed paraffin-embedded (FFPE) tumor samples from 203 MPM patients receiving standard treatment without immunotherapy were collected from 5 European centers. PD-L1 and PD-1 expression of tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) were measured by immunohistochemistry and correlated with clinical parameters and long-term outcome. RESULTS: High (>10%) PD-L1 TC and PD-1 TILs expressions were found in 18 (8%) and 39 (24%) patients, respectively. PD-L1 was rarely expressed by TILs [≥1%, n=13 (8%); >10%, n=1]. No significant associations were found between the PD-L1 or PD-1 expression of TCs or TILs and clinicopathological parameters such as stage or histological subtype. Notably, patients with high (>10%) TC-specific PD-L1 expression exhibited significantly worse median overall survival (OS) (6.3 vs. 15.1 months of those with low TC PD-L1 expression; HR: 2.51, P<0.001). In multivariate cox regression analysis adjusted for clinical parameters, high TC PD-L1 expression (>10%) proved to be an independent negative prognostic factor for OS (HR: 2.486, P=0.005). There was no significant correlation between PD-L1 or PD-1 expression of TILs and OS. CONCLUSIONS: In this multicenter cohort study, we demonstrate that high (>10%) PD-L1 expression of TCs independently predicts worse OS in MPM. Further studies are warranted to investigate the value of PD-L1/PD-1 expression as a marker for treatment response in MPM patients receiving immunotherapy.

P14/ARF-Positive Malignant Pleural Mesothelioma: A Phenotype With Distinct Immune Microenvironment.

INTRODUCTION: The CDKN2A gene plays a central role in the pathogenesis of malignant pleural mesothelioma (MPM). The gene encodes for two tumor suppressor proteins, p16/INK4A and p14/ARF, frequently lost in MPM tumors. The exact role of p14/ARF in MPM and overall its correlation with the immune microenvironment is unknown. We aimed to determine whether there is a relationship between p14/ARF expression, tumor morphological features, and the inflammatory tumor microenvironment. METHODS: Diagnostic biopsies from 76 chemo-naive MPMs were evaluated. Pathological assessments of histotype, necrosis, inflammation, grading, and mitosis were performed. We evaluated p14/ARF, PD-L1 (tumor proportion score, TPS), and Ki-67 (percentage) by immunohistochemistry. Inflammatory cell components (CD3+, CD4+, CD8+ T lymphocytes; CD20+ B-lymphocytes; CD68+ and CD163+ macrophages) were quantified as percentages of positive cells, distinguishing between intratumoral and peritumoral areas. The expression of p14/ARF was associated with several clinical and pathological characteristics. A random forest-based machine-learning algorithm (Boruta) was implemented to identify which variables were associated with p14/ARF expression. RESULTS: p14/ARF was evaluated in 68 patients who had a sufficient number of tumor cells. Strong positivity was detected in 14 patients (21%) (11 epithelioid and 3 biphasic MPMs). At univariate analysis, p14/ARF-positive epithelioid mesotheliomas showed higher nuclear grade (G3) (p = 0.023) and higher PD-L1 expression (≥50%) (p = 0.042). The percentages of CD4 and CD163 in peritumoral areas were respectively higher and lower in p14/ARF positive tumors but did not reach statistical significance with our sample size (both p = 0.066). The Boruta algorithm confirmed the predictive value of PD-L1 percentage for p14/ARF expression in all histotypes. CONCLUSIONS: p14/ARF-positive epithelioid mesotheliomas may mark a more aggressive pathological phenotype (higher nuclear grade and PD-L1 expression). Considering the results regarding the tumor immune microenvironment, p14/ARF-negative tumors seem to have an immune microenvironment less sensitive to immune checkpoint inhibitors, being associated with low PD-L1 and CD4 expression, and high CD163 percentage. The association between p14/ARF-positive MPMs and PD-L1 expression suggests a possible interaction of the two pathways. Confirmation of our preliminary results could be important for patient selection and recruitment in future clinical trials with anticancer immunotherapy.

T2-high Asthma, Classified by Sputum mRNA Expression of IL4, IL5, and IL13, is Characterized by Eosinophilia and Severe Phenotype.

Asthma is a common chronic disease, with different underlying inflammatory mechanisms. Identification of asthma endotypes, which reflect a variable response to different treatments, is important for more precise asthma management. T2 asthma is characterized by airway inflammation driven by T2 cytokines including interleukins IL-4, IL-5, and IL-13. This study aimed to determine whether induced sputum samples can be used for gene expression profiling of T2-high asthma classified by IL4, IL5, and IL13 expression. Induced sputum samples were obtained from 44 subjects, among them 36 asthmatic patients and eight controls, and mRNA expression levels of IL4, IL5, and IL13 were quantified by RT-qPCR. Overall, gene expression levels of IL4, IL5, and IL13 were significantly increased in asthmatic patients' samples compared to controls and there was a high positive correlation between expressions of all three genes. T2 gene mean was calculated by combining the expression levels of all three genes (IL4, IL5, and IL13) and according to T2 gene mean expression in controls, we set a T2-high/T2-low cutoff value. Twenty-four (67%) asthmatic patients had T2-high endotype and those patients had significantly higher eosinophil blood and sputum counts. Furthermore, T2-high endotype was characterized as a more severe, difficult-to-treat asthma, and often uncontrolled despite the use of inhaled and/or oral corticosteroids. Therefore, the majority of those patients (15 [63%] of 24) needed adjunct biological therapy to control their asthma symptoms/exacerbations. In conclusion, we found that interleukins IL4, IL5, and IL13 transcripts could be effectively detected in sputum from asthmatic patients. Implementation of T2 gene mean can be used as sputum molecular biomarker to categorize patients into T2-high endotype, characterized by eosinophilia and severe, difficult-to-treat asthma, and often with a need for biological treatment.