Circulating trefoil factors in relation to lung cancer, age and lung function: a cross-sectional study in patients referred for suspected lung cancer.

The trefoil factor family proteins: TFF1, TFF2 and TFF3 are secreted by epithelial cells in the respiratory tract. Here, we explore circulating concentrations of the trefoil factors in relation to lung cancer, age and lung function. We included 751 patients suspected of lung cancer. Lung cancer diagnosis was based on data reported to a national database. Serum TFF1, TFF2 and TFF3 concentrations were measured by ELISA, and spirometry was performed within ±3 days of blood sampling. Forced expiratory volume in the first second (FEV1) in relation to forced vital capacity (FVC), FEV1/FVC (a parameter used to quantify reduced lung function) was recorded. Lung cancer was diagnosed in 163 (22%) patients. Circulating concentrations of TFF3 (p = .021), but not TFF1 and TFF2, were significantly elevated in cancer patients. All three trefoil factors showed an increase in concentration with increasing age (p < .001) and declining lung function (p < .004). In the present cohort, concentrations of all three peptides were elevated compared with previous results published for healthy individuals. In conclusion, we report higher concentrations of TFF3 in patients with lung cancer, while increasing age and reduced lung function are associated with increasing concentrations of all trefoil factors in this specific patient population. The results emphasize that age and lung function should be taken into consideration when evaluating concentrations of trefoil factors in patients. However, the increases in trefoil factor concentrations were relatively small, and consequently, it is unlikely that circulating trefoil factor concentrations may have a role in the diagnosis of lung cancer and lung function impairment.

Integration of cryobiopsies for interstitial lung disease diagnosis is a valid and safe diagnostic strategy-experiences based on 250 biopsy procedures.

BACKGROUND: Transbronchial cryobiopsies has become increasingly used in the diagnostic workup in patients suspected of having interstitial lung disease. The procedure is associated with less complications, morbidity and mortality compared to surgical lung biopsies although with a diagnostic yield that is not as high, but close to that of surgical lung biopsies. The aim of the present study was to describe the complications and diagnostic yield and their prognostic factors. METHODS: All patients undergoing transbronchial cryobiopsies at the Department of Respiratory Diseases and Allergy, Aarhus University Hospital, were included in this prospective observational cohort study. RESULTS: A total of 250 patients were included [61% male, mean age 66 years (range, 22-81 years)]. Pneumothorax was detected in 70 (28%) of the patients, moderate hemorrhage in 53 (21%) and severe hemorrhage in 2 (1%) of the patients. Hemorrhage was associated with central biopsies, but not with anticoagulant therapy. None of the complications were related to lung function, exercise capacity, biopsy or probe size. Only one patient experienced an acute exacerbation. Three-month mortality was 0.4% (1 patient), caused by cancer and unrelated to the procedure. Cryobiopsies contributed to the final diagnosis in 72% of the patients and after multidisciplinary team discussion, a consensus diagnosis was obtained in 82% of the patients. The gender, the total sum of biopsy sizes, number of biopsies and presence of more than 50% alveolar tissue in biopsies increased the diagnostic yield. CONCLUSIONS: Our study confirms that using cryobiopsies in the diagnostic setup for interstitial lung diseases is safe with a limited risk of acute exacerbations and mortality. Cryobiopsies contribute to the diagnosis in the majority of patients.

SARS-CoV-2 evades immune detection in alveolar macrophages.

Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV-2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of pro-inflammatory cytokines later during infection.

Pregnancy-Associated Plasma Protein-A2 Is Associated With Mortality in Patients With Lung Cancer.

Pregnancy-associated plasma protein-A (PAPP-A) and its homolog PAPP-A2 are enzymes that modulate the availability and mitogenic activity of insulin-like growth factor-I (IGF-I). PAPP-A has been implicated in numerous cancers but reports on PAPP-A2 in malignancy are non-existent. In a prospective observational study of 689 patients under suspicion of lung cancer, we examined levels of PAPP-A and PAPP-A2 and their relationship with mortality. Serum PAPP-A and PAPP-A2 concentrations were determined in pre-diagnostic blood samples using ELISA, and immunohistochemical staining of PAPP-A and PAPP-A2 was performed in malignant tissue from five operable patients. A total of 144 patients were diagnosed with lung cancer, whereas the diagnosis was rejected in 545 subjects, who served as a control group. PAPP-A2 concentrations were higher in patients with lung cancer [median (IQR): 0.33 (0.21-0.56) ng/mL] than in controls [0.27 (0.17-0.39) ng/mL], p < 0.001, whereas PAPP-A levels did not differ. Presence of PAPP-A and PAPP-A2 were confirmed in tumor specimens, and staining occurred in a heterogeneous pattern. Patients were observed for a median (range) of 7 (6; 8) years, during which 114 patients (79.2%) died. Patient mortality differed according to PAPP-A2 tertile (p < 0.001). PAPP-A2 was associated with mortality with an unadjusted hazard ratio (95% CI) per doubling in protein concentration of 1.30 (1.12; 1.53), p = 0.001. In a multivariable model adjusted for age, sex, and BMI, PAPP-A2 remained predictive of the endpoint with a hazard ratio per doubling in protein concentration of 1.25 (1.05; 1.48), p = 0.013. Collectively, PAPP-A2, but not PAPP-A, is elevated in patients with lung cancer and associated with mortality. This novel role of PAPP-A2 in cancer warrants further functional studies as well as validation in external cohorts.

[Medical thoracoscopy in the elucidationof recurrent pleural effusion].

Medical thoracoscopy allows the respiratory physician access to inspection and biopsy of the pleura. The method has several names, such as pleuroscopy, non-intubated thoracoscopy and exploratory thoracoscopy, all of which cover the same procedure. The main indication for medical thoracoscopy is the diagnosis of recurrent pleural effusion, where pleural biopsy is needed. Medical thoracoscopy differs from thoracic surgical surgery as patients are sedated rather than in general anaesthesia. This is a review of the state of the art of pulmonological investigations of recurrent pleural effusion and the important role of medical thoracoscopy.

Development of a novel assay for IGFBP-2 complexed with IGF-I and-II in human serum.

BACKGROUND: Insulin-like growth factor binding-protein 2 (IGFBP-2) was originally identified as an IGF-carrier, governing IGF half-life, tissue accessibility and biological effects. Later, IGFBP-2 was discovered to possess IGF-independent effects. IGFBP-2 circulates in several forms, as free protein, complexed with IGF-I or IGF-II, or as IGFBP-2 fragments. The various IGFBP-2 forms are all included when measuring serum IGFBP-2 concentrations by immunoassay (i.e., immunoreactive (ir-)IGFBP-2). In this study, we describe a novel method to measure the amount of IGF that circulates bound to IGFBP-2. METHOD: IGFBP-2 was immunoprecipitated from human serum using magnetic beads, which were subsequently eluted by acidification. After neutralization, eluates were assayed for ir-IGFBP-2, IGF-I and IGF-II and compared to serum concentrations. This allowed measurement of IGFBP-2-compexed IGF-I and IGF-II, respectively. To test the method clinically, serum from 146 patients with lung cancer, 151 patients with non-cancer pulmonary diseases and 28 healthy controls were analyzed. RESULTS: We immuno-precipitated 97 ± 3.3% of serum IGFBP-2 and recovered > 75% of IGFBP-2-complexed IGFs, with intra- and inter-assay coefficient of variations (CVs) averaging < 5% and < 13%, respectively. No co-precipitation with IGFBP-1, -3 or - 4 was detected. Serum levels of ir-IGFBP-2 (median [25;75%]) differed between groups (cancer patients vs. non-cancer patients vs. healthy controls): 342 [260;480] vs. 262 [189;388] vs. 190 [141;269] µg/l (p < .0001). In parallel with this, concentrations of IGF-II carried by IGFBP-2 averaged: 45.0 [33.3;52.5] vs. 34.2 [25.4;46.1] vs. 19.8 [14.1;26.0] µg/l (p < .0001), and concentrations of IGF-I 8.0 [5.2;11.8] vs. 5.4 [3.6;7.3] vs. 7.0 [3.8;13.0] µg/l (p < .0001). Thus, IGFBP-2 carried more IGF-II than IGF-I in all groups (p < .0001). When expressed relative to IGF-concentrations, IGFBP-2 carried 9.0 [5.3;15.5] % of the IGF-I and 4.8 [2.9;5.8] % of the IGF-II in serum from healthy subjects. Notably, in patients, IGFBP-2 carried relatively less IGF-I, but more IGF-II (p < .0001). CONCLUSION: Using our novel assay, we demonstrate: that IGFBP-2 carries ≈10% of circulating IGF-I and ≈5% of circulating IGF-II in healthy subjects; that IGF-II is the primary ligand for IGFBP-2; and that IGFBP-2 carries even more IGF-II in patients than in healthy subjects. Thus, our assay may provide information on IGFBP-2 beyond what is achievable by simply measuring ir-IGFBP-2.

Systematic intrafraction shifts of mediastinal lymph node targets between setup imaging and radiation treatment delivery in lung cancer patients.

BACKGROUND AND PURPOSE: Internal target motion results in geometrical uncertainties in lung cancer radiotherapy. In this study, we determined the intrafraction motion and baseline shifts of mediastinal lymph node (LN) targets between setup imaging and treatment delivery. MATERIAL AND METHODS: Ten lung cancer patients with 2-4 fiducial markers implanted in LN targets received intensity-modulated radiotherapy with a daily setup cone-beam CT (CBCT) scan used for online soft-tissue match on the primary tumor. At a total of 122 fractions, 5 Hz fluoroscopic kV images were acquired orthogonal to the MV treatment beam during treatment delivery. Offline, the 3D trajectory of the markers was determined from their projected trajectory in the CBCT projections and in the intra-treatment kV images. Baseline shifts and changes in the respiratory motion amplitude between CBCT and treatment delivery were determined from the 3D trajectories. RESULTS: Systematic mean LN baseline shifts of 2.2 mm in the cranial direction (standard deviation (SD): 1.8 mm) and 1.0 mm in the posterior direction (SD: 1.2 mm) occurred between CBCT imaging and treatment delivery. The mean motion amplitudes during CBCT and treatment delivery agreed within 0.2 mm in all directions. CONCLUSIONS: Systematic cranial and posterior intrafraction baseline shifts between CBCT and treatment delivery were observed for mediastinal LN targets. Intrafraction motion amplitudes were stable.

Insulin-Like Growth Factor Bioactivity, Stanniocalcin-2, Pregnancy-Associated Plasma Protein-A, and IGF-Binding Protein-4 in Pleural Fluid and Serum From Patients With Pulmonary Disease.

Context: Members of the insulin-like growth factor (IGF) system are primarily produced in the liver and secreted into the circulation, but they are also produced, recruited, and activated locally in tissues. Objective: To compare activity and concentrations of IGF system components in pleural fluid and blood. Design: Pathological pleural fluid, secondary to lung cancer or nonmalignant disease, and matching blood samples were collected from 24 patients ages 66.7 to 81.9 years. Methods: IGF-related proteins and cytokine levels were measured by immunoassays or immunoblotting. Bioactive IGF was measured by an IGF-1 receptor phosphorylation assay. Results: Total IGF-1 concentration did not differ between the compartments, but concentrations of free IGF-1 and bioactive IGF were more than threefold higher in pleural fluid than in corresponding serum samples (P = 0.0004), regardless of etiology. Median pregnancy-associated plasma protein-A (PAPP-A) and interleukin (IL)-6 levels were increased 47-fold and 143-fold, respectively, in pleural fluid compared with plasma (P < 0.0001). PAPP-A and IL-6 concentrations correlated positively (r = 0.46; P = 0.02). In pleural fluid, levels of PAPP-A-generated IGF binding protein-4 fragments correlated inversely with that of stanniocalcin-2 (r ≤ -0.42; P ≤ 0.05), a PAPP-A inhibitor; such correlations were absent in plasma. Conclusion: Pathological pleural fluid is characterized by increased in vitro IGF bioactivity and elevated concentrations of PAPP-A, an IGF-activating proteinase. Thus, the tissue activity of the IGF system may differ substantially from that of the circulating IGF system. The correlation between IL-6 and PAPP-A indicates that inflammation plays a role in promoting local tissue IGF activity.

Introduction of cryobiopsies in the diagnostics of interstitial lung diseases - experiences in a referral center.

Introduction: Transbronchial cryobiopsies (cTBB) has emerged as a new method for obtaining lung tissue biopsies in the diagnosis of interstitial lung diseases (ILDs). Until now, it has been used in a few highly specialized interventional centers and has shown promising results in obtaining a definite diagnosis of ILDs. Method: All patients undergoing a cTBB between November 2015 and June 2016 were included in this case series study. Data on patient demographics, high-resolution computed tomography patterns, size and number of biopsies, histology patterns, the contribution to a confident diagnosis and complications were registered. Results: Thirty-eight patients underwent cTBB in the period. cTBB contributed to the diagnosis in 28 (74%) of the 38 patients. Only few complications were observed; pneumothorax was the most frequent complication (10 patients, 26%). In six patients, local bleeding occurred during the procedure and was easily controlled by a Fogarty catheter balloon and in some cases tranexamic acid. Conclusion: Performing cTBB in the diagnostics of ILDs is a safe and feasible procedure. cTBB resulted in a confident diagnosis in 74% of cases.

Mediastinitis: A Serious Complication of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration.

Mediastinitis is a rare but a serious complication of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). We present 3 cases of mediastinitis following these diagnostic procedures. In 2 of the patients oropharyngeal bacteria were found in the cultures from the mediastinal abscess. All 3 cases were treated successfully with thoracotomy and drainage of the abscess together with intravenous antibiotics. On the basis of these cases and an updated review of the literature we discuss the most likely etiology for mediastinitis in association with EBUS-TBNA and EUS-FNA procedures and propose how to reduce the risk for this serious complication. The possibility of mediastinitis should always be kept in mind when a patient complains of fever over a long period of time as well as chest pain and malaise after an EBUS-TBNA or EUS-FNA procedure.

Cardiac and respiration induced motion of mediastinal lymph node targets in lung cancer patients throughout the radiotherapy treatment course.

BACKGROUND AND PURPOSE: Involved mediastinal lymph nodes (LNs) are often included in the radiotherapy target for lung cancer patients. Their motion may differ from the primary tumor motion, possibly undermining the loco-regional control. This study determines the detailed differential target motion throughout the treatment course. MATERIAL AND METHODS: Ten lung cancer patients with 2-4 fiducial markers implanted in LN targets received IMRT with a daily pre-treatment cone-beam CT (CBCT) scan. Offline, the 3D trajectory of the markers was determined from their projected trajectory in the CBCT projections. Frequency analysis was performed to separate the intrafraction motion into a respiratory and cardiac component. The mean setup error of the markers and the motion range were used to calculate margins required for LN targets when setup is based on soft-tissue match. RESULTS: Respiration motion was largest in the CC direction and more prominent for more caudal LNs. Cardiac motion was often (73%) largest in the AP direction and tended to be largest for more cranial LNs. Margins for intrafraction motion and daily baseline shifts of LNs were 4.8mm (LR), 6.0mm (CC) and 6.7mm (AP). CONCLUSIONS: Detailed mapping showed that LN motion was in general governed by breathing, but some LNs had substantial cardiac induced motion.

Gene Expression of the EGF System-a Prognostic Model in Non-Small Cell Lung Cancer Patients Without Activating EGFR Mutations.

OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non-small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands is a likely explanation. The aim of this study is to demonstrate that the combined network of receptors and ligands from the EGF system is a prognostic marker. MATERIAL AND METHODS: Gene expression of the receptors EGFR, HER2, HER3, HER4, and the ligands AREG, HB-EGF, EPI, TGF-α, and EGF was measured by quantitative polymerase chain reaction in tumor samples from 100 NSCLC patients without EGFR activating mutations. Results were dichotomized into high or low levels of target expression. Coexpression of the ligands and receptors was observed, and a score was developed based on the summed effect of receptors and ligands. Akaike's information criteria selected the optimal score. Results were correlated with age, sex, stage, histology, performance status, and overall survival. RESULTS: Patients were randomly split 1:1 to create test and validation cohorts. In multivariate analyses, the only individual prognostic marker was EPI (hazard ratio [HR] 0.38 [0.20-0.72], P = .003). The optimal score in the test cohort was validated as a marker of inferior survival in the validation cohort and by bootstrapping. Multivariate analysis confirmed the combined score as a prognostic marker of inferior survival (HR 3.75 [2.17-6.47], P < .00001). CONCLUSION: Our study has developed a model that takes the complexity of the EGF system into account and shows that this model is a strong prognostic marker in NSCLC patients.

Exosomal Proteins as Diagnostic Biomarkers in Lung Cancer.

INTRODUCTION: Exosomes have been suggested as promising biomarkers in NSCLC because they contain proteins from their originating cells and are readily available in plasma. In this study, we explored the potential of exosome protein profiling in diagnosing lung cancers of all stages and various histological subtypes in patients. METHODS: Plasma was isolated from 581 patients (431 with lung cancer and 150 controls). The extracellular vesicle array was used to phenotype exosomes. The extracellular vesicle array contained 49 antibodies for capturing exosomes. Subsequently, a cocktail of biotin-conjugated CD9, CD81, and CD63 antibodies was used to detect and visualize captured exosomes. Multimarker models were made by combining two or more markers. The optimal multimarker model was evaluated by area under the curve (AUC) and random forests analysis. RESULTS: The markers CD151, CD171, and tetraspanin 8 were the strongest separators of patients with cancer of all histological subtypes versus patients without cancer (CD151: AUC = 0.68, p = 0.0002; CD171: AUC = 0.60, p = 0.0002; and TSPAN8: AUC = 0.60, p = 0.0002). The multimarker models with the largest AUC in the cohort of patients with all lung cancer histological subtypes and in the cohort of patients with adenocarcinoma only covered 10 markers (all cancer: AUC = 0.74 [95% confidence interval: 0.70-0.80]; adenocarcinoma only: AUC = 0.76 [95% confidence interval: 0.70-0.83]). In squamous cell cancer and SCLC, multimarker models did not exceed CD151 as an individual marker in separating patients with cancer from controls. CONCLUSION: We have demonstrated exosome protein profiling to be a promising diagnostic tool in lung cancer independently of stage and histological subtype. Multimarker models could make a fair separation of patients, demonstrating the perspectives of exosome protein profiling as a biomarker.

Ultra-micro samples can be used for mRNA quantification of lung cancer biomarkers.

BACKGROUND: Isolating sufficient material for molecular testing remains challenging in non-small cell lung cancer (NSCLC). The use of new ultra-microsamples (uMS) is proven sufficient for DNA and mRNA detection, but whether uMS are useful for quantifying mRNA expression is unknown. We investigated if uMS from lung cancer patients can be used to generate quantitative data on mRNA expression. METHODS: uMS were collected from primary tumors and lymph nodes from patients suspected of having lung cancer. mRNA was isolated, reverse-transcribed into cDNA and quantified with quantitative PCR assays for hepatocyte growth factor receptor (MET), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR) and amphiregulin (AREG) mRNA. The fraction of tumor cells to normal cells was estimated in each sample. RESULTS: MET, HGF, EGFR, and AREG expression were evaluated in 90 samples (30 containing cancer cells and 60 without cancer cells). MET and EGFR expression were negligible in samples without cancer cells. In samples containing cancer cells, MET and EGFR could be quantified in 13 samples each. Adjustment for tumor-cell fraction made it possible to obtain a quantitative result for the tumor-cell mRNA expression of MET and EGFR. In contrast, AREG and HGF were expressed in samples without tumor cells. These samples were used to establish the AREG and HGF mRNA expression in normal cells. Seven out of 14 AR-positive and two out of eight HGF-positive samples with tumor cells were above a cut-off of the mean + 2SD established in samples without tumor cells. CONCLUSION: We demonstrate that uMS contain high-quality mRNA, and quantitative studies can be performed when the tumor-cell fraction is considered.

Ultrasound-assisted aspiration of loculated pneumothorax: A new technique.

In this report, we describe a new sonographic (US) technique that can assist in the aspiration of a loculated pneumothorax. Patients may develop loculated pneumothorax as a result of such conditions as pleural malignancy or pleural infection or after undergoing thoracic surgery. Often the loculated pneumothorax is outside of safe areas, and chest tubes need to be placed near vital structures. This report presents the cases of three patients with iatrogenic loculated pneumothorax that required aspiration. We used US to assist in the placement of chest tubes, and we describe our technique of US-assisted aspiration of loculated pneumothorax. The procedure is a new approach to a common problem in chest medicine that may increase the safety of treatment. © 2015 Wiley Periodicals, Inc. J Clin Ultrasound 44:326-330, 2016.

Reduced specialist time with direct computed tomography for suspected lung cancer in primary care.

INTRODUCTION: Lung cancer (LC) is the most common cause of cancer death in Denmark, and triaging patients through fast-track diagnostic pathways is recommended to improve patient outcome. Data on the most efficient triage organisation of such pathways are limited. The aim of this study was to test a strategy of a straight-to-test model for patients referred to the fast-track pathway. Outcomes were number of computed tomographies (CT) performed, use of specialist time and staff acceptability. MATERIAL AND METHODS: We performed a randomised controlled study enrolling 493 patients who were referred from general practice to fast-track LC evaluation (1 January-1 December 2012). Half of the patients were randomly assigned to the intervention and went straight to a chest CT before chest-physician evaluation. Time was measured for patients at random days. Acceptability was examined in a focus group interview. RESULTS: In the intervention group, 95.5% of patients had a CT performed compared with 97.2% in the control group. There was no difference in the number of CTs between the groups (risk difference (RD) = 1.3% (95% confidence interval (CI): 4.4-2.0; p = 0.454)). In the intervention group, chest-physician time was 13.3 min. (min.-max.: 7.7-19.5 min.) lower per referred patient than in the control group. CONCLUSION: Giving general practitioners direct access to a CT did not change the number of CTs performed and significantly reduced chest-physician time per patient. In addition, the strategy was associated with high levels of staff acceptability. FUNDING: The project was supported by the Danish Cancer Research Foundation, the Danish Cancer Society and the Novo Nordisk Foundation. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01779726.