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The Lung Screen Uptake Trial tested a novel invitation strategy to improve uptake and reduce socioeconomic and smoking-related inequalities in lung cancer screening (LCS) participation. It provides one of the first UK-based ‘real-world’ LCS cohorts. Of 2012 invited, 1058 (52.6%) attended a ‘lung health check’. 768/996 (77.1%) in the present analysis underwent a low-dose CT scan. 92 (11.9%) and 33 (4.3%) participants had indeterminate pulmonary nodules requiring 3-month and 12-month surveillance, respectively; 36 lung cancers (4.7%) were diagnosed (median follow-up: 1044 days). 72.2% of lung cancers were stage I/II and 79.4% of non-small cell lung cancer had curative-intent treatment.

The National Optimal Lung Cancer Pathway recommends rapid progression from abnormal chest X-rays (CXRs) to CT. The impact of the more rapid reporting on the whole pathway is unknown. The aim of this study was to determine the impact of immediate reporting of CXRs requested by primary care by radiographers on the time to diagnosis of lung cancer.MethodPeople referred for CXR from primary care to a single acute district general hospital in London attended sessions that were prerandomised to either immediate radiographer (IR) reporting or standard radiographer (SR) reporting within 24 hours. CXRs were subsequently reported by radiologists blind to the radiographer reports to test the reliability of the radiographer report. Radiographer and local radiologist discordant cases were reviewed by thoracic radiologists, blinded to reporter.Results8682 CXRs were performed between 21 June 2017 and 4 August 2018, 4096 (47.2%) for IR and 4586 (52.8%) for SR. Lung cancer was diagnosed in 49, with 27 (55.1%) for IR. The median time from CXR to diagnosis of lung cancer for IR was 32 days (IQR 19, 70) compared with 63 days (IQR 29, 78) for SR (p=0.03).8258 CXRs (95.1%) were reported by both radiographers and local radiologists. In the 1361 (16.5%) with discordance, the reviewing thoracic radiologists were equally likely to agree with local radiologist and radiographer reports.ConclusionsImmediate reporting of CXRs from primary care reduces time to diagnosis of lung cancer by half, likely due to rapid progress to CT. Radiographer reports are comparable to local radiologist reports for accuracy.Trial registrationInternational Standard Randomised Controlled Trial NumberISRCTN21818068. Registered on 20 June 2017.

The results of the Dutch-Belgian low-dose CT (LDCT) screening trial (NELSON) have been eagerly awaited since the US National Lung Screening Trial (NLST), published in 2011, demonstrated annual LDCT screening of the chest led to a 20% decrease in lung cancer mortality compared with chest X-ray screening.1 While the US Preventative Service Task Force approved lung cancer screening in 2014,2 Europe and the rest of the globe have been paralysed by fear of implementation costs and the feasibility of introducing national LDCT screening programmes. The consensus from healthcare payers outside of the USA has been that we should wait for the results of the NELSON trial which, while smaller in size, would give us the confidence of a second randomised controlled trial and proof of effect in a population outside of the US healthcare system. Indeed, a recent Health Technology Assessment of LDCT screening in the UK specifically named the NELSON trial as an important source of future information and explicitly stated the results were required in order to make a decision about its efficacy and cost-effectiveness.3 It was on this background of hope and perhaps, dare we say it, healthcare payer fear that the NELSON trial preliminary results were released at the World Conference on Lung Cancer in Toronto in October. Despite NELSON being smaller in size than NLST and having a preponderance of male participants, the results were clear: LDCT screening compared with no screening leads to a statistically significant lung cancer mortality reduction of 26% for men and numbers hint that the benefit could be even greater in women (between 40% and 60%). With this new data the UK, and indeed the world outside of the USA, now needs to cast aside concerns over efficacy, as well as procrastination over implementation, and concentrate more …

Malignant pleural mesothelioma is a rare but devastating cancer of the pleural lining with no effective treatment. The tumour is often diffusely spread throughout the chest cavity, making surgical resection difficult, while systemic chemotherapy offers limited benefit. Bone marrow-derived mesenchymal stem cells (MSCs) home to and incorporate into tumour stroma, making them good candidates to deliver anticancer therapies. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic molecule that selectively induces apoptosis in cancer cells, leaving healthy cells unaffected. We hypothesised that human MSCs expressing TRAIL (MSCTRAIL) would home to an in vivo model of malignant pleural mesothelioma and reduce tumour growth. Human MSCs transduced with a lentiviral vector encoding TRAIL were shown in vitro to kill multiple malignant mesothelioma cell lines as predicted by sensitivity to recombinant TRAIL (rTRAIL). In vivo MSC homing was delineated using dual fluorescence and bioluminescent imaging, and we observed that higher levels of MSC engraftment occur after intravenous delivery compared with intrapleural delivery of MSCs. Finally, we show that intravenous delivery of MSCTRAIL results in a reduction in malignant pleural mesothelioma tumour growth in vivo via an increase in tumour cell apoptosis.

Since the publication of the National Lung Screening Trial (NLST) in 2011 demonstrating a 20% reduction in lung cancer-specific mortality by annual low-dose CT (LDCT),1 implementation of screening has continued apace in the USA. Other developed nations, perhaps mindful of the potential cost implications of national screening programmes, have been more reticent, with many awaiting the final outcome data from the largest European randomised trial—the Dutch-Belgian Lung Cancer Screening trial (NELSON) study which remains in follow-up.2 Despite the convincing mortality reduction in NLST, questions remain about implementation of LDCT screening for lung cancer. Unresolved issues include how to best identify high-risk individuals for screening, what is the most appropriate screening interval to achieve an optimal balance between mortality reduction and cost-effectiveness and how to minimise possible harms associated with screening—particularly overdiagnosis. This issue of Thorax includes two papers which provide important additions to accumulating evidence in this field. Paci and colleagues3 report lung cancer incidence and mortality rates from the Italian Lung Cancer Screening Trial (ITALUNG) randomised lung cancer screening trial conducted in Tuscany, Italy, commencing in 2004. From an invited population of over 71 232 subjects aged 55–69 years, 1613 were randomised to four rounds of annual LDCT screening and 1593 to usual care. Like many of the other European screening studies, ITALUNG was not powered to demonstrate mortality reduction in isolation, and pooled analysis with other similarly designed randomised trials is planned. As expected, lung cancer incidence was higher in the intervention group during the 4-year screening period (55% excess diagnoses vs usual care group). However, this was followed by a compensatory reduction in diagnoses during the 5-year follow-up period, such that there was no significant difference in incidence rates between the groups at the end of the study. After a median follow-up of 9 years, the authors report …

Background Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as an important tool for the diagnosis and staging of lung cancer but its role in the diagnosis of tuberculous intrathoracic lymphadenopathy has not been established. The aim of this study was to describe the diagnostic utility of EBUS-TBNA in patients with intrathoracic lymphadenopathy due to tuberculosis (TB). Methods 156 consecutive patients with isolated intrathoracic TB lymphadenitis were studied across four centres over a 2-year period. Only patients with a confirmed diagnosis or unequivocal clinical and radiological response to antituberculous treatment during follow-up for a minimum of 6 months were included. All patients underwent routine clinical assessment and a CT scan prior to EBUS-TBNA. Demographic data, HIV status, pathological findings and microbiological results were recorded. Results EBUS-TBNA was diagnostic of TB in 146 patients (94%; 95% CI 88% to 97%). Pathological findings were consistent with TB in 134 patients (86%). Microbiological investigations yielded a positive culture of TB in 74 patients (47%) with a median time to positive culture of 16 days (range 3–84) and identified eight drug-resistant cases (5%). Ten patients (6%) did not have a specific diagnosis following EBUS; four underwent mediastinoscopy which confirmed the diagnosis of TB while six responded to empirical antituberculous therapy. There was one complication requiring an inpatient admission. Conclusions EBUS-TBNA is a safe and effective first-line investigation in patients with tuberculous intrathoracic lymphadenopathy.

Introduction and objectives Chronic obstructive pulmonary disease (COPD) and emphysema are considerably under-diagnosed conditions. Low dose CT (LDCT) for lung cancer screening, if implemented, may provide an opportunity for earlier diagnosis of smoking-related conditions, in addition to lung cancer. Data gathered in a lung cancer screening demonstration pilot was analysed to look at COPD-related radiological changes and their relationship with patients’ pre-existing diagnoses, spirometry results and smoking status. The aims were to better understand these interlinked conditions and identify the potential for earlier diagnosis and management of smoking cessation interventions for these conditions. Methods Data were collected as part of the Lung Screen Uptake Trial. Smokers and recent former smokers (quit Results 275 patients responded to an invitation to attend a ‘lung health check’ in the first six months of recruitment. 149 (54.2%) had values consistent with COPD on spirometry. 106 [71.1%, (95% CI: ± 7.3%)] of these individuals were not aware of a diagnosis of COPD, and 81 (76.4%) were current smokers (Figure 1). Of the 103 individuals who had emphysema and no suspicious lesion on LDCT, 74 [71.8%, (95% CI: ± 8.7%)] were not aware of a diagnosis of COPD or emphysema. 55 (74.3%) of these were current smokers, and 33 (44.6%) had preserved spirometry (Table 1). Conclusion Our data demonstrate the considerable burden of undiagnosed COPD in at-risk groups (38.5% of all screened individuals), and show the prevalence of emphysematous change in those patients without a pre-existing, self-reported diagnosis of COPD or with preserved spirometry. Early diagnosis and CT evidence of smoking damage may provide the opportunity to co-implement supportive smoking cessation interventions, earlier medical intervention and prevention of further progression of disease through improved management of these conditions.

There has been almost a 1000 ‘lung cancer screening’ papers and abstracts over the past 10 years, with half published in the last two. Avoiding the topic of lung cancer screening at respiratory, radiology and oncology conferences is becoming increasingly challenging. Is it realistic that we are going to screen for lung cancer in the UK? We know, despite our continued efforts, that the UK health system serves its patients with lung cancer poorly. Current UK lung cancer statistics demonstrate a 13% 5-year survival attributed largely to late-stage presentation.1 Approximately 70% of lung cancer is diagnosed at stage III and IV where options for curative treatment are greatly diminished.2 There can be no doubt, therefore, that earlier diagnosis is crucial to improving lung cancer outcomes, and nowhere more so than the UK. However, the data supporting low radiation dose CT screening (LDCT) for lung cancer are from the USA where medicine is practised rather differently and resources not so limited. So, until this issue of Thorax , there has been a paucity of evidence from Britain to support the extrapolation of available data to our patients. The UK Lung Cancer Pilot Screening Trial (UKLS) reports the findings of its Wald single screen design randomised controlled pilot providing data that address many questions, but raise others.3 The first issue is that of determining eligibility for screening. One can reduce patient eligibility by increasing the risk threshold required to screen, thereby enriching the lung …

Background Our laboratory has a unique cohort of patients with pre-invasive lung squamous cell carcinoma (SqCC) lesions, within which there is a clear discrepancy between the prevalence of pre-invasive lesions and the incidence of lung cancer, suggesting that not all pre-invasive lesions progress to cancer. Using gene expression microarrays we identified 1846 genes significantly differentially expressed between progressive and regressive pre-invasive SqCC lesions. The macrophage metalloelastase MMP12 gene was found to be highly expressed in progressive lesions, and we hypothesised that it plays a role in epithelial-to-mesenchymal transition (EMT). Conversely, the actin binding protein LIM-domain only 7 (LMO7) gene was highly expressed in regressive lesions, and we postulated that it may be protective against EMT due to its role in the maintenance of epithelial architecture. Initial studies using three SqCC cell lines (A431, H357 and H376) with MMP12-shRNA knockdown showed a significant decrease in migration and invasion compared to non-silencing shRNA controls. LMO7-shRNA knockdown in HBECS was found to significantly increase migration. The aim of this study is to further characterise the function and signalling of MMP12 and LMO7 in lung SqCC development. Methods Eight-week-old NOD/SCID mice were used for tumorigenesis experiments. Adhesion assays were carried out to assess the roles of MMP12-knockdown or LMO7-overexpression on cell adhesion. Cell signalling mechanisms were assessed using western blotting, qPCR and immunostaining. Results We observed that MMP12-knockdown decreases tumorigenicity in an immunocompromised mouse model. Both A431- and H357 MMP12-knockdown cells produced significantly smaller tumours compared with non-silencing shRNA cells. We found that MMP12-knockdown decreases cell adhesion, which is currently being further investigated along with effects on integrin signalling pathways. Levels of EMT markers were assessed in MMP12-knockdown and LMO7 overexpressing cells using qPCR, western blotting and immunostaining. Results indicate that higher MMP12 expression is associated with a mesenchymal phenotype, whereas higher LMO7 expression is associated with an epithelial phenotype. Conclusions Our results suggest that MMP12 is a key driver of migration and invasion in SqCC and its high expression may contribute to EMT, whereas LMO7 is a putative tumour suppressor with a crucial role in maintaining epithelial cell architecture. MMP12 and LMO7 may be potential early stage therapeutic markers for lung cancer.

Screening for the early detection of lung cancers should increase the percentage of operable tumours, thus improving cure rates. A large randomised US trial showed that CT screening moderate/heavy smokers is effective but expensive, with a high false-positive rate. We designed LungSEARCH in 2006 to target screening in higher-risk subjects. Because most tumours in the UK were of squamous-histology, we hypothesised that sputum cytology plus cytometry would be an effective initial screen, only offering more intensive/expensive tests to those with abnormal sputum. Eligibility criteria were: current/former smokers (≥20 pack-years and/or smoked ≥20 years), GOLD-defined COPD, no prior cancer. Subjects were randomised to surveillance or a control group, and each followed for 5 years. Screened subjects provided sputum for central assessment, and those with abnormal results (cytology: low/high-grade squamous intraepithelial lesions, and/or cytometry: abnormal ploidy) were referred for annual low-dose CT and autofluorescence bronchoscopy (AFB) for the remainder of the trial, with diagnostic investigations when cancer suspected by abnormal CT/AFB. Sputum-negatives provided annual sputum samples only. Control subjects had a chest X-ray when they reached 5 years. Primary objective: to show a higher proportion of early stage cancers using surveillance than controls. 1568 subjects were recruited (target 1300) from GPs or chest clinics around 10 UK centres (August 2007–March 2011): 785 screened, 783 controls. Mean age 63 years; males 52%; current (56%), former (44%) smokers; mild (25%), moderate (75%) COPD; from GPs (79%). >90% screened subjects provided sputum samples in their first year. After 5 years, the overall sputum-positive rate is 33%; 30% (236/785) had a CT scan and 25% (193/785) had an AFB at any time. Of those who had a CT scan 19% (45/236) were abnormal (lung nodule(s) ≥9 mm); and of those who had AFB 3% (5/193) had severe dysplasia or worse. 79 lung cancers have been identified to date via the centres/national registry: 43 surveillance and 36 control. But awaiting staging details for 6 surveillance and 14 control cases. Preliminary results are promising: 57% (surveillance) versus 41% (controls) of cancers were diagnosed with stage I/II non-small-cell-lung cancer or limited disease small-cell-lung cancer. Final data available later in 2016.

Background Aberrations of EGFR signalling drive cancer development. In squamous cell lung cancer (SqCLC), EGFR is overexpressed. LRIG1 is a negative regulator of EGFR and patient pre-invasive SqCLC samples show LRIG1 loss, suggesting involvement in early disease pathogenesis. In skin and gut homeostasis, LRIG1 regulates stem cells. In the upper airway, basal cells act as stem cells and are the putative origin of SqCLC. We hypothesise LRIG1 has a key role in airway homeostasis and its loss promotes pre-invasive SqCLC development. Methods Lrig1 EGFP-ires-CreERT2 mice were used to delineate airway LRIG1 expression. Flow sorted LRIG1-positive and -negative murine basal cells were used in 2D and 3D colony-forming, spheroid and proliferation assays. A murine SqCLC model was set up through application of N-Nitrosotris-(2-chloroethyl)urea (NTCU). Pre-invasive lesions and tumour development were compared between wild-type (WT), heterozygous and LRIG1-knockout (KO) animals. Human basal cells obtained from bronchoscopy were sorted according to LRIG1 expression and used directly in colony-forming assays or maintained in primary culture to assess the effect of shRNA knockdown of LRIG1. LRIG1-knockdown cells were assessed in colony-forming and proliferation assays, and differentiation and invasion were assessed using organotypic models. Results LRIG1 is expressed by 40% of airway basal cells. LRIG1-expressing murine basal cells exhibit increased colony-forming capacity (p = 0.0286), spheroid formation (p = 0.0043) and proliferation (p = 0.0043) compared with LRIG1-negative cells. Similarly, LRIG1-expressing human airway basal cells isolated from endobronchial brush biopsy samples exhibit increased colony-forming capacity (p = 0.0469). Topical application of NTCU to mice recapitulates the development of human pre-invasive and SCLC lesions after 23 weeks. Results show lesions in LRIG1-KO mice to be larger than those of WT animals. Knock down of LRIG1 in cultured human airway basal cells alters cell phenotype, leading to an increased colony-forming efficiency and greater proliferation at cell confluence. Conclusions LRIG1 has an important role in stem cell homeostasis of the human and murine airway epithelium. Loss of LRIG1 promotes pre-cancerous lesion development in a murine SqCLC mouse model and behaviour of human epithelial cells in culture, indicating a potential target for chemoprevention of SqCLC in humans.

Lung cancer is the most lethal cancer type worldwide. In order to improve patient survival it is important to enhance our understanding of the early changes associated with lung cancer progression. UCLH has a unique cohort of patients with pre-invasive lung squamous cell carcinoma (SCC) lesions. Within this cohort there is a discrepancy between the prevalence of pre-invasive lesions and the incidence of invasive lung cancer, which suggests that not all pre-invasive lesions progress to invasive carcinomas. The aim of this study was to identify and characterise key genes involved in the early pathogenesis of lung SCC. We performed genome-wide gene expression Illumina Whole-Genome DASL® arrays in 19 regressive and 20 progressive pre-invasive lung SCC lesions. The expression of matrix metallopeptidase 12 (MMP12) and LIM domain 7 (LMO7) was also determined in the 39 pre-invasive lung cancer lesions by immunostaining analysis. The functional role of MMP12 and LMO7 in cell migration and invasion was demonstrated by MMP12 and LMO7-knockdown in different squamous cell carcinoma cell lines and human bronchial epithelial cells (HBECs), respectively. We found 939 genes significantly differently expressed between the progressive and the regressive pre-invasive lung SCC lesions. We identified a remarkably elevated expression of a spectrum of genes in the progressive lung SCC lesions involved in different related cancer pathways including chromosome instability, p53 signalling and Wnt/β-catenin signalling. MMP12 and LMO7 were found within the highest significantly differently expressed genes and were therefore chosen to pursue studies focused on understanding the potential mechanisms leading to the development of lung SCC. In agreement with the gene expression data the expression of MMP12 and LMO7 proteins were up-regulated and down-regulated, respectively, in progressive when compared with regressive lesions. Inhibiting MMP12 by MMP12 knockdown significantly reduced the migration and invasion of different squamous cell carcinoma cell lines (A431, H357 and H376). We also established HBECs knockdown targeting LMO7. We observed a significant increase in the migration and invasion of HBECs cells in the LMO7 shRNA knockdown compared to control. Our results suggest that MMP12 and LMO7 may be potential therapeutic markers for lung cancer at early stage.

Background Computerised Tomography (CT) has been shown to be the only lung cancer screening modality to be effective in reducing lung cancer specific mortality. 1 A minimally invasive technique to stratify those at greatest risk within the population of adult smokers may help to target CT screening more effectively. Rationale Tobacco smoke exposure causes a field of injury to the airways (including nose and mouth) that if detected may inform an individual’s risk of lung cancer. 2 Infrared spectroscopy (IR) is a technique that can detect subtle biochemical alterations in macroscopically normal cells. Methods Buccal cells were exfoliated from 76 patients including 38 smokers without and 38 with lung cancer (matched for age, gender and pack years). The cells were fixed onto IR windows and spectra recorded using synchrotron radiation (Diamond facility, Oxford). Data was acquired using x36 objective and 15 × 15 µm aperture in transmission mode; 256 interferograms at 4 cm -1 resolution were recorded for 50 cells per sample. All samples analysed were confirmed to be cytologically normal. Outlying data was removed using principal component analysis and a prediction model built using partial least squares discriminant analysis. Results Smokers with lung cancer could be differentiated from matched smokers without lung cancer with a diagnostic accuracy of 80%. The spectral region showing greatest difference between groups was in the 1200–900 cm -1 region; comparison to reference spectra shows that this is likely to represent a metabolic change caused by an increased abundance of glycogen or its derivatives. Conclusions We have shown for the first time that IR spectroscopy of macroscopically normal upper respiratory tract cells may have a role to play in the early detection of lung cancer. Future work will validate these findings and aim to develop this non-invasive test for use as a pre-screen to select those at greatest risk who may be enrolled economically into surveillance programs. References The National Lung Screening Trial Research Team. Reduced lung-cancer mortality with low-dose computed tomographic screening. N Engl Med 2011;365(5):395–409 Sridhar et al . Smoking-induced gene expression changes in the bronchial airway are reflected in nasal and buccal epithelium. BMC Genomics 2008;9(1):259

Congenital diaphragmatic hernia (CDH) is a developmental diaphragmatic anomaly resulting in pulmonary hypoplasia and consequent pulmonary hypertension and respiratory failure sequelae. Despite advances in treatment, CDH remains associated with high morbidity and mortality rates. Reduced levels of vascular endothelial growth factor (VEGF) have been implicated in CDH pathogenesis. Animal studies have shown that intrauterine VEGF replacement enhances pulmonary vascularisation and lung epithelial cell proliferation. This study aimed to deliver VEGF through the engineering of a biocompatible and slow releasing nanodiamond (ND) platform, in a rat model of CDH. NDs were either fluorescently labelled (ND-FL) or conjugated to recombinant VEGF164 (ND-VEGF; 2 μg/mL VEGF164). Nitrofen was administered to pregnant Wistar rats at E9 (term=E22) to induce fetal CDH. At E19, maternal hysterotomy was performed, and NDs (75 μg/mL in 50 μL vehicle/saline) were administered intratracheally followed by fetal tracheal occlusion (TO). Blinded assessment of lung-to-body weight ratio (LBWR) and lung morphometric parameters was performed at E21.5 in CDH offspring. Prenatal ND administration did not have overt adverse effects. ND-FL biodistribution indicated that NDs localised in type II pneumocytes. ND-VEGF+TO was associated with improved lung growth (LBWR: 5.9 ± 0.2%), which was greater than that observed in VEGF+TO (3.5 ± 0.4%; p This is the first study to show that nanoparticle-mediated prenatal delivery of VEGF induces significant lung growth in CDH and suggests that sustained cargo release is pivotal in mimicking the temporal expression of VEGF in normal lung development.

Introduction Lung cancer is the leading cause of cancer mortality worldwide, with squamous cell carcinomas commonly arising in the central airways and accounting for nearly 30% of cases. Progression from normal bronchial epithelium to carcinoma in-situ (CIS) has been well described, and is found at the resection margin after lobectomy in up to 2.5% of cases; however, its fate has not been defined. Method Cases referred to the autofluoresence bronchoscopy (AFB) surveillance programme at this institution were analysed retrospectively from 1999–2012, for all those shown to have CIS at the resection margin following surgery for TxN0M0 squamous cell carcinoma. Patients underwent longitudinal assessment of the tracheobronchial tree to (a) confirm CIS at the resection margin and track its fate over time (b) characterise development of other preinvasive lesions. Results Twenty-two cases were identified with a median interval of 6 months (range 3–9) from surgical resection to first AFB. Thirteen patients (59%) were confirmed to have CIS on biopsy at the bronchial resection margin during the first AFB. Eleven (85%) of these progressed to invasion over a median interval of 37 months (range 4–85). A subgroup of these (5 patients) developed 8 invasive cancers at sites distant to the anastomotic site and 9 patients had >1 CIS lesion at a distant site. Two patients (9%) found to have CIS after initial post-resection AFB, persisted after follow-up of 36–45months. Although no progression has been seen, both have developed CIS at distant sites to the resection margin. Nine patients (41%) were found to have no evidence of CIS at the resection margin and during a median surveillance period of 37 months (range 19–126), all were found to have normal bronchial epithelium. One patient in this group developed a second primary lung cancer that was surgically resected. Conclusion CIS at the bronchial resection margin is a strong indicator of its fate to progression to invasive carcinoma. Its persistence sets precedent for the development of multiple, consecutive CIS lesions and invasive squamous cell carcinomas, and highlights the importance of routine AFB surveillance following surgery in these cases.

Introduction NICE guidance for the diagnosis and treatment of lung cancer recommends choosing “investigations that give the most information about diagnosis and staging with the least risk to the patient”. [1] EBUS-TBNA is expanding as an important diagnostic modality in lung cancer and provides simultaneous information on lung cancer phenotype, genotype and nodal staging. The impact of the introduction of EBUS-TBNA on the use of diagnostic modalities for tissue acquisition in patients with lung cancer is unknown. Methods A retrospective review of 407 consecutive patients diagnosed with lung cancer at a university teaching hospital in 2007, 2009 and 2011. Data were collected on age, gender, FEV1, performance status, diagnostic modality, and pathological subtype. Patients where only a clinical diagnosis was made (n=21) were excluded. Data were analysed using the SPSS version 17 (Chicago, IL, USA). For comparison between categorical variables, Chi-square or Fisher’s exact test were used as appropriate. All reported p -values are two-tailed, and are considered statistically significant when p Results 386 patients were included in the analysis. The mean (SD) age (years) and FEV1 (L/min) were 69 (12) and 1.81 (0.80) for 2007, 67 (10) and 1.81 (0.79) for 2009 and 68 (12) and 2.07 (0.68) for 2011 respectively. In 2007, 2009 and 2011 57.9%, 51.4% and 62.7% were males. The results on diagnostic modalities, performance status, histological subtype and staging are listed in Table 1. Comparing 2007 to 2011 there has been a significant reduction in standard bronchoscopy (P=0.0001), CT guided biopsy of peripheral lesions (P=0.0008) and mediastinoscopy (P=0.0382). The proportion of cases diagnosed by EBUS-TBNA significantly increased from 0% in 2007 to 21.4% in 2009 and 25.4% in 2011 (P Conclusions The use of diagnostic modalities that provide information on diagnosis and staging in a single intervention are increasing. At our hospital, the use of EBUS-TBNA has led to a significant reduction in CT guided biopsies, standard bronchoscopies and mediastinoscopies. These changes in practise have implications for future service provision, training and commissioning. Reference NICE Lung cancer. 2011.http://publications.nice.org.uk/lung-cancer-cg121/key-priorities-for-implementation.

Background Patients with bronchial pre-invasive lesions represent a significant management challenge due to the risk of lung cancer both at the site of known dysplasia and at remote sites within their lungs. The role of combined positron emission tomography/CT (PET/CT) in those patients is unknown. Aims To evaluate the diagnostic and clinical utility of 18F-FDG PET/CT in a surveillance programme for patients with pre-invasive endobronchial lesions. This was defined as the ability of abnormal 18F-FDG uptake to detect invasive bronchial carcinomas adjacent to known pre-invasive lesions or at remote sites in the lung. The prognostic value of 18F-FDG uptake at dysplasia sites was also assessed with surveillance. Can 18F-FDG uptake predict progression of pre-invasive lesions or cancer elsewhere? Methods 39 patients with pre-invasive endobronchial lesions underwent 18F-FDG PET/CT examination prior to autofluorescence bronchoscopy. Pre-invasive lesions were classified as either high-grade (carcinoma in situ or severe dysplasia) or low grade (mild to moderate dysplasia). The degree of uptake of 18F-FDG was analysed without knowledge of the bronchoscopic or other clinical findings. Results 8/39 patients (all with high grade dysplasia) had increased 18F-FDG uptake at known dysplasia sites. Of these 8 patients 1 had surgical resection of invasive carcinoma and two patients were diagnosed and treated as invasive cancer based on imaging and follow-up. Eight patients had 18F-FDG uptake at sites remote from known dysplasia; 2/8 patients had synchronous invasive lung carcinoma (pT1N0M0), 2/8 recurrent cancer in hilar and mediastinal nodes, and 4/8 patients had inflammatory uptake in lung, mediastinal or hilar nodes. During surveillance of up to 3 years, 3/5 patients with positive 18F-FDG uptake developed biopsy proven invasive cancer at site of dysplasia. 3/31 patients with negative 18F-FDG uptake developed invasive cancer at high grade dysplasia sites during surveillance. No low grade lesion showed 18F-FDG uptake or progressed to invasive cancer during surveillance. Conclusions PET/CT was able to detect early synchronous cancers in patients with pre-invasive endobronchial lesions. PET/CT was also able to detect 18F-FDG uptake in a proportion of patients at known dysplasia sites suggesting adjacent or underlying occult invasive carcinoma.

Introduction Isolated mediastinal lymphadenopathy (IML) is a common presentation to respiratory physicians and mediastinoscopy is traditionally considered the gold-standard investigation when a pathological diagnosis is required. EBUS-TBNA is established as an alternative to mediastinoscopy in patients with lung cancer. However, the utility and healthcare costs of EBUS-TBNA in patients with IML is unknown. Methods This prospective clinical trial recruited 77 consecutive patients with IML who were referred for mediastinoscopy from five centres between April 2009 and March 2011. All patients underwent EBUS-TBNA. If the results from EBUS-TBNA were not conclusive, patients underwent mediastinoscopy. The co-primary endpoints were the proportion of mediastinoscopies saved and NHS costs. The Bonferroni correction was applied to the type 1 error to account for multiple significance testing. Economic evaluation of the EBUS-TBNA strategy (where negative EBUS-TBNA is followed by mediastinoscopy) vs mediastinoscopy alone from an NHS perspective was carried out using a decision tree model and univariate threshold sensitivity analysis. Results EBUS-TBNA prevented 87% of mediastinoscopies (97.5% CI 78 to 96%, p Conclusions EBUS-TBNA is a safe, highly sensitive and cost-saving initial investigation in patients with IML being referred for mediastinoscopy. The low negative predictive value of EBUS-TBNA in this setting indicates that mediastinoscopy should be performed in cases of negative EBUS-TBNA. Trial registration clinicaltrials.gov NCT00932854.

Bone marrow derived mesenchymal stem cells (MSC) are promising tools for lung cancer therapy considering their tendencies for tumour homing and low immunogenicity. Tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a pro-apoptotic protein that induces selective apoptosis of tumour cells, while sparing normal cells. Therefore, it is expected that MSCs engineered to produce TRAIL will home to and kill cancer cells. In this study, two lentiviral vectors were constructed to express the full-length (flT) or a truncated soluble form of TRAIL (sT) driven by a CMV promoter/enhancer. A secretion targeting sequence and an isoleucine zipper (ILZ) peptide were sequentially added to the N-terminal of the soluble TRAIL to produce secreted and trimerised TRAIL. TRAIL lentiviruses were prepared and human BM-MSCs were transduced with a multiplicity of infection (MOI) of 2. FACS analysis by anti-TRAIL antibody staining demonstrated that over 99% of flT or sT viruses transduced cells are positive for TRAIL expression. TRAIL expression was further confirmed by Western blotting and ELISA assays. The flT or sT expressing MSCs both showed similar level of cellular TRAIL expression (~350 ng TRAIL per 1 mg of total cellular protein). Co-culture of cancer cells with transduced MSCs determined the cancer killing efficacy of MSCs expressing flT or sT. Twenty cancer cell lines were tested and classified into four TRAIL response groups; high, medium, low, and no sensitivity to recombinant TRAIL (rTRAIL) at the concentration of 50 ng/ml. At the co-culture ratio of 4:1 cancer to MSC cells, MSC-sT treatment showed no or only marginal cancer cell killing effect, in contrast, MSC-flT showed promising effects on all tested cell lines, with an apoptosis induction rate ranging between 35–75%. In groups designated as high, moderate and low, MSC-flT are as effective as rTRAIL and induced marked cell death (p In conclusion, these results demonstrate MSC-flT is a promising cell therapy and have great potential for clinical treatment of lung cancers and pleural metastases.

Introduction Endobronchial ultrasound and transbronchial needle aspiration (EBUS-TBNA) has been embraced as a breakthrough in the diagnosis and mediastinal staging of lung cancer. However, its utility in determining a diagnosis of lymphoma is not well defined, and is currently not recommended by the British Thoracic Society. Aim Evaluate the role of EBUS-TBNA in the diagnosis and subtyping of haematological malignancies. Method Patients referred with mediastinal lymphadenopathy for EBUS-TBNA in whom lymphoma was suspected, were identified retrospectively in 3 tertiary centres in the UK between 2008 and 2013. EBUS was performed using a linear array ultrasonic bronchoscope and specimens taken with a 21 or 22 gauge needle. The diagnostic accuracy, avoidance of mediastinoscopy and surgical biopsy in cases of primary and relapsing haematological malignancy was recorded. Where EBUS-TBNA was negative, patients subsequently underwent surgical biopsy or clinical and radiological surveillance. Results Twenty-four patients (10 female and 14 male) with a mean age of 55.5 years underwent EBUS-TBNA for evaluation of mediastinal lymphadenopathy. Clinical and radiological diagnosis was of either, isolated mediastinal lymphadenopathy of unknown cause (n = 15) or suspected mediastinal recurrence of lymphoma (n = 8). Five patients (62.5%) were found to have a relapse of their haematological malignancy, whilst a new diagnosis of lymphoma was made in 11 cases (73.3%) where the presentation was of isolated mediastinal lymphadenopathy. One patient in this group was found to have tuberculosis. Tissue obtained from nodal aspiration was sufficient to subtype the disease in detail in 14 patients with immunohistochemistry. Overall, 17 patients (70%) were prevented from having mediastinoscopy and other biopsies, and the diagnostic accuracy and sensitivity of EBUS-TBNA in primary and relapsing lymphoma was 74%. Conclusion EBUS-TBNA is a safe and important approach to mediastinal lymphadenopathy in suspected lymphoma, whereby detection of either primary or relapsing haematological malignancy prevents need for other more invasive biopsies.

Mesenchymal stromal cells (MSCs) are inherently tumour-homing and can be isolated, expanded and transduced, making them viable candidates for cell therapy. This tumour-tropism has been used to deliver anti-cancer therapies to various tumour models in several organs. In a previous study we have shown that MIF is the key director of MSC migration and infiltration towards tumour cells. We have shown this major role for MIF (mainly via CXCR4), using in vitro migration and invasion assays, in presence of different receptor inhibitors and achieving a drastic decrease in both processes using MIF inhibitor. Importantly we show that knock down of either CXCR4 or MIF abrogates MSC homing to tumours in an in vivo pulmonary metastasis model, confirming the in vitro 2D and 3D assays. In this study we define the mechanism behind MIF stimulation of MSC homing to tumours. We show that MIF upregulates other cytokines involved in chemotaxis, such as IL6, IL8 and CCL2 and upregulates MIF as well, amplifying the initial trigger and generating a positive feedback loop. However when inhibiting those cytokines individually, we never achieved a decrease in migration as drastic as for MIF inhibition. This suggests that the up-regulation of this set of cytokines would lead to chemoattraction of leucocytes to the site of the tumour, which was observed in a 3D model. Therefore, MIF trigger is amplified by its own upregulation in MSCs via a positive feedback loop, confirming again our previous findings and its key role as a regulator of MSC homing to tumours. This improved understanding of MSC tumour tropism will further enable development of novel cellular therapies for cancers.

Introduction Early detection is the key to survival in non-small cell lung cancer (NSCLC) where surgical resection can be undertaken. However, stage I and II disease combined account for only 25–30% of patients presenting with lung cancer. Although, clinical pathways from primary care exist to facilitate expeditious management of patients, the role of other referral pathways to diagnosis of surgically treatable lung cancers is not known. Methods Patients suitable for surgical resection for curative intent for primary lung cancer were identified between 2007 and 2011 at this institution. Patients diagnosed ‘incidentally’ on radiology were compared to those detected through the standard ‘two week’ wait target referral system. Specific data on demographics, diagnostics utilised, pathological stage, and lung cancer mortality were recorded. Results Eighty-four patients were treated with surgical resection for non-small cell cancer. The ‘two-week’ wait referrals accounted for one quarter (n = 21; 95% CI 17–35%) of the all the referrals, whilst 61% (n = 51; 95% CI 50–70%) of patients were found to have lung cancer detected incidentally through investigations performed by other specialties in the hospital. The presentation of patients to hospital with cancer related symptoms whether they had operable disease or not, had significantly higher lung cancer specific mortality (p = 0.02; see figure). Conclusion We demonstrate that patients who have cancer-related symptoms have a worse outlook. Whilst asymptomatic patients diagnosed by chance have better prospects for cure by surgical resection, thus highlighting the promise of CT screening for lung cancer in patients with high risk factors.

Introduction Super paramagnetic iron oxide nanoparticles (SPION) offer attractive possibilities in biomedicine. Hyperthermia treatment of cancer involves introducing SPION into tumours and applying an alternating magnetic field (AMF). The AMF causes the SPION to heat, resulting in cell death. It has been shown previously that mesenchymal stem cells (MSCs) can be labelled with SPION, with no effect on cell survival, and that they will migrate to and integrate into lung metastases in vivo, following systemic administration. Furthermore, SPION can be used to follow the fate of labelled cells in the body as they cause a marked shortening in T2* on MRI. Therefore, MSCs labelled with SPION offer a promising delivery mechanism for treating lung metastases with hyperthermia therapy. In this preliminary study, the distribution of SPION labelled MSCs and the anti-tumour effect of hyperthermia treatment was evaluated in vitro and in a subcutaneous murine tumour model. Methods MSCs were obtained from Tulane University, New Orleans. Cells were incubated overnight in 0.5 mg/ml of the SPION Ferucarbotranfor labelling. Subcutaneous tumours were induced by co-injecting 5 million OVCAR cells and 0.5 million SPION-labelled MSCs. Images of cells within mice were obtained on a 9.4Thorizontal bore Varian NMR system. A 10 mT 1.05 MHz AMF was generated using a copper solenoid coil, diameter 3 cm. Mice were placed inside the coil under anaesthesia and each hyperthermia session lasted 20 min. Post-heating histology was obtained. Results Hyperthermia treatment caused a rise in temperature up to 60°C in vitro and a rise in tumour temperature of up to 4°C above body temperature in vivo (Abstract S89 Figure 1), detected by thermal imaging and with fibre optic probes. Iron was confirmed in the tumour using MR imaging and histology. Discussion This study demonstrates the potential of SPION to act as an imaging agent and cancer therapy. SPION-labelled MSCs can be imaged in vivo in very low cell numbers. We have demonstrated that the application of an alternating magnetic field causes a temperature rise in these cells, both in vitro and in vivo . In the future, we will optimise SPION as an imaging and hyperthermia agent, for the targeted treatment of lung metastases.

Introduction Intra-thoracic lymphadenopathy is commonly observed in patients infected with HIV. The broad range of aetiologies and the non-specific clinical picture constitute a diagnostic challenge which has not been previously addressed. Therefore we aimed to identify the causes of mediastinal lymphadenopathy in HIV infected patients presenting to our specialist unit and to characterise their mode of diagnosis. Methods We conducted a retrospective analytical cohort study of HIV positive patients who underwent CT scanning of the chest between January 2004 and December 2008. Each patient was followed clinically for at least 12 months. Results 234 consecutive patients underwent chest CT in the study period. Employing the conventional CT cut-off for significant lymphadenopathy of 10 mm in short-axis, mediastinal lymphadenopathy was present in 49 (21%) patients. In these 49 patients, the mean age was 40 (range 19–69) years. 43 patients were male and 24 of the male patients were homosexual. 25 (51%) of patients were receiving antiretroviral therapy and 24 patients had a CD4 lymphocyte count below 250 cells ×106/l. A specific diagnosis was made in 42 cases. Lymphoma was the most common diagnosis with 9 cases, followed by TB with seven cases. Six patients had reactive lymphadenopathy secondary to pneumonia. There were four cases of Castleman9s disease, four of malignancy and three of non-tuberculous mycobacterial infection. In total, 13 patients (31%) were diagnosed with cancer. Extra-thoracic lymphadenopathy documented by CT was present in 26 (53%) of patients, including in seven cases of lymphoma and in all cases of Castleman9s disease. The diagnosis was established by histopathology in 57% of cases, by clinical follow-up in 19% and by microbiology in 7%. USS guided peripheral lymph node biopsy was the preferred biopsy method in 16 cases. Surgical biopsy was performed in seven cases and EBUS-TBNA in two cases. Conclusions Mediastinal lymphadenopathy is common in HIV positive patients undergoing CT scanning of the thorax and can be associated with a wide range of conditions. However, the prevalence of malignancy is high in this setting, emphasising the need for a pathological diagnosis.

In recent years, the development of lineage tracing approaches has provided quantitative new insights into tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and alterations in disease is not known. We demonstrate that the statistical analysis of pathologically neutral somatic mitochondrial mutations that are accumulated over time can provide access to clonal fate behaviour at single cell resolution in human, providing a direct means to explore mechanisms of cell fate and tissue maintenance. Employing this approach, we define the progenitor cell population and the cellular hierarchy of the major human airways. By applying a novel quantitative approach to lineage tracing data, we conclude that, in normal homeostasis, the lining of human lung epithelium is maintained by an equipotent progenitor cell population of basal cells, in which the chance loss of cells due to commitment is perfectly compensated by the duplication of neighbouring cells, leading to neutral drift dynamics of the clone population. Further, we show that in airways of smokers, this process is accelerated leading to intensified clonal consolidation and a fertile background for tumorigenesis. This study provides the benchmark for the use of somatic mutations to quantitatively explore patterns of homeostatic growth in human tissues, and a platform to explore factors leading to homeostatic dysregulation and disease.

Introduction and Objectives Differentiating tuberculosis and sarcoidosis can be difficult, particularly in the context of mediastinal lymphadenopathy, because both diseases are characterised by overlapping clinical phenotypes and histologically similar granulomatous inflammation. Currently, diagnosis relies heavily on microbiological confirmation of tuberculosis which is only available in

Introduction Malignant pleural mesothelioma (MPM) is a highly aggressive, incurable, chemoresistant tumour. Recent studies have shown that Mesenchymal stem cells (MSC) can home to and incorporate into the tumour stroma. Their tumour tropism can be used to deliver Tumour necrosis factor related apoptosis inducing ligand (TRAIL), a transmembrane protein that selectively induces apoptosis in transformed cells. However, not all tumours are sensitive to TRAIL. TRAIL works through triggering the extrinsic apoptotic pathway while conventional chemotherapeutic agents act by triggering the intrinsic apoptotic pathway. We hypothesised the crosstalk between these two pathways could be exploited by combining chemotherapy and MSC-TRAIL in MPM tumour cell lines. Methods MSC were engineered to express TRAIL using a lentiviral plasmid vector. A Tetracycline (Tet)-inducible system was used as a backbone to control the expression of TRAIL. Apoptosis induced by recombinant TRAIL, MSC-TRAIL in MPM cell lines on combination with Vorinostat, a chemotherapeutic agent, was measured by Annexin-V/DAPI based flow cytometry. Results The combination of recombinant TRAIL and Vorinostat act synergistically to induce apoptosis in MPM cell lines. Recombinant TRAIL and Vorinostat, as monotherapies induce 7.17% and 51.35% apoptosis in an MPM cell line JU77 respectively. In CRL2081 and ONE58 cell lines, recombinant TRAIL induces 56.75% and 13.41% apoptosis while Vorinostat leads to 78.95% and 43.97% apoptosis respectively. The combination of recombinant TRAIL and Vorinostat shows an increased amount of apoptosis in JU77, CRL2081 and ONE58 cell lines at 80.77%, 96.6% and 77.27% respectively (Table 1). Similar synergistic affect was observed when TRAIL expressing MSCs were co-cultured with Vorinostat treated MPM cell lines. MSC-TRAIL induced apoptosis in JU77 (48.73%), CRL2081 (57.63%) and ONE58(53.8%). Combined treatment of Vorinostat and MSC-TRAIL significantly increased apoptosis to 77.7% in JU77, 90.93% in CRL2081 and 77.8% in ONE58 cells (Table 1). Conclusion The combination of Vorinostat and recombinant TRAIL acts synergistically to induce apoptosis in malignant plural mesothelioma cells. Similar affect is observed with the combination of MSC-TRAIL and Vorinostat. This study indicates that Mesenchymal stem cells can be used as vectors for delivery of TRAIL and upon combination with Vorinostat, could be a potential therapy for malignant pleural mesothelioma.

Background Bronchial pre-invasive lesions represent the earliest stages of the stepwise progression of squamous carcinogenesis, they predominantly affect the large airways and are readily detectable using autofluoresence bronchoscopy (AFB) however very little is known about the natural history of these lesions and no randomised data exists to determine whether intervention before progression to invasion improves outcome. Methods A total of 94 patients with bronchial dysplasia were enrolled into an on-going surveillance cohort at University College London Hospital running prospectively since 1999. Lesions were biopsied longitudinally and kept under regular surveillance with AFB and low dose annual CT scanning until resolution or progression to invasive disease occurred. Retrospective analysis of lesional destiny was undertaken to determine the proportions of progressive vs. regressive lesions that occur in low grade dysplasia (LGD- squamous metaplasia, mild and moderate dysplasia) vs. high grade dysplasia (HGD- severe dysplasia (SD) and carcinoma-in-situ). A lesion was considered to have progressed/ regressed if it crossed between groups (LGD, HGD, invasive cancer). Results A total of 117 separate lesions that were biopsied on more than one occasion were identified of which 61 were HGD and 56 LGD. Of the low grade lesions 54/56 (96%) regressed or remained static, 1 (2%) progressed to CIS and 1 (2%) to invasive carcinoma both of these lesions progressed from moderate dysplasia. Of the high grade lesions there were 13 SD and 48 CIS, overall 35/61 (57%) of HGD progressed to invasive cancer 9/61 (15%) regressed and 17/61 (28%) remained static. There was a trend toward higher progression to cancer (62% vs 56%) and lower rates of regression (8% vs. 17%) for SD versus CIS in the HGD cohort although the numbers are too small to be statistically significant (see fig. 1). In the HGD group median time to invasion was 9.5 months (range 3–49), static lesions were documented to have remained as such for a median of 17 months (range 4–60). Conclusions In our cohort we see very few lesions following the traditional stepwise progression and LGD remains relatively indolent. There is a significant proportion of HGD that progresses to invasive cancer and further studies are required to test the role of endobronchial intervention to prevent progression and to determine the most efficacious modality of treatment.

Introduction Lung cancer is the second most common cancer in the UK with about 42,000 people being diagnosed in 2010 alone (Office of National Statistics, 2012). With a tendency to form invasive metastases coupled with its frequent late stage diagnosis, lung cancer attributes to the largest cause of cancer related mortality worldwide. Despite advances in treatment and care, the five-year mortality rate remains at 90%. There is a desperate need to improve patient survival, which can be achieved partly through improved screening techniques and more importantly by expanding our understanding of the molecular changes associated with lung cancer development and progression. We investigate the role of a tumour suppressor gene, first identified in lung cancer, tumour suppressor in lung cancer 1 (TSLC1) or cell adhesion molecule 1 (Cadm1) in regulating squamous cell carcinoma (SqCC) growth and metastases. Methods Cadm1 expression levels were examined using q-PCR analysis on human pre-invasive airway and normal lung tissue collected as part of an on-going UCL/CRUK longitudinal-tracking study (Lung-Surveillance and Lung-SEARCH trials). Cadm1 was introduced into an established SqCC cell line (A431) and in vitro functional assays performed to investigate its effect on tumour growth, progression and invasion. Pre-clinical mice models were used to study the effect of Cadm1 expression in tumour growth and metastatic potential. Results q-PCR analyses demonstrated that loss of Cadm1 expression is a frequent early event in pre-invasive human airway compared to normal tissue (p = 0.001). Functional assays using A431, with Cadm1 reintroduced, showed Cadm1 expression levels directly associated with a significant decrease in cell proliferation (p = 0.001) over 10 days and significant reduction in invasion (p = 0.001) over 72 hours compared to control A431 cells without Cadm1. Pre-clinical xenograft tumourigenecity experiments in mice showed that Cadm1 expression significantly inhibited tumour growth (p = 0.01) together with a significant reduction in the number of metastases observed (p = 0.01) when compared with the control group. Conclusion These data suggest that restoration of Cadm1 expression in human squamous cell carcinomas play an important role in regulation of tumour growth and metastasis. Understanding the mechanism through which Cadm1 expression is able to modulate cancer progression maybe therapeutically beneficial.

Background Malignant pleural mesothelioma (MPM) is an aggressive fatal cancer with no effective treatments. Mesenchymal stem cells (MSCs) migrate and incorporate into tumour stroma making them good vehicles for the delivery ofanti-cancer therapies. TNF-relatedapoptosis inducing ligand (TRAIL) selectively induces apoptosis in malignant cells without affecting healthy tissues. This study aimed to test whether MSCs modified to express TRAIL (MSCTRAIL) could be a successful cell therapy for MPM. Methods Human MSCs were transduced with a lentiviral vector containing TRAIL IRES-GFP under the control of a tetracycline dependent promoter. Successful transduction was measured using flow cytometry and TRAIL expression was confirmed by immuno-blotting and ELISA. The biological activity of MSCTRAIL was determined using co-culture experiments. DiI stained MPM cells were plated in a 1:1 ratio with MSCTRAIL cells. TRAIL production was activated and cells were treated for 48 hours. Both cells and supernatant were collected and stained with Annexin V and DAPI to detect apoptosis and death respectively on flow cytometry. To test the effect of MSCTRAIL in vivo a bioluminescent tumour model was established. Plasmid containing fireflyluciferase and YFP was expanded and the correct sequences confirmed by restriction digest. Lentivirus was produced, viral titres were determined using flow cytometry and MPM cells were transduced (MPMLuc). A pure population was generated using hygromycin selection. 80,000 MPMLuc cells were injected into the pleural cavity of NOD/SCID mice and their growth was assessed using an IVIS Lumina system to detect bioluminescence. 1 million MSCTRAIL cells were delivered via tail vein injections on days 5, 9, 12, 15 and 18 post tumour inoculation and bioluminescence was measured twice weekly. Results MSCs were successfully transduced with TRAIL with 96% efficiency and TRAIL production was confirmed by ELISA. Seven human MPM cell lines were tested with 6/7 (86%) being sensitive to MSCTRAIL. In vivo delivery of MSCTRAIL resulted in a significant reduction in MPM tumour growth. Conclusions Delivery of TRAIL via MSCs causes a significant reduction in MPM tumour growth and is a potential novel cellular therapy for this currently incurable disease.

Introduction Pulmonary fibrosis evolves in response to epithelial injury in a number of lung diseases, and carries a poor prognosis; novel therapies are urgently needed. The epithelial mitogen keratinocyte growth factor (KGF) has been shown to prevent fibrosis in a number of animal models however its therapeutic utility is limited by its short half-life. There is a growing interest in cell therapy approaches, and we hypothesised that macrophages could be used as vehicles to target KGF therapy to injured lung. Methods Lentiviral vectors expressing luciferase, KGF and GFP (control) were generated and used to transduce the IC-21 macrophage cell line. Appropriate transgene expression was confirmed. KGF macrophages were co-cultured with primary mouse tracheal epithelial cells in a proliferation bioassay. Luciferase-expressing macrophages were tracked longitudinally using bioluminescence imaging after oropharyngeal delivery to the lungs of mice given bleomycin to induce injury, or saline control. Immunostaining was used to localise macrophages within lung sections. KGF and GFP-macrophages were delivered during bleomycin-induced lung injury; endpoint measures included lung histology, micro-CT analysis, and quantification of inflammatory cell infiltrates, vascular leak, lung collagen by HPLC, and inflammatory and fibrotic mediators by ELISA and qPCR. Results Exogenously delivered macrophages were retained in the lungs of bleomycin-injured mice, but not uninjured controls, when given during either the inflammatory or fibrotic phases of injury, and localised to injured lung parenchyma. KGF-transduced macrophages induced proliferation of mouse tracheal epithelial cells during co-culture, but delivery to bleomycin-injured mice was not associated with overall improvements in endpoints when delivered during either the inflammatory or fibrotic phases of injury. Delivery of macrophages per se was associated with an increase in inflammatory mediators consistent with classical M1 macrophage activation, which may have off-set any beneficial effects of KGF-transduced macrophages. Conclusions Exogenously delivered macrophages are preferentially retained in injured lung and show potential as vehicles to target therapeutic transgenes by localising to damaged areas. Whilst KGF-transduced macrophages induced epithelial proliferation in vitro, any protective effects in vivo may have been negated by the exacerbatory effects of macrophage delivery. Future work will determine whether ex vivo manipulation of macrophage phenotype can confer therapeutic benefit.

Human lung cancers including squamous cell carcinoma (SCC) are a leading cause of death, and while evidence suggests that basal stem cells drive SCC initiation and progression, the mechanisms regulating these processes remain unknown. In this study we show that β-catenin signalling regulates basal stem cell fate and subsequent SCC progression. In a cohort of preinvasive SCCs we established that elevated basal stem cell β-catenin signalling is positively associated with increased disease severity, epithelial proliferation, and reduced intercellular adhesiveness. We demonstrate that transgene-mediated β-catenin inhibition within keratin 14-expressing basal stem cells delayed normal airway repair while basal cell-specific β-catenin activation increased cell proliferation, directed differentiation, and promoted an epithelial-to-mesenchymal transition (EMT) that included increased Snail transcription and reduced E-cadherin-mediated adhesiveness. These effects were recapitulated in normal human bronchial epithelial cells in vitro following both pharmacological β-catenin activation and E-cadherin inhibition, and mirrored our findings in preinvasive SCCs. Overall this data shows that airway stem cell β-catenin modulates cell adhesiveness to determine cell fate and its mis-expression is a key step in the development of human lung cancer.

Background Malignant pleural mesothelioma (MPM) is an aggressive fatal cancer caused by asbestos exposure. Current treatments are ineffective with an average survival of 4–18 months. Mesenchymal stem cells (MSCs) migrate to tumours and incorporate into tumour stroma making them good vehicles for the delivery of anti-cancer therapies. TNF-related apoptosis inducing ligand (TRAIL) is a transmembrane protein that selectively induces apoptosis in malignant cells without affecting healthy tissues. In this study I tested the hypothesis that MSCs modified to express TRAIL (MSCTRAIL) would cause MPM cell death. Methods Human MSCs were transduced with a lentiviral vector containing TRAIL IRES-GFP under the control of a tetracycline dependent promoter. Successful transduction was measured using flow cytometry and immunoblotting. The biological activity of MSCTRAIL was determined using co-culture experiments. 5×10 5 MPM cells were stained withDiI and plated with 5×10 5 MSCTRAIL cells. After 24 h doxycycline (10 μg/ml) was added to induce TRAIL production and left for 48 h. Both cells and supernatant were collected and stained with Annexin V and DAPI to detect apoptosis and death respectively onflow cytometry. Results MSCs were successfully transduced with TRAIL with 96% showing GFP positivity on flow cytometry. Seven human MPM cell lines were tested with 6/7 (86%) being sensitive to MSCTRAIL. JU77 was highly sensitive with an increase in apoptosis from 10.32±2.34 to 48.73±4.3 (percentage ± SEM, p≤0.0001), while MSTO-211H and ONE 58 showed increases from 3.46±0.81to 27.68±1.1 and 8.92±0.05 to 32.93±1.08 respectively (p≤0.001). Conclusions MSCs can be successfully transduced to produce TRAIL and can induce significant levels of apoptosis in the majority of MPM cell lines tested.

Introduction Injury to the alveolar epithelium underlies a number of important lung diseases, exemplified by the syndromes of acute lung injury and acute respiratory distress syndrome, which currently have a poor prognosis. Keratinocyte growth factor (KGF) is a mitogen for, and exerts beneficial effects on, type II alveolar epithelial cells. Pre-treatment with KGF is associated with improvements in outcomes in animal models of lung injury, but the use of recombinant KGF as a clinical therapy is limited by its short bioavailability and lack of specificity. We sought to determine whether macrophages could be used as vehicles to deliver KGF therapy to the injured lung. Methods Macrophages from a murine macrophage cell line (IC-21) were transduced with a lentiviral vector expressing KGF and the reporter gene GFP. Mice were given oropharyngeal (OP) bleomycin to induce lung injury. On days one and three after induction of lung injury, mice were given 3×10 6 KGF-transduced IC-21 cells (or controls) by OP instillation. Mice were sacrificed on day 5, and bronchoalveolar lavage fluid (BALF) was harvested and lungs were processed for histology. For in vivo tracking experiments, IC-21 macrophages were transduced with a lentiviral vector expressing luciferase and mice were imaged longitudinally using real-time bioluminescence imaging. Results KGF-expression was confirmed in KGF-lentivirus-transduced macrophages, however delivery of these cells was not associated with improvements in measures of alveolar-capillary membrane permeability (BALF albumin) or inflammation (total and differential cell counts) after lung injury. Cells expressing GFP were recovered in BALF, and immunohistochemistry showed groups of cells close to conducting airways. Longitudinal imaging of mice after OP delivery of luciferase-transduced IC-21 cells suggested that cells initially localised to the lungs, but did not persist at 48 h after delivery. Conclusions KGF-expressing macrophages can be generated using lentiviral vectors, but therapeutic delivery of these cells to the lungs did not improve measured outcomes in the mouse bleomycin lung injury model. Longitudinal imaging suggested that the lack of therapeutic efficacy of KGF-transduced macrophages may be due to their limited survival, and future work should focus on optimising macrophage delivery and survival in vivo.

A 19-year-old student who had never smoked presented with a 6-month history of cough, wheeze, sputum production and generalised fatigue. His exercise tolerance was unaffected and he continued to play rugby competitively. He had a history of childhood asthma and received treatment with inhaled salbutamol with no effect. A chest x-ray revealed a round density at the base of the right lung. A CT chest was performed (figure 1) and subsequently a 68Ga-DOTATATE (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-d-Phe(1),Tyr(3)-octreotate) PET-CT (positron emission tomography-CT) was arranged, demonstrating that the mass had a maximum standardised uptake value of 6.6 (figure 2). He was referred for a right lower lobectomy. Histological evaluation of the lobectomy …

“Crack lung” is an acute pulmonary syndrome with a spectrum of clinical and histopathological findings. Smoking crack, a potent and highly addictive crystalline form of cocaine, usually involves deep inspiration followed by the valsalva manoeuvre and coughing. A 30 year old man presented 8 hours after smoking crack with severe chest pain and coughing. His chest radiograph showed …