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1.  Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors 
eLife  2013;2:e00966.
Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease.
DOI: http://dx.doi.org/10.7554/eLife.00966.001
eLife digest
As air flows into our lungs, the lining of the nasal cavity, the throat and the rest of the respiratory tract prevents microbes, bacteria, dust and other small particles from entering the lungs. The lining of these airways is made up of many different types of cells, which must be continuously replaced as they become damaged. Experiments in mice have shown that cells called basal cells act as progenitor cells to keep the lining supplied with new cells. Progenitor cells are similar to stem cells: they divide to make, on average, one copy of themselves and one mature cell of another type (such as a secretory cell). This ensures that healthy supply of progenitor cells is maintained for the future. However, it is not clear whether this process takes place at the level of individual progenitor cells or as an average for a population of cells.
Teixeira et al. have now performed a study which shows that basal cells achieve this balance as a result of averaging. The study took advantage of the fact that cellular organelles called mitochondria have their own DNA, which gradually accumulates mutations over time. This makes it possible to identify groups of cells that are descended from a single progenitor cell because they will all contain the same mitochondrial mutation.
By studying lung tissue from seven individuals, Teixeira et al. were able to identify clusters of related cells and found that, as expected, the size of the clusters increased with age. And by applying a mathematical model across all the cells in the study, it was discovered that whenever one basal progenitor cell committed to a particular fate, another progenitor cell duplicated itself: however, this balancing process happened in a random manner across a large number of cells, and not at the level of individual progenitor cells. Interestingly, it was found random cell division happened among smokers too, but was accelerated. This leads to clusters of identical cells forming more quickly in smokers than in non-smokers. In addition to providing further insights into the origins of lung cancer, the statistical methods developed by Teixeira et al. could be used to analyse the behaviour of many other types of stem or progenitor cells.
DOI: http://dx.doi.org/10.7554/eLife.00966.002
doi:10.7554/eLife.00966
PMCID: PMC3804062  PMID: 24151545
Human lineage tracing; mtDNA mutations; lung basal progenitor stem cells; stochastic homeostasis; airways; Human
2.  Suitability of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration Specimens for Subtyping and Genotyping of Non–Small Cell Lung Cancer 
Rationale: The current management of advanced non–small cell lung cancer (NSCLC) requires differentiation between squamous and nonsquamous subtypes as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the subclassification and genotyping of NSCLC.
Objectives: To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC.
Methods: Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across five centers in the United Kingdom between 2009 and 2011.
Measurements and Main Results: The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% confidence interval [CI], 73–80). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted odds ratio, 0.50; 95% CI, 0.28–0.82; P = 0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value, and diagnostic accuracy of EBUS-TBNA in patients with NSCLC were 88% (95% CI, 86–91), 72% (95% CI, 66–77), and 91% (95% CI, 89–93), respectively.
Conclusions: This large, multicenter, pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for subtyping of NSCLC and EGFR mutation analysis and that the use of immunohistochemistry reduces the rate of NSCLC-NOS.
doi:10.1164/rccm.201202-0294OC
PMCID: PMC3378660  PMID: 22505743
endobronchial ultrasound; non–small cell lung cancer; adenocarcinoma; EGFR mutation; NSCLC-NOS
3.  Suitability of EBUS-TBNA Specimens for Subtyping and Genotyping of NSCLC: A Multi-Centre Study of 774 Patients 
Rationale
The current management of advanced non-small cell lung cancer (NSCLC) requires differentiation between squamous and non-squamous sub-types as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the sub-classification and genotyping of NSCLC.
Objectives
To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC.
Methods
Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across 5 centres in the United Kingdom between 2009 and 2011.
Measurements and Main Results
The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% CI 73% - 80%). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted OR 0.50 95% CI 0.28 – 0.82, P=0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value and diagnostic accuracy of EBUS-TBNA in patients with NSCLC was 88% (95% CI 86% - 91%), 72% (95% CI 66% - 77%) and 91% (95% CI 89% - 93%) respectively.
Conclusions
This large multi-centre pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for sub-typing of NSCLC and EGFR mutation analysis and that use of immunohistochemistry reduces the rate of NSCLC-NOS.
doi:10.1164/rccm.201202-0294OC
PMCID: PMC3378660  PMID: 22505743
Endobronchial ultrasound; non-small cell lung cancer; adenocarcinoma; EGFR mutation; squamous cell carcinoma; NSCLC-NOS
4.  Combination of endobronchial ultrasound-guided transbronchial needle aspiration with standard bronchoscopic techniques for the diagnosis of stage I and stage II pulmonary sarcoidosis 
Respirology (Carlton, Vic.)  2011;16(3):467-472.
Background and objective
Standard bronchoscopic techniques (transbronchial lung biopsy and endobronchial biopsy) provide a diagnosis in 70% of patients with pulmonary sarcoidosis. Previous data suggest that endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has a high sensitivity in patients with sarcoidosis. The feasibility and utility of combining EBUS-TBNA with standard bronchoscopic techniques is unknown. The aim of this study was to evaluate the feasibility, safety and efficacy of combined EBUS-TBNA and standard bronchoscopic techniques in patients with suspected sarcoidosis and enlarged mediastinal or hilar lymphadenopathy.
Methods
Forty consecutive patients with suspected pulmonary sarcoidosis and enlarged mediastinal or hilar lymph nodes (radiographical stage I and stage II) underwent EBUS-TBNA followed by transbronchial biopsies and endobronchial biopsies under conscious sedation.
Results
Thirty-nine out of 40 patients successfully underwent combined EBUS-TBNA and standard bronchoscopy. Twenty-seven patients were diagnosed with sarcoidosis, eight had tuberculosis, two had reactive lymphadenopathy, two had lymphoma and one had metastatic adenocarcinoma. In patients with sarcoidosis, the sensitivity of EBUS-TBNA for detection of noncaseating granulomas was 85%, compared with a sensitivity of 35% for standard bronchoscopic techniques (P < 0.001). The diagnostic yield of combined EBUS-TBNA and bronchoscopy was 93% (P < 0.0001).
Conclusions
Combination of EBUS-TBNA with standard bronchoscopic techniques is safe and feasible, and optimizes the diagnostic yield in patients with pulmonary sarcoidosis and enlarged intrathoracic lymphadenopathy.
doi:10.1111/j.1440-1843.2011.01933.x
PMCID: PMC3361303  PMID: 21261784
endobronchial ultrasound; mediastinal lymphadenopathy; sarcoidosis; transbronchial biopsy
5.  Utility of endobronchial ultrasound-guided transbronchial needle aspiration in patients with tuberculous intrathoracic lymphadenopathy: a multicentre study 
Thorax  2011;66(10):889-893.
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.
doi:10.1136/thoraxjnl-2011-200063
PMCID: PMC3361304  PMID: 21813622
6.  Off-Pump Coronary Artery Bypass Leads to a Regional Hypercoagulable State Not Detectable Using Systemic Markers 
Innovations (Philadelphia, Pa.)  2006;1(5):232-238.
Objective:
It is believed that off-pump coronary artery bypass grafting (OPCAB) leads to hypercoagulability, but efforts to document such a state have been unrevealing. We hypothesized that OPCAB increases the risk of developing a regional hypercoagulable state.
Methods:
Blood was obtained from the aorta and coronary sinus (CS) after CABG performed off- (N=69) or on-pump (N=35) to determine the transcardiac gradients of F1.2 (thrombin production), XIIa (coagulation activation), myoglobin (ischemia) and IL-6, IL-8 using ELISA and platelet-derived microparticles using FACS. Platelet function was measured using aggregometry. Regional myocardial pH and SVG flow were recorded intraoperatively. SVG biopsies were analyzed for endothelial integrity (EI) using immunohistochemistry and graft patency was determined by predischarge CT angiography.
Results:
Compared with on-pump, OPCAB provoked significantly higher transcardiac F1.2 (117±200 v. 31±38%), FXII-a (14±29 v. 2±4%), microparticles (14±−9.5% v. 6.4±−4.1%), IL-6 (119±183 v. 28±39%), and a trend toward increased IL-8 (67±94 v. 24±46%, P = 0.077). Myoglobin release after OPCAB, also greater than on-pump CABG (54±89 v. 8±14%, P < 0.01), correlated with regional pH change (R=−0.96, P < 0.0001), and F1.2 release (R=0.55, P = 0.0002). In contrast, systemic changes in these markers were all less after OPCAB. SVG flow was significantly reduced in OPCAB (39.4 versus 66.5 mL/min, P = 0.0002), but EI and graft patency rates were the same.
Conclusions:
Through the use of transcardiac assays, we illustrated that regional coagulation was enhanced after off- compared with on-pump CABG. If the findings of this pilot study are confirmed, OPCAB may require additional antithrombotic therapies to respond to this local hypercoagulable state.
PMCID: PMC2614863  PMID: 19132144
Off-pump coronary artery bypass graft surgery; Ischemia; Thrombin; Saphenous vein graft
7.  Cell migration leads to spatially distinct but clonally related airway cancer precursors 
Thorax  2014;69(6):548-557.
Background
Squamous cell carcinoma of the lung is a common cancer with 95% mortality at 5 years. These cancers arise from preinvasive lesions, which have a natural history of development progressing through increasing severity of dysplasia to carcinoma in situ (CIS), and in some cases, ending in transformation to invasive carcinoma. Synchronous preinvasive lesions identified at autopsy have been previously shown to be clonally related.
Methods
Using autofluorescence bronchoscopy that allows visual observation of preinvasive lesions within the upper airways, together with molecular profiling of biopsies using gene sequencing and loss-of-heterozygosity analysis from both preinvasive lesions and from intervening normal tissue, we have monitored individual lesions longitudinally and documented their visual, histological and molecular relationship.
Results
We demonstrate that rather than forming a contiguous field of abnormal tissue, clonal CIS lesions can develop at multiple anatomically discrete sites over time. Further, we demonstrate that patients with CIS in the trachea have invariably had previous lesions that have migrated proximally, and in one case, into the other lung over a period of 12 years.
Conclusions
Molecular information from these unique biopsies provides for the first time evidence that field cancerisation of the upper airways can occur through cell migration rather than via local contiguous cellular expansion as previously thought. Our findings urge a clinical strategy of ablating high-grade premalignant airway lesions with subsequent attentive surveillance for recurrence in the bronchial tree.
doi:10.1136/thoraxjnl-2013-204198
PMCID: PMC4033139  PMID: 24550057
Lung Cancer; Airway Epithelium

Results 1-7 (7)