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Background Pulmonary hypoplasia (PH) and congenital lobar emphysema (CLE) are very rare congenital pulmonary anomalies in veterinary medicine. PH refers to the incomplete pulmonary development due to embryologic imbalance of bronchial development between the lung buds, while CLE is defined as alveolar hyperinflation due to bronchial collapse during expiration caused by bronchial cartilage dysplasia, external bronchial compression, and idiopathic etiology. CLE may develop into pulmonary blebs or bullae that may rupture and induce a spontaneous pneumothorax. There are no reports on concurrent PH and CLE in animals. Case presentation A 7-month-old castrated male Italian Greyhound weighing 5.5 kg presented with vomiting and acute onset of severe dyspnea without any previous history of disease. After emergency treatment including oxygen supplementation and thoracocentesis, plain radiology and computed tomography scanning were performed and lobar emphysema with multiple bullae in the left cranial lung lobe associated with tension pneumothorax was identified. Since the pneumothorax was not resolved despite continuous suction of intrathoracic air for 3 days, a complete lobectomy of the left cranial lung lobe was performed. The excised lobe was not grossly divided into cranial and caudal parts, but a tissue mass less than 1 cm in size was present at the hilum and cranial to the excised lobe. Postoperatively, the dog recovered rapidly without air retention in the thoracic cavity. Histopathologically, the mass was identified as a hypoplastic lung tissue with collapsed alveoli, bronchial dysplasia, and pulmonary arterial hypertrophy. Additionally, the excised lung lobe presented CLE with marked ectasia of alveoli, various blebs and bullae, and general bronchial cartilage dysplasia. According to gross and histopathologic findings, the dog was diagnosed with concurrent PH and CLE in the left cranial lung lobe. During 16 months of follow-up, the dog was well and without any respiratory problems. Conclusions This case report confirmed the clinical and histologic features of two different types of rare congenital pulmonary anomalies, PH and CLE, which occurred concurrently in a single lung lobe of a young dog. The condition was successfully managed with lobectomy.

Improving the early detection and chemoprevention of lung cancer are key to improving outcomes. The pathobiology of early squamous lung cancer is poorly understood. We have shown that amplification of sex-determining region Y-box 2 (SOX2) is an early and consistent event in the pathogenesis of this disease, but its functional oncogenic potential remains uncertain. We tested the impact of deregulated SOX2 expression in a novel organotypic system that recreates the molecular and microenvironmental context in which squamous carcinogenesis occurs.(1) To develop an in vitro model of bronchial dysplasia that recapitulates key molecular and phenotypic characteristics of the human disease; (2) to test the hypothesis that SOX2 deregulation is a key early event in the pathogenesis of bronchial dysplasia; and (3) to use the model for studies on pathogenesis and chemoprevention.We engineered the inducible activation of oncogenes in immortalized bronchial epithelial cells. We used three-dimensional tissue culture to build an organotypic model of bronchial dysplasia.We recapitulated human bronchial dysplasia in vitro. SOX2 deregulation drives dysplasia, and loss of tumor promoter 53 is a cooperating genetic event that potentiates the dysplastic phenotype. Deregulated SOX2 alters critical genes implicated in hallmarks of cancer progression. Targeted inhibition of AKT prevents the initiation of the dysplastic phenotype.In the appropriate genetic and microenvironmental context, acute deregulation of SOX2 drives bronchial dysplasia. This confirms its oncogenic potential in human cells and affords novel insights into the impact of SOX2 deregulation. This model can be used to test therapeutic agents aimed at chemoprevention.

Lung cancer is a deadly disease, killing 160,000 a year in the US. The 5-year survival rate of lung cancer patients is invariably low, partially due to a lack of early diagnoses and limited access to CT screening programs during the early, most-curable stages of the disease. The urgent need for alternative early-stage diagnostics has been addressed in previous work, which focused on smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells. These lung cancer associated alterations have been shown to serve as a clinically relevant biomarker for the early detection of lung cancer. In this study, we have expanded upon this work by leveraging gene expression from airway epithelial cells collected in the presence of bronchial premalignant lesions. Specifically, we conducted network-based analyses to further explore alterations in bronchial gene coexpression (GCE) patterns in the uninvolved large airway of patients with tobacco-induced premalignancy and those with lung cancer. By studying airway GCE patterns present in patients with lung cancer and premalignancy, but absent in disease-free smokers, we hope to discover the earliest changes in process of lung carcinogenesis that may serve as novel targets for lung cancer treatment and prevention. Coexpression networks use expression-profile correlations to cluster genes to provide system-wide context for single genes implicated in a disease and elucidate the influence of interconnectedness of cellular components on phenotype. Here, we employed Weighted Gene Coexpression Network Analysis (WGCNA) to build GCE networks derived from cytologically normal airway epithelial cells obtained from current and former smokers with lung cancer (n=409), dysplastic airway lesions (n=121), and neither condition (n=412). Samples were collected from subjects undergoing white-light bronchoscopy as part of the AEGIS trial by Allegro Dx, or from subjects undergoing autofluorescence bronchoscopy in the British Columbia Lung Health Study. RNA was extracted from cells and profiled on microarrays or by mRNA-Seq. In order to detect and quantify reconfiguration of networks under different disease states, the Modular Differential Connectivity (MDC) measure was calculated to determine the degree of topological similarity between pairs of GCE modules. In addition, Fisher's Exact Test (FET) was used to calculate significance of gene-member overlap between pairs of GCE modules originating from disease networks to distinguish modules conserved in premalignancy and cancer. To identify modules conserved between disease states, but dissimilar from the healthy smoker modules, we focused on those with (a) significant overlap by FET, (b) non-differentiating premalignancy vs. cancer MDC and (c) differentiating normal vs. disease MDC. Hub genes within each network were identified based on intra-modular connectivity, a measure of module essentiality. We prioritized genes with increased connectivity upon entering the premalignant state that remained strong in the lung cancer network. We identified 16 module pairs conserved between disease states, i.e. cancer modules similarly connected (MDC FDR Integration of differential connectivity and GCE network analyses represents a novel approach to discovering pathways associated with early lung carcinogenesis. Our results suggest the occurrence of transcriptomic alterations that arise in patients with premalignancy and remain present in patients with lung cancer. Future work will include experimental validation of these findings. The computational approach outlined above may aid in the identification of candidate master regulators of early lung carcinogenesis that have potential utility both as indicators of increased lung cancer risk and as early-stage drug targets for chemoprevention or therapy. Citation Format: Anna Tassinari, Bin Zhang, Katrina Steiling, Duncan Whitney, Kate Porta, Stephen Lam, Marc Lenburg, Avrum Spira, Jennifer Beane. Airway gene-coexpression network rewiring in the presence of bronchial dysplasia and lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-17.

Rationale Improving the early detection and chemoprevention of lung cancer are key to improving outcomes. The pathobiology of early squamous lung cancer is poorly understood. We have shown that amplification of SOX2 is an early and consistent event in the pathogenesis of this disease but its functional oncogenic potential remains uncertain. We tested the impact of deregulated SOX2 expression in a novel organotypic system that recreates the molecular and microenvironmental context in which squamous carcinogenesis occurs. Objectives 1) To develop an in vitro model of bronchial dysplasia that recapitulates key molecular and phenotypic characteristics of the human disease 2) To test the hypothesis that SOX2 deregulation is a key early event in the pathogenesis of bronchial dysplasia 3) To use the model for studies on pathogenesis and chemoprevention Methods We engineer the inducible activation of oncogenes in immortalised bronchial epithelial cells. We use 3-dimensional tissue culture to build an organotypic model of bronchial dysplasia. Measurements and Main Results We recapitulate human bronchial dysplasia in vitro. SOX2 deregulation drives dysplasia, and loss of TP53 is a co-operating genetic event that potentiates the dysplastic phenotype. Deregulated SOX2 alters critical genes implicated in hallmarks of cancer progression. Targeted inhibition of AKT prevents the initiation of the dysplastic phenotype. Conclusion In the appropriate genetic and microenvironmental context acute deregulation of SOX2 drives bronchial dysplasia. This confirms it’s oncogenic potential in human cells and affords novel insights into the impact of SOX2 deregulation. This model can be used to test therapeutic agents aimed at chemoprevention.

Resume A cote de l'endoscopie en autofluorescence, de nouvelles techniques endoscopiques ont vu le jour au cours des 5 dernieres annees qui pourraient modifier profondement l'exercice de l'endoscopie broncho-pulmonaire. Ces techniques, qui incluent la video-endoscopie en autofluorescence, le narrow band imaging , l'OCT (tomographie de coherence optique), et la micro-endoscopie confocale fibree, etendent le champ des explorations endoscopiques de la bronche proximale aux territoires alveolaires et de la macroscopie a l'imagerie microscopique in vivo. Dans un avenir proche, ces techniques pourront permettre l'evaluation microscopique in vivo en temps reel de nombreuses pathologies bronchiques et parenchymateuses pulmonaires, et en particulier des lesions precancereuses et des cancers superficiels.

Lung cancer is the leading cause of cancer death in the world. In the United States, lung cancer causes more death per annum than colorectal, breast, and prostate cancers combined. While smoking prevention and cessation are essential strategies against lung cancer, they are often ineffective, and former smokers remain at lower, yet persistent risks. The magnitude of the tobacco epidemic and burden of lung cancer will continue to escalate globally, underscoring the importance of advancing chemoprevention research. Recently, exciting results from phase 2b randomized control trials are beginning to emerge. Advancements in understanding clinical tumor biology, diagnostic technology, and bioinformatics will continue to propel significant progress in the field. This update will provide a general overview of the background and rationale for lung cancer chemoprevention, review important results from historical trials, summarize key findings from recently completed studies, and discuss ongoing clinical trials and future directions for lung cancer chemoprevention.

Summary The diagnosis of precancerous lesions of the bronchial tree remains a challenge for WKH FKHVW SK\VLFLDQV ,Q WKH ODVW \HDUV WKH GHYHORSPHQW RI DXWRAXRUHVFHQFH EDVHG techniques greatly impacted the detection of precancerous lesions compared with white light endoscopy. New techniques have been recently developed to get over the limitations RI DXWRAXRUHVFHQFH DOORZLQJ WKH OXQJ H[SORUDWLRQ ERWK GLVWDOO\ DQG DW WKH PLFURVFRSLF level in vivo. 7KHVH PHWKRGV LQFOXGH DXWRAXRUHVFHQFH YLGHRHQGRVFRS\ QDUURZ EDQG imaging (NBI), endobronchial ultrasonography (EBUS), optical coherence tomography (OCT) and probe based confocal laser endomicroscopy (MCFF). The histopathologic analysis is still currently the gold standard, however these new techniques could allow optical biopsies, then improving the diagnosis and the therapeutic management.© 2011 SPLF. Published by Elsevier Masson SAS. All rights reserved. Diagnostic endoscopique des lesions precoces Endoscopic diagnosis of early lesions

To assess the multiple steps involved in carcinogenesis we evaluated p53 gene mutation frequencies, and loss of heterozygosity (LOH) of chromosomes 3p, 9p and 17p in hyperplasia, squamous metaplasia, dysplasia, squamous cell carcinoma in-situ and early squamous cell carcinoma of the bronchial epithelium, using immunohistochemical, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and microsatellite analysis. Overexpression of p53 oncoprotein was observed in 31 % of mild dysplasia, 50% of moderate and 67% of severe dysplasia. Mutation of the p53 gene, however, was confirmed only in 2 severe dysplasias. LOH of 3p, 9p and 17p were detected in 8% of mild and 6% of moderate dysplasias. Among other types of bronchial lesions, two columnar cell hyperplasias showed LOH of 3p and 17p, respectively. These results suggest that LOH of 3p or 9p may be a rare but early event in the development of squamous cell carcinoma of the lung. In particular, deletion of 3p21.3 is a relatively common event in premalignancy. The results also suggest that p53 gene abnormalities occur during the following stage of carcinogenesis.

A high level of apoptotic activity and an independence of apoptosis from the expression of p53 and bcl-2 have been observed in non-small-cell lung carcinoma. We examined 44 samples of normal, metaplastic and premalignant (i.e. mild, moderate and severe dysplasias and carcinoma in situ) bronchial epithelia to evaluate whether differences in the apoptotic activity could already be seen in the stages preceding squamous cell carcinoma of the lung (SQCLC). Apoptotic cells and bodies were visualized by 3′ end labelling. The expression of p53 and members of the bcl-2 gene family, such as bcl-2, bax and mcl-1, were determined immunohistochemically with specific antibodies. The relative number of apoptotic cells and bodies [apoptotic index (AI%)] was already increased threefold as the normal bronchial epithelium changed to squamous metaplasia, and the AIs of the dysplastic lesions were about four times higher than those of the normal epithelium. Apoptosis was significantly associated with cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) immunohistochemistry. However, the extent of apoptosis did not correlate with the expression of p53, bcl-2, bax and mcl-1. We conclude that, in the metaplasia–dysplasia–carcinoma sequence in the lung, the elevation of the AI% is an early event associated with cell proliferation activity, but is independent of the expression of p53, bcl-2, mcl-1 and bax. © 1999 Cancer Research Campaign

OBJECTIVES--To investigate whether sputum cytology can be used to monitor epithelial cell changes in groups at high risk of lung cancer from exposure to radiation. METHODS--Dysplasia of bronchial cells was investigated by means of sputum cytology in a group of 434 underground miners. 100 of them were not exposed, and 334 were exposed to 222Rn progeny at cumulative exposures < 450 working level months. RESULTS--The frequency of dysplasia in the exposed group was significantly higher than that in the not exposed group (P < 0.0001), and an exposure-response relation was found. This relation was different for smokers and non-smokers. CONCLUSIONS--Possibly the frequencies of dysplasia could be used to assess past exposures of groups of miners. This approach could be applied to cases where data on radiation monitoring are not available or are very scarce.

The term ‘field cancerization’ is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

7070 Background: Lung cancer is preceded by a premalignant phase during which intervention could decrease associated morbidity and mortality. Molecular characterization of factors involved in controlling progression of bronchial dysplasia (BD) will provide markers of premalignant change and identify targets for chemoprevention. Methods: Immunohistochemical (IHC) analysis of EGFR, HER2, Ki67 and MCM2 expression in BD was undertaken in 268 bronchoscopically obtained biopsies from 134 consecutive subjects recruited to University of Colorado protocols as study subjects with prior sputum atypia and >20 pack years tobacco use or FEV1 < 75% predicted or as never- or healthy-smoker controls. IHC on the worst and best histologic biopsies from each subject were scored and linear regression tests were used to determine significance of correlation between marker expression and histology or proliferation. Results: Analysis of biopsies with the most severe diagnosis from each subject showed a linear relationship between increasing marker expression and severity of dysplastic change for EGFR (p < 0.001), Ki67 (p < 0.001) and MCM2 (p = 0.001) but not HER2 (p = 0.102). Increased expression of either EGFR or HER2 was associated with increased expression of proliferation markers Ki67and MCM2, and combined overexpression of these receptors was associated with the highest levels of proliferation. Finally, in a comparison of subjects that were grouped according to the degree of difference between their best and worst biopsy histology, the absence of an associated trend toward increased EGFR expression with increased difference in histology suggested a field effect in the induction of EGFR expression. Conclusions: Our results 1) demonstrate a direct correlation between levels of EGFR expression and degree of BD, 2) suggest a field effect in the induction of EGFR expression in the airways of subjects with BD and 3) indicate a potential synergistic effect on epithelial proliferation in lesions with increased expression of both EGFR and HER2. These findings suggest that EGFR plays a prominent role in the development and progression of BD and could provide a promising target for chemoprevention of lung cancer. [Table: see text]

Digitized images of 1,556 squamous metaplastic cells from the sputum of 15 patients with confirmed squamous-cell carcinoma of the lung and 13 subjects without apparent neoplasia were analyzed to determine if features existed within these relatively normal cells that could be used to differentiate between the two populations. Results indicate that such marker features do exist. A cross-validation study using an additional 465 cells confirmed the conclusion that squamous metaplastic cells from patients with squamous-cell carcinoma of the lung do differ from those of subjects without cancer. While such marker features alone are not reliable enough to assist pathologists in the diagnosis of squamous-cell carcinoma of the lung, they might, in sufficient numbers, be used to identify patients for whom follow-up testing would be advised.

SUMMARY AND CONCLUSIONS Hygroscopic, heated, saline-aerosol induction of sputum provides specimens which are superior to spontaneously raised sputum or bronchial aspirates for the cytologic diagnosis of bronchial carcinoma. This new technic not only increases the sensitivity of the cytologic diagnosis of bronchogenic carcinoma, but provides the means for studying cells from bronchial dysplasia and early neoplasia. Studies are needed to determine the feasibility of using this procedure for detecting premalignant or early malignant changes in the bronchial mucosa. The method is recommended for routine hospital, out-patient, and office practice. It may very well become a part of routine periodic examination as are roentgenograms of the chest and electrocardiographic examination of the heart.