Bronchial Brushing Research Articles

Sputum transcriptomics reveal upregulation of IL-1 receptor family members in patients with severe asthma

Sputum analysis in asthmatic patients is used to define airway inflammatory processes and might guide therapy. We sought to determine differential gene and protein expression in sputum samples from patients with severe asthma (SA) compared with nonsmoking patients with mild/moderate asthma. Induced sputum was obtained from nonsmoking patients with SA, smokers/ex-smokers with severe asthma, nonsmoking patients with mild/moderate asthma (MMAs), and healthy nonsmoking control subjects. Differential cell counts, microarray analysis of cell pellets, and SOMAscan analysis of sputum analytes were performed. CRID3 was used to inhibit the inflammasome in a mouse model of SA. Eosinophilic and mixed neutrophilic/eosinophilic inflammation were more prevalent in patients with SA compared with MMAs. Forty-two genes probes were upregulated (>2-fold) in nonsmoking patients with severe asthma compared with MMAs, including IL-1 receptor (IL-1R) family and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NRLP3) inflammasome members (false discovery rate<0.05). The inflammasome proteins nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 1 (NLRP1), NLRP3, and nucleotide-binding oligomerization domain (NOD)-like receptor C4 (NLRC4) were associated with neutrophilic asthma and with sputum IL-1β protein levels, whereas eosinophilic asthma was associated with an IL-13-induced TH2 signature and IL-1 receptor-like 1 (IL1RL1) mRNA expression. These differences were sputum specific because no activation of NLRP3 or enrichment of IL-1R family genes in bronchial brushings or biopsy specimens in patients with SA was observed. Expression of NLRP3 and of the IL-1R family genes was validated in the Airway Disease Endotyping for Personalized Therapeutics cohort. Inflammasome inhibition using CRID3 prevented airway hyperresponsiveness and airway inflammation (both neutrophilia and eosinophilia) in a mouse model of severe allergic asthma. IL1RL1 gene expression is associated with eosinophilic SA, whereas NLRP3 inflammasome expression is highest in patients with neutrophilic SA. TH2-driven eosinophilic inflammation and neutrophil-associated inflammasome activation might represent interacting pathways in patients with SA.

Human β-defensin-2 production upon viral and bacterial co-infection is attenuated in COPD.

Viral-bacterial co-infections are associated with severe exacerbations of COPD. Epithelial antimicrobial peptides, including human β-defensin-2 (HBD-2), are integral to innate host defenses. In this study, we examined how co-infection of airway epithelial cells with rhinovirus and Pseudomonas aeruginosa modulates HBD-2 expression, and whether these responses are attenuated by cigarette smoke and in epithelial cells obtained by bronchial brushings from smokers with normal lung function or from COPD patients. When human airway epithelial cells from normal lungs were infected with rhinovirus, Pseudomonas aeruginosa, or the combination, co-infection with rhinovirus and bacteria resulted in synergistic induction of HBD-2 (p<0.05). The combination of virus and flagellin replicated this synergistic increase (p<0.05), and synergy was not seen using a flagella-deficient mutant Pseudomonas (p<0.05). The effects of Pseudomonas aeruginosa were mediated via interactions of flagellin with TLR5. The effects of HRV-16 depended upon viral replication but did not appear to be mediated via the intracellular RNA helicases, retinoic acid-inducible gene-I or melanoma differentiation-associated gene-5. Cigarette smoke extract significantly decreased HBD-2 production in response to co-infection. Attenuated production was also observed following co-infection of cells obtained from healthy smokers or COPD patients compared to healthy controls (p<0.05). We conclude that co-exposure to HRV-16 and Pseudomonas aeruginosa induces synergistic production of HBD-2 from epithelial cells and that this synergistic induction of HBD-2 is reduced in COPD patients. This may contribute to the more severe exacerbations these patients experience in response to viral-bacterial co-infections.

A Correlational Study of Bronchial Brushing With Bronchoscopic Biopsy in Diagnosis of Malignant Lung Neoplasms

A Correlational Study of Bronchial Brushing With Bronchoscopic Biopsy in Diagnosis of Malignant Lung Neoplasms

Comparative Analysis for Diagnostic Yield of Small Cell Lung Cancer by Cytology and Histology During the Same Bronchoscopic Procedure

BackgroundBiopsy, brushing, and transbronchial needle aspiration (TBNA) are the most common methods used for the diagnosis of small cell lung cancer during the same diagnostic bronchoscopic procedure. However, it is not clear which method provides better results. Patients and MethodsA retrospective analysis was performed of 140 patients who had undergone video bronchoscopy for diagnostic purposes. Bronchial brushings were obtained from all subjects. Biopsy specimens were also obtained from all subjects, except for 6 cases that could not be sampled; the TBNA method was used for some special lesions. The results were analyzed separately by histology and cytology. ResultsThe diagnostic yield of cytology was significantly greater than that of histology (P < .01) and that of conventional smear preparations in cytology was obviously greater than that of hematoxylin and eosin stains in histology (P < .01). The false-negative results were significantly lower with cytology than with histology (P < .01). Also, the cases of sampling site restriction with cytology were distinctly less than those with histology (P < .05). Stretch deformation of the tissue structure and cell morphology was the main reason for the false-negative results in the histologic diagnosis. The use of TBNA resolved all 4 cases of hilar adenopathy and 2 cases of lesions outside the bronchus. Multiple brushings of the tissue adjacent to cancer tissue and liquid-based preparations of cancerous necrotic tissue can significantly reduce the false-negative results from biopsy. ConclusionsThe diagnostic yield of cytologic examination of brushings and TBNA for small cell lung cancer was superior to that of histologic examination of hematoxylin and eosin stains and immunohistochemistry.

Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases.

IA01.04 Does Cytological Material Fit All - Lessons from EBUS/Bronchoscopy

In the diagnosis of lung cancer the primary diagnostic approach is microscopic evaluation of tissue biopsy. However, in many cases the only material for microscopic evaluation is cytological one. There are numerous types of cytological material in evaluation of lung tumors including sputum, bronchial brush, bronchial washing, transthoracic and transbronchial fine needle aspirates. Historically sputum was the most common diagnostic material but at present the most common types of cytological specimen are bronchial brush, and transthoracic or transbronchial FNAB. Nowadays worked out and recognized cytological criteria allows not only to diagnose carcinoma but in majority of cases to specify the histologic type of a tumor. This approach to microscopic diagnosis of lung carcinoma was incorporated to the latest WHO classification of lung tumors (ref). The key reason that cytodiagnosic criteria and terminology were included into WHO classification was that in about 30% cases of lung tumors the cytological specimen is the only material for microscopic evaluation. Similarly to evaluation of small tissue biopsies in cytopathology, in doubtful cases, immunocytochemistry may be implemented to determine histologic type of tumor. Most useful in evaluation of cytological specimen are IHC antibodies with nuclear presentation (e.g. p40 for squamous cell carcinoma and TTF1 for adenocarcinoma). Cytological material may be utilized in evaluation and in differential diagnosis between primary and metastatic lung tumor. The panel of the antibodies (e.g. CDX2, PSA, Melan A) may be used to indicate location of primary tumors mainly adenocarcinomas. Since cytological smears are of limited diagnostic value for immunocytochemistry, the so-called cell blocs technique is recommended. This technique allows to use larger panel of antibodies for immunohistochemical evaluation. Cytology become very useful for clinical staging of lung carcinoma routinely utilizing EBUS and EUS technique for obtaining material from parahilar and mediastinal lymph nodes. Cytological criteria are similar to that used for specimens obtained by transthoracic FNAB. In our center practice cooperation with adequately trained thorax surgeons provides adequate material for microscopic evaluation from EBUS and EUS obtained specimens in 94% cases. Currently one of the important task for pathologist in evaluating material from lung carcinoma is adequate selection of the material for molecular test. In case of lung carcinoma or to be specific lung adenocarcinoma â material is selected for evaluation of EGFR and K-RAS mutation. In our experience cytological material particularly fine needle aspirates - fulfill such demands. Percentages of tumors cells in the sample is often even higher than in tissue sections especially in fine needles of peripherally location lesions. Cytological material may be useful in evaluation of EGFR and K-RAS mutation as well as in determination of presence of translocations of ALK and ROS1 using FISH technique. Evaluation of ALK and ROS1 translocation remained in the hands of cytopathologists since the crucial point is the location of translocation in nuclei of tumors cells. Latest challenge for pathologists evaluating lung carcinoma specimen is to determine predictive criteria for immunotherapy in those tumors. At the moment it seems that cytological material may be not satisfactory for adequate evaluation of immunocytochemical expression of PD-L1 and PD-1. In summary cytological material from lung carcinoma is useful in establishing the firm microscopic diagnosis of malignancy, to determine histologic type and to be used for molecular tests. It also allows to differentiated between primary and metastatic lung malignancies as well as determined primary location of metastatic lung carcinoma. Cytological specimens obtained by EBUS and EBUS techniques are very useful in clinical staging of lung carcinoma. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG eds. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. 4th ed. Lyon, France: IARC Press; 2015. cytology, lung carcinoma, molecular testing

‘Bronchoscopic Characterization of Lesions’: Significant impact on lung cancer diagnosis with use of Transbronchial needle aspiration (TBNA) in Comparison to conventional diagnostic techniques (CDTs)

Background: Lung Cancer is the leading cause of cancer deaths around the world. Lung cancer accounts for 13.8% of all cancer diagnosis in India. Aims: (1) To find the role of bronchoscopic characterization of lesions and its impact on outcome in yield in lung cancer with use of conventional diagnostic techniques. (2) To observe additional yield of transbronchial needle aspiration (TBNA) in comparison to other conventional diagnostic techniques (CDTs) such as bronchial wash (BW), bronchial brush (BB), and forcep biopsy. Settings and Design: This is a prospective muticentric study conducted during June 2013–December 2016 at bronchoscopy unit of Venkatesh Chest Hospital Latur and Pulmonary Medicine, MIMSR Medical College Latur India. Materials and Methods: The study included 210 patients on the basis of clinical and radiological features of malignancy after inclusion and exclusion criteria and institutional review board approval. Fiberoptic bronchoscopic abnormalities were categorized as Exophytic endobronchial lesions, submucosal lesions, peribronchial lesions, and no abnormality. TBNA and other CDTs such as forcep biopsy, BB, and BW were performed during bronchoscopy procedure. Histopathological and cytological examinations of specimens were performed at pathology department. Statistical Analysis Used: The statistical analysis was done using Chi-square test. Results: In exophytic endobronchial lesions, yield of TBNA, CDTs, and TBNA plus CDTs was 62.60%, 79.67%, and 84.55%, respectively (P 0.8). In peribronchial lesions, TBNA has significant yield, i.e., 63.41% individually as compared to forcep biopsy 26.82% and CDT 39.02% (P < 0.001). Overall diagnostic yield of fiberoptic bronchoscopy in confirming the diagnosis was 71.95%. Conclusions: Bronchoscopic characterization of lesions and use of technique accordingly during bronchoscopy has a significant outcome in the form of yield also it will decrease the need for repeat bronchoscopy.

Interleukin-26 Production in Human Primary Bronchial Epithelial Cells in Response to Viral Stimulation: Modulation by Th17 cytokines

Interleukin (IL)-26 is abundant in human airways and this cytokine is involved in the local immune response to a bacterial stimulus in vivo. Specifically, local exposure to the toll-like receptor (TLR) 4 agonist endotoxin does increase IL-26 in human airways and this cytokine potentiates chemotactic responses in human neutrophils. In addition to T-helper (Th) 17 cells, alveolar macrophages can produce IL-26, but it remains unknown whether this cytokine can also be produced in the airway mucosa per se in response to a viral stimulus. Here, we evaluated whether this is the case using primary bronchial epithelial cells from the airway epithelium in vitro and explored the signaling mechanisms involved, including the modulatory effects of additional Th17 cytokines. Finally, we assessed IL-26 and its archetype signaling responses in healthy human airways in vivo. We found increased transcription and release of IL-26 protein after stimulation with the viral-related double stranded (ds) RNA polyinosinic-polycytidylic acid (poly-IC) and showed that this IL-26 release involved mitogen-activated protein (MAP) kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The release of IL-26 in response to a viral stimulus was modulated by additional Th17 cytokines. Moreover, there was transcription of IL26 mRNA and expression of the protein in epithelial cells of bronchial brush and tissue biopsies respectively after harvest in vivo. In addition, the extracellular IL-26 protein concentrations in bronchoalveolar lavage (BAL) samples did correlate with increased epithelial cell transcription of an archetype intracellular signaling molecule downstream of the IL-26-receptor complex, STAT1, in the bronchial brush biopsies. Thus, our study suggests that viral stimulation causes the production of IL-26 in lining epithelial cells of human airways, structural cells that constitute a critical immune barrier and that this production is modulated by Th17 cytokines.

Flexible fiberoptic bronchoscopy in respiratory care: Diagnostic yield, complications, and challenges in a Nigerian Tertiary Center.

Flexible fiberoptic bronchoscopy (FOB) is a key diagnostic and therapeutic procedure in pulmonology. Experience with fiberoptic bronchoscopy is scanty in most developing countries. The goal of this study was to report our experience and clinical utility of fiberoptic bronchoscopy. A review of bronchoscopy requests, services, and reports performed over a 5-year period was performed. Demographic characteristics were extracted. Indications for the procedures, type of bronchoscopic sampling done, final diagnosis, and complications were reported. Sensitivities, specificities, and overall diagnostic yield of the procedures were determined. About 163 diagnostic bronchoscopies were performed during the study. Ninety-nine patients with complete data were analyzed. Mean age was 54.8 ± 19.2 years, with males constituting the majority, 56.6%. Suspected bronchial cancer and pleural effusion were the main indications for bronchoscopy (33% and 19.1%, respectively). A total of 80, 39, and 99 bronchial washings, brushings, and bronchial biopsies were performed, respectively. Bronchial cancer was confirmed in 51.5% and was diagnostic in 57% of suspected pleural effusion. Pulmonary tuberculosis was confirmed in 50% of suspected cases and additional 8 cases were diagnosed. The overall diagnostic yield of bronchoscopy was 62%. Specificities of bronchial brushing and washing cytology for excluding bronchial cancer were 90.9 and 83%, respectively, and sensitivities of detecting bronchial cancer were 64.3% and 59%, respectively, P< 0.05 each. Serious complication occurred in about 1%. There was no mortality. These results show that FOB is a useful and safe procedure with a low complication rate in our setting.

Utility of Various Bronchoscopic Modalities in Lung Cancer Diagnosis

Background:Bronchoscopy and different techniques to obtain tissue sample form the cornerstone of lung cancer diagnostics. The utility, specificity and sensitivity of various techniques are compared against each other and also in relation to the tumour type, location and morphology.Aim:To assess and compare the utility of various bronchoscopic procedures in lung malignancies. Study also compares the utility of these techniques on tumour site, morphology and cell type.Methods:One hundred and fifty six patients with suspected malignancies in whom bronchoscopy was deemed as the primary diagnostic procedure were selected. These patients underwent bronchoscopic lavage, brushings and biopsy. Samples were assessed by microbiological, cytology and histopathological analysis.Results:Bronchoscopic procedures have a high diagnostic accuracy of 81.25% in confirming lung malignancies in central tumours and also in non-accessible peripheral tumours. Bronchial brushings had the highest yield in central tumours (55.9%) and a reasonably good yield even in perpipheral tumours (40.8%). When all the modalities were compared against each other, brushings was the single most decisive technique as it alone yielded a diagnosis in a significant 33% of cases, whereas the comparative diagnosis by biopsy alone was in a minority 7.6% of cases.

Filariasis in Bone Marrow: Presenting as Pancytopenia

Filariasis is caused by Wuchereria bancrofti or Brugia malayi, both of which are transmitted through Culex mosquito. It is one of the major public health hazards in endemic areas of the India. The patients present with pyrexia, lymphadenopathy and elephantiasis. It is demonstrated in the peripheral blood, body fluids, fine needle aspirates and in the bronchial brushings. The occurrence of microfilaria in the bone marrow is a rare presentation. We hereby report one such case of a geriatric patient who presented as a case of pyrexia of unknown origin and pancytopenia.

The pitfalls in cytology diagnosis of poorly differentiated neuroendocrine carcinoma of lung and their treatment response.

Lung is the most common site of small cell carcinoma (SCLC) - a poorly differentiated neuroendocrine carcinoma (PDNEC). SCLC comprises 15-20% of the invasive cancers of the lung. This study was conducted to appraise the accuracy and pitfalls of the diagnosis of PDNEC on cytology along with treatment responses if available. Retrospective study for 2 years yielded 21 cases on cytology. Slides of fine-needle aspiration of lymph nodes, the tumor, bronchial brush, and bronchoalveolar lavage specimens were used. The histological correlation was obtained as were treatment responses. Eighteen SCLCs were confirmed on review. Of these, 13 initial reports were concordant and five, discordant. The rest three cases which initially reported as SCLC were found to be negative (2) and combined SCLC (1). One SCLC with concordant initial and reviewed diagnoses failed to confirm on histopathology. The patients, all heavy smokers, were predominantly males in the seventh to eighth decade age group. The sensitivity and specificity of reviewed diagnoses were better than that of the original. The difference between histopathology and cytology diagnoses (reviewed and original) was statistically insignificant. All patients were categorized as "extensive stage" by positron emission tomography-computerized tomography, and five were treated with etoposide and cisplatin with/without radiotherapy. Age group (61-70) and gender (males) distribution were statistically significant. Intermediate variants of SCLC may be misdiagnosed as adenocarcinoma. Similarly, combined SCLC may be missed on cytology if the observer does not sustain a high index of suspicion. Unequivocal cytology diagnosis opposed to negative histopathology report demands repeat biopsy.

Multiple guided technologies based on radial probe endobronchial ultrasound for the diagnosis of solitary peripheral pulmonary lesions: a single-center study.

Objective To evaluate the value of multiple guided technologies based on radial probe endobronchial ultrasound (R-EBUS) in the application of the diagnosis of solitary pulmonary peripheral lesions (PPLs).Methods Analysis of diagnostic yield in 4 groups patients with different combined multiple guided technologies based on R-EBUS, 94 patients with 94 solitary PPLs from Mar, 2013 to Nov, 2014 in Changhai Hospital.Results The overall diagnostic yield was 75% (70/94), the diagnostic yield of Group A (R-EBUS) was 62%(16/26), Group B (R-EBUS with guided sheath, EBUS-GS) was 76% (34/45), Group C (EBUS-GS with fluoroscopy) was 82% (9/11), Group D (virtual bronchoscopic navigation guided EBUS-GS with fluoroscopy) was 92% (11/12). The overall histopathological diagnostic yield was 56% (53/94. Better histopathological diagnostic yield was associated with application of multiple guided technologies based on EBUS-GS, lesions located in non-lower lobes, lesion's diameter > 2cm, radial probe within the lesions and lidocaine nebulization combined with intravenous anesthesia. There were no severe complications in all the 94 cases. A ultrasonic radial probe was broken when exploring a lesion located in the apical-posterior segment of left upper lobe.Conclusion Multiple guided technologies based on R-EBUS were safe and effective in the diagnosis of solitary PPLs. In practice, the diagnosis yield improved with the application of forcep biopsies combined with bronchial brushing and washing.

MA05.01 Virtual Bronchoscopic Navigation-Guided Ultrathin Bronchoscopy for Diagnosing Peripheral Pulmonary Lesions

Diagnosis of peripheral pulmonary lesions with ultrathin bronchoscopy (UTH) has fewer complications than transthoracic needle aspiration (TTNA). However, diagnostic yield with UTH is lower. Virtual bronchoscopic navigation (VBN) might increase diagnostic performance of UTH. The main objective was to compare diagnostic yield of UTH with and without VBN. Prospective case-control study paired 1:2 for lesion size and localization, bronchus sign, sex and final diagnosis. LungPoint (Broncus, USA) was used to plan and navigate based upon online image analysis, putting endoscopic and virtual images in correspondence. Fluoroscopy was used in all. Sampling included bronchial washing and brushing (if no direct vision) or biopsy (if lesion directly visualized). Statistical analyses R-3.2.3. Total of 63 patients (VBN and non-VBN, 21:42). Clinical characteristics in table 1. Diagnostic yield was 75% vs 43.9% (p=0.029). Factors associated to positive diagnosis in table 2. Further diagnostic techniques were needed in 14% vs 52% (p=0.001). No differences seen in procedure duration or complications.View Large Image Figure ViewerDownload Hi-res image Download (PPT) VBN significantly improves the diagnostic yield of ultrathin bronchoscopy for diagnosing peripheral pulmonary lesions, especially those located in the utmost periphery and fluoroscopically not visible. Therefore, use of VBN reduces the need for further diagnostic procedures. Funded by La MaratóTV3-20133510, FIS-ETES PI09/90917, FUCAP and SEPAR.

P1.03-056 Implementation of a Prospective Biospecimen Collection Study in an Established Lung Cancer Screening Program

Complexities such as addressing indeterminate pulmonary nodules (IPNs) are an inherent part of a lung screening program, and defining which individuals will benefit from invasive intervention is not always known. With the goal of combining non-invasive biomarkers with imaging to more definitively stratify patients, we initiated an investigational biospecimen collection process into our lung screening program. Ultimately, these biomarkers may improve specificity within the screening population, thereby reducing the overall cost and potential morbidity from false positive results of low-dose computed tomography scan (LDCT) screening. Studies like this are important to the ongoing improvement of lung cancer screening. NCCN high-risk individuals enrolled in a high volume clinical lung screening program were introduced to our IRB approved research biospecimen study at the time of the scheduling of their LDCT. Patients were consented, and routine biospecimens were collected by research staff at the time of their LDCT scan, including nasal epithelial brushings, buccal swabs and blood. When available, additional biospecimens consisting of bronchial airway brushings and tumor samples were collected from subjects who underwent diagnostic interventions for suspicion of malignancy. Since 2012 there have been 3856 patients enrolled and 8776 LDCT scans to date with 100 lung cancer diagnoses within our lung screening program. In 2014, funding (LUNGevity and Robert E Wise grants) was obtained for biospecimen collection from subjects enrolled in our institutional LDCT screening program. Initial prospective collection of biospecimens was slower than anticipated due to various factors. After review of the process, a number of adjustments were made, which significantly increased enrollment. This included implementation of a multidisciplinary taskforce consisting of research and clinical staff committed to patient outreach and participation. To date, samples from approximately 1420 subjects have been collected. Of these, 268 (19%) were found to have newly detected IPNs measuring 6-20mm on LDCT, and 28 samples were from patients with subsequent diagnoses of lung cancer. Successful coordination of biospecimen collection within a lung screening program is complex, but achievable with multidisciplinary coordination, and has great potential to help further stratify patients that may or may not benefit from invasive diagnostics and therapy. We demonstrate an established lung screening program that is successfully accruing to a prospective diagnostic study and share specific recommendations for how to successfully accrue in other programs.

Diagnostic Efficacy of Bronchial Brush and Bronchoalveolar Lavage Cytology in Bronchopulmonary Lesions

Diagnostic Efficacy of Bronchial Brush and Bronchoalveolar Lavage Cytology in Bronchopulmonary Lesions

P2.03b-080 A Comprehensive Test of Cancer Treatment-Related Genes for the Clinical Samples of Non-Small Cell Lung Cancer (NSCLC): Topic: Biomarkers

Molecular targeted therapies are one of the key drugs for NSCLC. For all advanced NCSLC patients to receive precise molecular targeted therapies, mutation analysis should be comprehensively analyzed from tissue and/or cytological samples with high sensitivity and cost-effectiveness. To satisfy these requirements, we established a system called MINtS(Mutation Investigation System using a Next-generation Sequencer). According to our basic data, by deep sequencing, MINtS enables us to detect genetic alterations from the clinical specimens of which cancer cell content is just over 1%. The lists of mutations are EGFR, KRAS, BRAF, HER2, and ALK/RET/ROS1 fusion genes. We report the data analyzed from the clinical cytological and tissue samples using MINtS. MINtS adapted amplicon sequencing strategy. For the EGFR, KRAS, BRAF and HER2 genes, the target regions are amplified by the multiplex PCR. For the ALK, RET and ROS1 fusion genes as well as OAZ1 housekeeping gene (internal control), the targets are amplified by the multiplex RT-PCR. The index sequences were then added for discriminating samples derived from different patients. Amplified fragments from multiple samples are combined, and run on a Next generation Sequencer. According to the index sequences, the sequencing reads obtained were de-multiplexed for each sample. Using MINtS system, we analyzed 65 tissue samples that were obtained surgically, and 76 cytological specimens that were obtained by bronchial brushing or pleural effusions from NSCLC patients. Our preliminary data indicated that mutations for EGFR, KRAS, BRAF, and HER2 were detected from DNA, and the test detected mutations from samples with cancer cell content > 1% with specificity and sensitivity >0.99. Fusion genes were detected from mRNA, and the test were considered to detect them from samples with cancer cell content >1%. Among 141samples, 140samples were successfully analyzed. EGFR mutations in 36 samples (25.5%), KRAS mutations in 10 samples (7%), BRAF mutations in 5 samples (3.5%), and HER2 mutations in 5 samples (3.5%). Regarding fusion genes, ALK fusion gene were 2 samples (1.4%), RET fusion gene were 2 samples (1.4%), and ROS fusion gene was 1 sample (0.7%). MINtS could analyze from both tissue and cytological samples. MINtS tests >100 samples in a single run. This fulfills clinical requirement of a high throughput testing at an affordable unit price; 30US dollars per sample. MINtS provides a protoytpe of mutation test applicable to cancers originating from organs other than lung, and may be also adaptable to liquid biopsy.

A Transcriptome-driven Analysis of Epithelial Brushings and Bronchial Biopsies to Define Asthma Phenotypes in U-BIOPRED.

Asthma is a heterogeneous disease driven by diverse immunologic and inflammatory mechanisms. Using transcriptomic profiling of airway tissues, we sought to define the molecular phenotypes of severe asthma. The transcriptome derived from bronchial biopsies and epithelial brushings of 107 subjects with moderate to severe asthma were annotated by gene set variation analysis using 42 gene signatures relevant to asthma, inflammation, and immune function. Topological data analysis of clinical and histologic data was performed to derive clusters, and the nearest shrunken centroid algorithm was used for signature refinement. Nine gene set variation analysis signatures expressed in bronchial biopsies and airway epithelial brushings distinguished two distinct asthma subtypes associated with high expression of T-helper cell type 2 cytokines and lack of corticosteroid response (group 1 and group 3). Group 1 had the highest submucosal eosinophils, as well as high fractional exhaled nitric oxide levels, exacerbation rates, and oral corticosteroid use, whereas group 3 patients showed the highest levels of sputum eosinophils and had a high body mass index. In contrast, group 2 and group 4 patients had an 86% and 64% probability, respectively, of having noneosinophilic inflammation. Using machine learning tools, we describe an inference scheme using the currently available inflammatory biomarkers sputum eosinophilia and fractional exhaled nitric oxide levels, along with oral corticosteroid use, that could predict the subtypes of gene expression within bronchial biopsies and epithelial cells with good sensitivity and specificity. This analysis demonstrates the usefulness of a transcriptomics-driven approach to phenotyping that segments patients who may benefit the most from specific agents that target T-helper cell type 2-mediated inflammation and/or corticosteroid insensitivity.

The airway epithelium undergoes metabolic reprogramming in individuals at high risk for lung cancer.

The molecular determinants of lung cancer risk remain largely unknown. Airway epithelial cells are prone to assault by risk factors and are considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into the molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushing specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified 2,869 proteins, of which 312 proteins demonstrated a trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for 7 months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-13C5] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results provide a rationale for the development of potentially new chemopreventive strategies and selection of patients for surveillance programs.

Features of the bronchial bacterial microbiome associated with atopy, asthma, and responsiveness to inhaled corticosteroid treatment

Compositional differences in the bronchial bacterial microbiota have been associated with asthma, but it remains unclear whether the findings are attributable to asthma, to aeroallergen sensitization, or to inhaled corticosteroid treatment. We sought to compare the bronchial bacterial microbiota in adults with steroid-naive atopic asthma, subjects with atopy but no asthma, and nonatopic healthy control subjects and to determine relationships of the bronchial microbiota to phenotypic features of asthma. Bacterial communities in protected bronchial brushings from 42 atopic asthmatic subjects, 21 subjects with atopy but no asthma, and 21 healthy control subjects were profiled by using 16S rRNA gene sequencing. Bacterial composition and community-level functions inferred from sequence profiles were analyzed for between-group differences. Associations with clinical and inflammatory variables were examined, including markers of type 2-related inflammation and change in airway hyperresponsiveness after 6weeks of fluticasone treatment. The bronchial microbiome differed significantly among the 3 groups. Asthmatic subjects were uniquely enriched in members of the Haemophilus, Neisseria, Fusobacterium, and Porphyromonas species and the Sphingomonodaceae family and depleted in members of the Mogibacteriaceae family and Lactobacillales order. Asthma-associated differences in predicted bacterial functions included involvement of amino acid and short-chain fatty acid metabolism pathways. Subjects with type 2-high asthma harbored significantly lower bronchial bacterial burden. Distinct changes in specific microbiota members were seen after fluticasone treatment. Steroid responsiveness was linked to differences in baseline compositional and functional features of the bacterial microbiome. Even in subjects with mild steroid-naive asthma, differences in the bronchial microbiome are associated with immunologic and clinical features of the disease. The specific differences identified suggest possible microbiome targets for future approaches to asthma treatment or prevention.