M.E. Tan

Reestablishment of Recipient-associated Microbiota in the Lung Allograft Is Linked to Reduced Risk of Bronchiolitis Obliterans Syndrome

RATIONALE Bronchiolitis obliterans syndrome (BOS) is the primary limiting factor for long-term survival after lung transplantation, and has previously been associated with microbial infections. OBJECTIVES To cross-sectionally and longitudinally characterize microbial communities in allografts from transplant recipients with and without BOS using a culture-independent method based on high-throughput sequencing. METHODS Allografts were sampled by bronchoalveolar lavage, and microbial communities were profiled using 16S rRNA gene amplicon pyrosequencing. Community profiles were compared using the weighted Unifrac metric and the relationship between microbial populations, BOS, and other covariates was explored using PERMANOVA and logistic regression. MEASUREMENTS AND MAIN RESULTS Microbial communities in transplant patients fell into two main groups: those dominated by Pseudomonas or those dominated by Streptococcus and Veillonella, which seem to be mutually exclusive lung microbiomes. Aspergillus culture was also negatively correlated with the Pseudomonas-dominated group. The reestablishment of dominant populations present in patients pretransplant, notably Pseudomonas in individuals with cystic fibrosis, was negatively correlated with BOS. CONCLUSIONS Recolonization of the allograft by Pseudomonas in individuals with cystic fibrosis is not associated with BOS. In general, reestablishment of pretransplant lung populations in the allograft seems to have a protective effect against BOS, whereas de novo acquisition of microbial populations often belonging to the same genera may increase the risk of BOS.

ADAM28: a potential oncogene involved in asbestos-related lung adenocarcinomas.

Asbestos‐related lung cancer accounts for 4–12% of all lung cancers worldwide. Since putative mechanisms of carcinogenesis differ between asbestos and tobacco induced lung cancers, tumors induced by the two agents may be genetically distinct. To identify gene expression biomarkers associated with asbestos‐related lung tumorigenicity we performed gene expression array analysis on tumors of 36 patients with primary lung adenocarcinoma, comparing 12 patients with lung asbestos body counts above levels associated with urban dwelling (ARLC‐AC: asbestos‐related lung cancer‐adenocarcinoma) with 24 patients with no asbestos bodies (NARLC‐AC: non‐asbestos related lung cancer‐adenocarcinoma). Genes differentially expressed between ARLC‐AC and NARLC‐AC were identified on fold change and P value, and then prioritized using gene ontology. Candidates included ZNRF3, ADAM28, PPP1CA, IRF6, RAB3D, and PRDX1. Expression of these six genes was technically and biologically replicated by qRT‐PCR in the training set and biologically validated in three independent test sets. ADAM28, encoding a disintegrin and metalloproteinase domain protein that interacts with integrins, was consistently upregulated in ARLC across all four datasets. Further studies are being designed to investigate the possible role of this gene in asbestos lung tumorigenicity, its potential utility as a marker of asbestos related lung cancer for purposes of causal attribution, and its potential as a treatment target for lung cancers arising in asbestos exposed persons.

MS4A1 Dysregulation in Asbestos-Related Lung Squamous Cell Carcinoma Is Due to CD20 Stromal Lymphocyte Expression

Asbestos-related lung cancer accounts for 4–12% of lung cancers worldwide. We have previously identified ADAM28 as a putative oncogene involved in asbestos-related lung adenocarcinoma (ARLC-AC). We hypothesised that similarly gene expression profiling of asbestos-related lung squamous cell carcinomas (ARLC-SCC) may identify candidate oncogenes for ARLC-SCC. We undertook a microarray gene expression study in 56 subjects; 26 ARLC-SCC (defined as lung asbestos body (AB) counts >20AB/gram wet weight (gww) and 30 non-asbestos related lung squamous cell carcinoma (NARLC-SCC; no detectable lung asbestos bodies; 0AB/gww). Microarray and bioinformatics analysis identified six candidate genes differentially expressed between ARLC-SCC and NARLC-SCC based on statistical significance (p<0.001) and fold change (FC) of >2-fold. Two genes MS4A1 and CARD18, were technically replicated by qRT-PCR and showed consistent directional changes. As we also found MS4A1 to be overexpressed in ARLC-ACs, we selected this gene for biological validation in independent test sets (one internal, and one external dataset (2 primary tumor sets)). MS4A1 RNA expression dysregulation was validated in the external dataset but not in our internal dataset, likely due to the small sample size in the test set as immunohistochemical (IHC) staining for MS4A1 (CD20) showed that protein expression localized predominantly to stromal lymphocytes rather than tumor cells in ARLC-SCC. We conclude that differential expression of MS4A1 in this comparative gene expression study of ARLC-SCC versus NARLC-SCC is a stromal signal of uncertain significance, and an example of the rationale for tumor cell enrichment in preparation for gene expression studies where the aim is to identify markers of particular tumor phenotypes. Finally, our study failed to identify any strong gene candidates whose expression serves as a marker of asbestos etiology. Future research is required to determine the role of stromal lymphocyte MS4A1 dysregulation in pulmonary SCCs caused by asbestos.

Peripheral compartment innate immune response to Haemophilus influenzae and Streptococcus pneumoniae in chronic obstructive pulmonary disease patients

Alterations in innate immunity that predispose to chronic obstructive pulmonary disease (COPD) exacerbations are poorly understood. We examined innate immunity gene expression in peripheral blood polymorphonuclear leukocytes (PMN) and monocytes stimulated by Haemophilus influenzae and Streptococcus pneumoniae. Thirty COPD patients (15 rapid and 15 non-rapid lung function decliners) and 15 smokers without COPD were studied. Protein expression of IL-8, IL-6, TNF-α and IFN-γ (especially monocytes) increased with bacterial challenge. In monocytes stimulated with S. pneumoniae, TNF-α protein expression was higher in COPD (non-rapid decliners) than in smokers. In co-cultures of monocytes and PMN, mRNA expression of TGF-β1 and MYD88 was up-regulated, and CD14, TLR2 and IFN-γ down-regulated with H. influenzae challenge. TNF-α mRNA expression was increased with H. influenzae challenge in COPD. Cytokine responses were similar between rapid and non-rapid decliners. TNF-α expression was up-regulated in non-rapid decliners in response to H. influenzae (monocytes) and S. pneumoniae (co-culture of monocytes and PMN). Exposure to bacterial pathogens causes characteristic innate immune responses in peripheral blood monocytes and PMN in COPD. Bacterial exposure significantly alters the expression of TNF-α in COPD patients, although not consistently. There did not appear to be major differences in innate immune responses between rapid and non-rapid decliners.

Lung Asbestos Content in Lungs Resected for Primary Lung Cancer

Introduction: The majority of Australias burden of lung cancer occurs in current or former tobacco smokers. To determine the possible contribution of asbestos exposure in Australians presenting with primary lung cancer, we measured lung asbestos content in cases resected consecutively at a single cardio-thoracic hospital. Methods: Asbestos bodies were quantified by lung tissue digestion, filtration, and light microscopy, and were correlated with exposure questionnaires and clinicopathological features. Results: We demonstrate high intrarater reproducibility and interrater reliability using these methods. In 463 patients with resected primary lung cancers, asbestos content ranged from 0 to 749 asbestos bodies per gram wet weight (AB/gww). Forty-eight percent of patients had no asbestos bodies identified. One-third had less than or equal to 20 AB/gww (a level previously found to be consistent with urban dwelling). Nineteen percent had lung content in excess of this level. Only 20 cases had AB >100/gww, approximately equivalent to the Helsinki threshold for attribution of lung cancer to asbestos. Median asbestos body counts were higher in patients who reported previous asbestos exposure than in those who reported no exposure. A subgroup of cases gave detailed exposure histories that did not predict presence or absence of asbestos bodies in men or women. In cases with cumulative tobacco exposure less than 20 pack-years, asbestos body counts exceeding 20 AB/gww were overrepresented. Conclusions: We found that the majority of patients with primary lung cancer at a single Australian center have detectable asbestos in resected lung tissue, but fiber burdens are generally low. The contributory role of this low-level asbestos exposure in causing lung cancer remains uncertain.

EGFR mutation testing in non-small cell lung cancer

Non-small cell lung cancer [NSCLC] is a major cause of cancer related deaths in the world. In a significant proportion of NSCLC cases, the Epidermal Growth Factor Receptor [EGFR] is over-expressed prompting the development of anti-EGFR therapies. Clinical studies of EGFR tyrosine kinase inhibitors [TKIs] demonstrated an overall response rate of 10% in NSCLC with higher response rates in females, never smokers, adenocarcinoma histology and East Asians. Mutations in exons 18-21 of the EGFR gene appear to be the most important molecular predictors of response to TKIs with response rates of >60% reported in most studies. Consequently screening NSCLC patients for tumours harbouring EGFR mutations will assist in selecting patients for TKI therapy. The gold standard for detecting EGFR mutations has been direct DNA sequencing however the sensitivity of this molecular technique is limited by the amount of tumour DNA is time and resource consuming. Recently several highly sensitive techniques have been developed to detect EGFR mutant DNA in different biological samples. In this review we aim to provide an overview of the clinical relevance of screening for EGFR mutations in NSCLC and recent molecular tests that have been developed for this purpose to allow the reader to critically evaluate the various methodologies that are available.