Lung cancer encompasses multiple malignant epithelial tumour types, each with specific targetable, potentially actionable mutations, such that precision management mandates accurate tumour typing. Molecular characterisation studies require high tumour cell content and low necrosis content, yet lung cancers are frequently a heterogeneous mixture of tumour and stromal cells. We hypothesised that there may be systematic differences in tumour cell content according to histological subtype, and that this may have implications for tumour banks as a resource for comprehensive molecular characterisation studies in lung cancer. To investigate this, we estimated tumour cell and necrosis content of 4267 samples resected from 752 primary lung tumour specimens contributed to a lung tissue bank. We found that banked lung cancer samples had low tumour cell content (33%) generally, although it was higher in carcinoids (77.5%) than other lung cancer subtypes. Tumour cells comprise a variable and often small component of banked resected tumour samples, and are accompanied by stromal reaction, inflammation, fibrosis, and normal structures. This has implications for the adequacy of unselected tumour bank samples for diagnostic and molecular investigations, and further research is needed to determine whether tumour cell content has a significant impact on analytical results in studies using tissue from tumour bank resources.

SUMMARY: The classification of adenocarcinoma was revised by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society in 2011. In the face of advances of medical therapy in advanced stage disease, these groups sought to improve prognostication and to standardize reporting protocols. A discussion of recent alterations in terminology is undertaken. Included with this is clarification of terminology used small biopsy and cytology specimens. In addition, it will be discussed how an architectural-based classification can be used to assess prognosis and how this applies staging and potential patient management. Several studies have confirmed the effectiveness and reproducibility of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification on lung adenocarcinoma. In addition, there is increasing evidence of clinical application in early stage lung adenocarcinoma. While there may be potential revision in the future, the classification provides better and more standardized information for both clinicians and researchers.

BACKGROUND:MicroRNAs (MiRNA) are small non-coding RNAs that regulate gene expression. The aim of this study was to identify miRNAs differentially expressed between mild and moderately emphysematous lung, as well as their functional target mRNAs. Resected lung from patients with COPD undergoing lung cancer surgery was profiled using miRNA (Agilent Human miRNA profiler G4470 V1.01) and mRNA (OperonV2.0) microarrays. Cells of lung origin (BEAS-2B and HFL1) were profiled using mRNA microarrays (Illumina HumanHT-12 V3) after in vitro manipulation.RESULTS:COPD patients had mean (SD) age 68 (6) years, FEV1 72 (17)% predicted and gas transfer (KCO) 70 (10)% predicted. Five miRNAs (miR-34c, miR-34b, miR-149, miR-133a and miR-133b) were significantly down-regulated in lung from patients with moderate compared to mild emphysema as defined by gas transfer (p < 0.01). In vitro upregulation of miR-34c in respiratory cells led to down-regulation of predicted target mRNAs, including SERPINE1, MAP4K4, ZNF3, ALDOA and HNF4A. The fold change in ex-vivo expression of all five predicted target genes inversely correlated with that of miR-34c in emphysematous lung, but this relationship was strongest for SERPINE1 (p = 0.05).CONCLUSION:Differences in miRNA expression are associated with emphysema severity in COPD patients. MiR-34c modulates expression of its putative target gene, SERPINE1, in vitro in respiratory cell lines and ex vivo in emphysematous lung tissue.

Lung adenocarcinoma (AC) and squamous cell carcinoma (SCC) tumours have a large variance in tumour cell content. This heterogeneity is a concern for genomic studies, as it is difficult to distinguish mutational differences between tumour and non-tumour if low percentage tumour is used for analysis. In addition to this, tumour samples are affected by the amount of necrosis present, as the overall number of viable cells is decreased. We assessed tumour and necrotic content in lung tumour specimens from AC and SCC patients and aimed to identify possible implications for the suitability of these samples in molecular characterisation studies using next generation sequencing technology.

Background: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma.Methods: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs.Results: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions.Conclusion: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma.

This review addresses the pathology of lung disease in which the predominant finding is diffuse cystic change. Although cysts may be found radiologically in a wide variety of disease states, the entities discussed are those most likely to be encountered in biopsies where the underlying aetiology is unclear. These include Langerhans cell histiocytosis, lymphangioleiomyomatosis and Birt-Hogg-Dubé syndrome, and recent advances in the molecular pathology of these entities are reviewed. Conditions in which cyst formation may occur but does not represent the predominant pathology are also considered, including alveolar septal amyloidosis, light chain disease, follicular bronchiolitis and lymphocytic interstitial pneumonia. Cystic metastases may present a differential diagnostic dilemma.

The last decade has seen significant advances in our understanding of lung cancer biology and management. Identification of key driver events in lung carcinogenesis has contributed to the development of targeted lung cancer therapies, heralding the era of personalised medicine for lung cancer. As a result, histological subtyping and molecular testing has become of paramount importance, placing increasing demands on often small diagnostic specimens. This has triggered the review and development of the first structured classification of lung cancer in small biopsy/cytology specimens and a new classification of lung adenocarcinoma from the IASLC/ATS/ERS. These have enhanced the clinical relevance of pathological diagnosis, and emphasise the role of the modern surgical pathologist as an integral member of the multidisciplinary team, playing a crucial role in clinical trials and determining appropriate and timely management for patients with lung cancer.

肺癌是全球肿瘤发病率和病死率的主因,且预后很差。加强对肿瘤生物学的认识对肺癌研究至关重要。被誉为"下一代测序技术"的NGS技术(next-generation sequencing)是一种针对全基因组鉴定的有力工具,可以对致癌体细胞突变进行全面检测。大多数的NGS技术是基于平台特异性DNA文库进行多重聚合酶链反应(polymerase chain reaction,PCR),从而对目的基因扩增后测序。这种技术适用于高通量测序,可以检测出肿瘤中出现的全部基因组变异。缺点是这种技术需要在时间、实验设备、计算机数据分析、生物信息技术等各方面的大量投入。NGS技术已广泛应用于全基因组、外显子组、转录组和表观基因组的研究中,为肺癌研究和医疗模式带来改变。这项新技术的开展将转变当前对致癌信号通路的认识,可为癌症诊疗提供新的分子靶点。肺癌体细胞突变已有NGS技术的分析报道,但大规模基因组研究仍在进行中。个体化治疗策略将改善那些潜在获益患者的治疗方式,避免"无辜"患者受无效治疗带来的高额费用和不良反应。NGS的组织化、计算机化和生物信息化技术推动了科技的进步,同时,患者知情权和数据发布的相关伦理问题也浮现出来。信号通路中,驱动基因(driver gene)突变和传递基因(passenger gene)突变的区别,需要对测序结果进行细致解读。解读准确与否取决于DNA提取的样本类型、样本处理技术和样本含量。肿瘤异质性也会降低肿瘤基因突变的检测效能。NGS技术将推动对肿瘤基因突变的基础和临床研究,而且,也可应用于单细胞和游离的循环DNA,未来还将用于从体液和肿瘤亚群中获取的DNA样本。如果能进一步降低费用、提高检验速度和精度,NGS技术无疑将会成为肺癌研究的绝佳选择。

Background The purpose of this study was to evaluate combined autofluorescence (AF) and narrow band imaging (NBI) for detection of mucosal lesions additional to known primary head and neck cancers and to determine impact on management. Methods Patients with head and neck cancer requiring preoperative screening or posttreatment surveillance had white light (WL), AF and NBI inspection of the head and neck and bronchus. Known primary cancers were not analyzed, only additional lesions. Moderate dysplasia or worse was considered significant. Results In all, 73 patients were recruited. Respectively, there were 24 and 18 additional lesions in the head and neck and bronchus that had significant histopathology. In both regions, AF and NBI were more sensitive than WL for detecting significant dysplasia with NBI demonstrating better specificity than AF (p = .003); 11 of 73 patients (15.1%) had additional findings detected by AF and NBI, which had an impact on management. Conclusion Combined AF and NBI inspection is highly specific at panendoscopy and can influence management. (c) 2012 Wiley Periodicals, Inc. Head Neck, 2013

Background: Primary tumor recurrence commonly occurs after surgical resection of lung squamous cell carcinoma (SCC). Little is known about the genes driving SCC recurrence.Methods: We used array comparative genomic hybridization (aCGH) to identify genes affected by copy number alterations that may be involved in SCC recurrence. Training and test sets of resected primary lung SCC were assembled. aCGH was used to determine genomic copy number in a training set of 62 primary lung SCCs (28 with recurrence and 34 with no evidence of recurrence) and the altered copy number of candidate genes was confirmed by quantitative PCR (qPCR). An independent test set of 72 primary lung SCCs (20 with recurrence and 52 with no evidence of recurrence) was used for biological validation. mRNA expression of candidate genes was studied using qRT-PCR. Candidate gene promoter methylation was evaluated using methylation microarrays and Sequenom EpiTYPER analysis.Results: 18q22.3 loss was identified by aCGH as being significantly associated with recurrence (p = 0.038). Seven genes within 18q22.3 had aCGH copy number loss associated with recurrence but only SOCS6 copy number was both technically replicated by qPCR and biologically validated in the test set. SOCS6 copy number loss correlated with reduced mRNA expression in the study samples and in the samples with copy number loss, there was a trend for increased methylation, albeit non-significant. Overall survival was significantly poorer in patients with SOCS6 loss compared to patients without SOCS6 loss in both the training (30 vs. 43 months, p = 0.023) and test set (27 vs. 43 months, p = 0.010).Conclusion: Reduced copy number and mRNA expression of SOCS6 are associated with disease recurrence in primary lung SCC and may be useful prognostic biomarkers.

BACKGROUND:The diagnosis of malignant pleural effusions (MPE) is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE.METHODS:We studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index)] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA.RESULTS:Based on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas) and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p<0.001). Cytology had a sensitivity of 55% in diagnosing MPE. If cytology and pleural fluid DNA integrity index were considered together, they exhibited 81% sensitivity and 87% specificity in distinguishing benign and malignant effusions. In cytology-negative pleural effusions (35 MPE and 28 benign effusions), elevated pleural fluid DNA integrity index had an 81% positive predictive value in detecting MPEs. In the detection of mesothelioma, at a specificity of 90%, pleural fluid DNA integrity index had similar sensitivity to pleural fluid and serum mesothelin (75% each respectively).CONCLUSION:Pleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice.

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.

Lung cancer is a leading cause of cancer related morbidity and mortality globally, and carries a dismal prognosis. Improved understanding of the biology of cancer is required to improve patient outcomes. Next-generation sequencing (NGS) is a powerful tool for whole genome characterisation, enabling comprehensive examination of somatic mutations that drive oncogenesis. Most NGS methods are based on polymerase chain reaction (PCR) amplification of platform-specific DNA fragment libraries, which are then sequenced. These techniques are well suited to high-throughput sequencing and are able to detect the full spectrum of genomic changes present in cancer. However, they require considerable investments in time, laboratory infrastructure, computational analysis and bioinformatic support. Next-generation sequencing has been applied to studies of the whole genome, exome, transcriptome and epigenome, and is changing the paradigm of lung cancer research and patient care. The results of this new technology will transform current knowledge of oncogenic pathways and provide molecular targets of use in the diagnosis and treatment of cancer. Somatic mutations in lung cancer have already been identified by NGS, and large scale genomic studies are underway. Personalised treatment strategies will improve care for those likely to benefit from available therapies, while sparing others the expense and morbidity of futile intervention. Organisational, computational and bioinformatic challenges of NGS are driving technological advances as well as raising ethical issues relating to informed consent and data release. Differentiation between driver and passenger mutations requires careful interpretation of sequencing data. Challenges in the interpretation of results arise from the types of specimens used for DNA extraction, sample processing techniques and tumour content. Tumour heterogeneity can reduce power to detect mutations implicated in oncogenesis. Next-generation sequencing will facilitate investigation of the biological and clinical implications of such variation. These techniques can now be applied to single cells and free circulating DNA, and possibly in the future to DNA obtained from body fluids and from subpopulations of tumour. As costs reduce, and speed and processing accuracy increase, NGS technology will become increasingly accessible to researchers and clinicians, with the ultimate goal of improving the care of patients with lung cancer.

Chronic obstructive pulmonary disease (COPD) is a major public health problem with increasing prevalence worldwide. The primary aim of this study was to identify genes and gene ontologies associated with COPD severity. Gene expression profiling was performed on total RNA extracted from lung tissue of 18 former smokers with COPD. Class comparison analysis on mild (n = 9, FEV(1) 80-110% predicted) and moderate (n = 9, FEV(1) 50-60% predicted) COPD patients identified 46 differentially expressed genes (p<0.01), of which 14 genes were technically confirmed by quantitative real-time-PCR. Biological replication in an independent test set of 58 lung samples confirmed the altered expression of ten genes with increasing COPD severity, with eight of these genes (NNMT, THBS1, HLA-DPB1, IGHD, ETS2, ELF1, PTGDS and CYRBD1) being differentially expressed by greater than 1.8 fold between mild and moderate COPD, identifying these as candidate determinants of COPD severity. These genes belonged to ontologies potentially implicated in COPD including angiogenesis, cell migration, proliferation and apoptosis. Our secondary aim was to identify gene ontologies common to airway obstruction, indicated by impaired FEV(1) and KCO. Using gene ontology enrichment analysis we have identified relevant biological and molecular processes including regulation of cell-matrix adhesion, leukocyte activation, cell and substrate adhesion, cell adhesion, angiogenesis, cell activation that are enriched among genes involved in airflow obstruction. Exploring the functional significance of these genes and their gene ontologies will provide clues to molecular changes involved in severity of COPD, which could be developed as targets for therapy or biomarkers for early diagnosis.

Areas covered: This review discusses the common mechanisms for susceptibility to lung cancer and COPD, which in addition to cigarette smoke, may involve inflammation, epithelial--mesenchymal transition, abnormal repair, oxidative stress, and cell proliferation. Furthermore, we discuss the underlying genomic and epigenomic changes (single nucleotide polymorphisms (SNPs), copy number variation, promoter hypermethylation and microRNAs) that are likely to alter biological pathways, leading to susceptibility to lung cancer and COPD (e.g., altered nicotine receptor biology).Expert opinion: Strategies to study genomics, epigenomics and gene-environment interaction will yield greater insight into the shared pathogenesis of lung cancer and COPD, leading to new diagnostic and therapeutic modalities.

Malignant pleural effusions (MPEs) are a common and important cause of cancer-related mortality and morbidity. Prompt diagnosis using minimally invasive tests is important because the median survival after diagnosis is only 4-9 months. Pleural fluid cytology is pivotal to current MPE diagnostic algorithms but has limited sensitivity (30-60%). Consequently, many patients need to undergo invasive diagnostic tests such as thoracoscopic pleural biopsy. Recent genomic, transcriptomic, methylation and proteomic studies on cells within pleural effusions have identified novel molecular diagnostic biomarkers that demonstrate potential in complementing cytology in the diagnosis of MPEs. Several challenges will need to be addressed prior to the incorporation of these molecular tests into routine clinical diagnosis, including validation of molecular diagnostic markers in well-designed prospective, comparative and cost-effectiveness studies. Ultimately, minimally invasive diagnostic tests that can be performed quickly will enable clinicians to provide the most effective therapies for patients with MPEs in a timely fashion.

Recurrence of the primary cancer after ‘curative’ surgical resection occurs in30-50% of early stage NSCLC patients. Genomic factors regulating recurrence, particularly squamous cell cancer (SCC) are poorly understood. Consistent and frequent copy number alterations (CNA) are likely to harbor regulatory cancer genes. We hypothesize that whole genome array comparative genomic hybridization (aCGH) will identify novel CNAs in recurrent SCCs.Methods: DNA from frozen tumour tissues was obtained from 58 patients with stage I-III primary SCC who underwent surgical resection;. 25 patients had recurrence (3-18 months) and 33 patients were recurrence free at 36 months. Two patients received neo-adjuvant radiotherapy, 1 patient received adjuvant chemotherapy and another received adjuvant radiotherapy. aCGH was performed using Agilent CGH 44B microarrays. CNAs were identified using the Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm developed at the Broad Institute. Fisher’s exact test was used to assess the relationship between CNAs and recurrence phenotype.Results: Regions of amplification occurred in 3q26.33, 8p11.23 and 19q13.2 while deletions occurred in 9p12.3, 3p12.1, 8p23.1, 5q13.3, 4q28.2, 10q23.31, 13q22.1, 1p21.1, 17p12, 16q23.3, 4p15.31 and 18q22.3. Deletions in 18q22.3 occurred more frequently in recurrence compared to non-recurrence samples (52% vs. 24%, P<0.001). This region contains 22 RefSeq genes, among which SOCS6, ZNF236, CD226, GALR1 and CNDP1 have been reported to function as tumour suppressor genes (TSGs). Conclusion: We have identified several CNAs in SCC and found 18q22.3 deletions occur more frequently in recurrent SCC. These findings will need to be validated using independent datasets and methodology.