Linda Passmore

MicroRNA-218 is deleted and downregulated in lung squamous cell carcinoma.

MicroRNAs (miRNAs) are a family of small, non-coding RNA species functioning as negative regulators of multiple target genes including tumour suppressor genes and oncogenes. Many miRNA gene loci are located within cancer-associated genomic regions. To identify potential new amplified oncogenic and/or deleted tumour suppressing miRNAs in lung cancer, we inferred miRNA gene dosage from high dimensional arrayCGH data. From miRBase v9.0 (http://microrna.sanger.ac.uk), 474 human miRNA genes were physically mapped to regions of chromosomal loss or gain identified from a high-resolution genome-wide arrayCGH study of 132 primary non-small cell lung cancers (NSCLCs) (a training set of 60 squamous cell carcinomas and 72 adenocarcinomas). MiRNAs were selected as candidates if their immediately flanking probes or host gene were deleted or amplified in at least 25% of primary tumours using both Analysis of Copy Errors algorithm and fold change (≥±1.2) analyses. Using these criteria, 97 miRNAs mapped to regions of aberrant copy number. Analysis of three independent published lung cancer arrayCGH datasets confirmed that 22 of these miRNA loci showed directionally concordant copy number variation. MiR-218, encoded on 4p15.31 and 5q35.1 within two host genes (SLIT2 and SLIT3), in a region of copy number loss, was selected as a priority candidate for follow-up as it is reported as underexpressed in lung cancer. We confirmed decreased expression of mature miR-218 and its host genes by qRT-PCR in 39 NSCLCs relative to normal lung tissue. This downregulation of miR-218 was found to be associated with a history of cigarette smoking, but not human papilloma virus. Thus, we show for the first time that putative lung cancer-associated miRNAs can be identified from genome-wide arrayCGH datasets using a bioinformatics mapping approach, and report that miR-218 is a strong candidate tumour suppressing miRNA potentially involved in lung cancer.

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.

Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

BackgroundThe 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.MethodsWe 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.ResultsBased 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).ConclusionPleural 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.

Phenotypes and Karyotypes of Human Malignant Mesothelioma Cell Lines

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.

Queensland Lung Cancer Screening Study: rationale, design and methods

Lung cancer is the leading cause of cancer‐related mortality in Australia. Screening using low‐dose computed tomography (LDCT) can reduce lung cancer mortality. The feasibility of screening in Australia is unknown. This paper describes the rationale, design and methods of the Queensland Lung Cancer Screening Study.

Brief Tailored Smoking Cessation Counseling in a Lung Cancer Screening Population is Feasible: A Pilot Randomized Controlled Trial.

INTRODUCTION Maximizing smoking abstinence in lung cancer screening participants is important to reduce individual risk of disease and improve screening cost-effectiveness; however, the optimal strategy remains undefined. We hypothesized that a single session of tailored face-to-face counseling on the day of screening CT scan, coupled with audio and printed cessation information would be feasible to deliver in a CT screening trial. METHODS We randomized volunteer smokers in the Queensland Lung Cancer Screening Study to intervention (counseling session, audio quit materials, printed quit materials, Quitline contact details) or control group (printed quit materials, Quitline contact details). Participants self-reported point prevalence quit rates at 1 year. RESULTS Fifty-five smokers were enrolled; 28 randomized to intervention and 27 controls. Median cigarette consumption was 25/day; 54/55 smoked at least 15 cigarettes per day. Median smoking duration was 46 years. Median Fagerström dependence score was 6. In total 58% did not report any quit attempt in the prior 12 months. Mean duration of counseling was 26.5 minutes. After 1 year, four participants (14.3%) in the intervention group and five participants (18.5%) in the control group had quit (P = .74). Combined annual point prevalence quit rate was 16.4%. CONCLUSIONS Although feasible to deliver a single session of tailored counseling on the day of screening this intervention had no discernible impact on cessation over and above printed materials and Quitline access. As participants exhibited hardcore smoking characteristics, more intensive strategies, in larger cohorts, should be explored. IMPLICATIONS The optimal smoking cessation strategy within a lung cancer screening program is not known. This study demonstrates that a single session of counseling can be feasibly delivered on the day of screening but may not have been intensive enough for long-term, hard-core smokers.

Screen-detected subsolid pulmonary nodules: long-term follow-up and application of the PanCan lung cancer risk prediction model

OBJECTIVE To report the long-term follow-up of subsolid nodules (SSNs) detected in participants of a prospective low-dose CT lung cancer screening cohort, and to investigate the utility of the PanCan model in stratifying risk in baseline SSNs. METHODS Participants underwent a baseline scan, two annual incidence scans and further follow-up scans for the detected nodules. All SSNs underwent a minimum of 2 years of follow-up (unless resolved or resected). Risk of malignancy was estimated using the PanCan model; discrimination [area under the receiver-operating characteristic curve (AUC)] and calibration (Hosmer-Lemeshow goodness-of-fit test) were assessed. The Mann-Whitney U-Wilcoxon test was used to compare estimated risk between groups. RESULTS 70 SSNs were detected in 41 (16.0%) out of 256 total participants. Median follow-up period was 25.5 months (range 2.0-74.0 months). 29 (41.4%) SSNs were transient. Five (7.1%) SSNs were resected, all found to be Stage I lung adenocarcinoma, including one SSN stable in size for 3.0 years before growth was detected. The PanCan model had good discrimination for the 52 baseline SSNs (AUC = 0.89; 95% confidence interval 0.76-1); the Hosmer-Lemeshow goodness-of-fit test was non-significant (p = 0.27). Estimated risk was significantly higher in the baseline SSNs found to be cancer vs those not found to be cancer after 2-6 years of follow-up (p < 0.01). CONCLUSION Our findings support a long-term follow-up approach for screen-detected SSNs for 3 years or longer. The PanCan model appeared discriminatory and well calibrated in this cohort. ADVANCES IN KNOWLEDGE The PanCan model may have utility in identifying low-risk SSNs which could be followed with less frequent CT scans.

Lung cancer screening feasibility in Australia

The National Lung Screening Trial (NLST) reported a 20% relative reduction in lung cancer-specific mortality using low-dose computed tomography (LDCT) screening [1]. US Preventative Services Task Force modelling [2] illustrates the potentially large benefits of screening, yet nationwide population-based screening has not been adopted. Controversial issues include high false positivity, and uncertain cost-effectiveness and relative applicability to different settings and countries [3–6]. The Queensland Lung Cancer Screening Study (QLCSS) is the first study to assess NLST screening protocol feasibility in Australia. Low-dose CT screening using the NLST protocol appears feasible in the Australian health setting http://ow.ly/JwtU2

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.

Genetic influences on right ventricular systolic pressure (RVSP) in chronic obstructive pulmonary disease (COPD)

BackgroundPulmonary hypertension (PH) is a complication of chronic obstructive pulmonary disease (COPD). This study examined genetic variations in mediators of vascular remodelling and their association with PH in patients with COPD. In patients with COPD, we genotyped 7 SNPs in 6 candidate PH genes (NOS3, ACE, EDN1, PTGIS, SLC6A4, VEGFA). We tested for association with right ventricular systolic pressure (RVSP), spirometry and gas transfer, and hypoxemia.MethodsIn patients with COPD, we genotyped 7 SNPs in 6 candidate PH genes (NOS3, ACE, EDN1, PTGIS, SLC6A4, VEGFA). We tested for association with right ventricular systolic pressure (RVSP), spirometry and gas transfer, and hypoxemia.Results580 COPD patients were recruited, 341 patients had a transthoracic echocardiogram, with RVSP measurable in 278 patients (mean age 69 years, mean FEV1 50% predicted, mean RVSP 44 mmHg, median history of 50 pack-years). Of the 7 tested SNPs, the NOS3-VNTR polymorphism was significantly associated with RVSP in a dose-dependent fashion for the risk allele: mean RVSP for a/a and a/b genotypes were 52.0 and 46.6 mmHg respectively, compared to 43.2 mmHg for b/b genotypes (P = 0.032). No associations were found between RVSP and other polymorphisms. ACE II or ID genotypes were associated with a lower FEV1% predicted than the ACE DD genotype (P = 0.028). The NOS3-298 TT genotype was associated with lower KCO % predicted than the NOS3-298 GG or GT genotype (P = 0.031).ConclusionsThe NOS3-VNTR polymorphism was associated with RVSP in patients with COPD, supporting its involvement in the pathogenesis of PH in COPD. ACE and NOS3 genotypes were associated with COPD disease severity, but not with the presence of PH. Further study of these genes could lead to the development of prognostic and screening tools for PH in COPD.