Deborah Courtney

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

Low tumour cell content in a lung tumour bank: Implications for molecular characterisation

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.

Intratumoral genomic heterogeneity of primary pulmonary adenocarcinoma in never smokers

Aim: Intratumoral genomic heterogeneity challenges personalized lung cancer care, especially where it relies upon small diagnostic samples. To explore genomic representation provided by tumor subsampling, we performed whole genome sequencing (WGS) of multiple regions of individual primary pulmonary adenocarcinomas (LUAC). Methods: An observational study was performed on three cases of never-smoking LUAC resected with curative intent. Post-diagnostic residual fresh tumor was procured with informed consent, along with constitutional samples from normal lung or blood. Selection criteria included: histologically confirmed LUAC; never-smoker [defined as fewer than 100 cigarettes consumed in a lifetime]; and no prior malignancy, cytotoxic therapy or thoracic radiotherapy. Tissue samples were procured by an anatomical pathologist and research scientist and snap frozen within 60 minutes of devascularization, then stored at -80 degrees celsius. Nine macrodissected subsamples met quality criteria of >40% tumor cellularity and WGS was performed on paired end libraries using Illumina9s HiSeq 2000 platform to 80x (tumor), 40x (normal lung) and 30x (blood) coverage. Reads were aligned to GRCh37 with BWA-MEM. Duplicates were removed using Picard and local INDEL realignment and base quality recalibration were performed with GATK. Single nucleotide variants (SNVs) were called by MuTect, Varscan, Strelka and SomaticSniper. Variants were considered ‘high priority’ if predicted by SNPEff to have ‘moderate’ or ‘high’ functional significance. Structural variants were detected from WGS data using Breakdancer and Pindel. Sample genotyping was performed using Illumina9s HumanOmni2.5-8 array and used to call copy number variations (CNVs) using the Genome Alteration Print tool. Results: All cases were Caucasian females. Case 1 consisted of a 37 year old with a well to moderately differentiated pathological stage IV (AJCC 7th Edition; T4 N1 M1a) tumor 75mm in maximal dimension for which DNA from 4 tumor regions and whole blood was available. Case 2 was an 80 year old with a 24mm, acinar predominant, moderately differentiated pathological stage 1A (T1b N0 M0) tumor for which DNA from 3 tumor regions and whole blood was available. Case 3 was an 82 year old with a 35mm, acinar predominant, pathological stage 1B (T2a N0 M0) tumor for which DNA from 2 tumor regions and non-tumor lung was available. Mean tumor cellularity (and mean sequencing coverage achieved) for regions 1, 2, 3 and 4 for case 1 were 50% (98x), 50% (100x), 73% (99x) and 58% (134x), respectively. Similarly, for regions 1, 2 and 3 of case 2, mean cellularity (and coverage) was 45% (93x), 45% (114x) and 40% (93x), respectively. Case 3 demonstrated 45% (107x) and 55% (97x) mean cellularity (and coverage) for regions 1 and 2, respectively. Less than 10% necrosis was observed in all tumor regions. Of 10275 SNVs detected in case 1, 3198 (3198/10275, 31%) were found in all 4 subsamples. 6911/15689 (44%) and 5595/9528 (59%) were shared among all subsamples in cases 2 and 3, respectively. The numbers of SNVs unique to each region relative to total SNVs observed for each region in case were: 869/5999 (14%), 1129/6437 (18%), 914/6969 (13%) and 517/5936 (9%). Similarly, the numbers of unique SNVs as a proportion of total SNVs for each region in case 2 were 1148/9835 (12%), 2556/11404 (22%) and 2632/10714 (25%); and for case 3 were 2293/7888 (29%) and 1640/7235 (23%). In case 1, 7 of 303 (2%) high priority variants were detected in all regions. Similarly, 44/303 (15%) and 29/302 (10%) high priority variants were detected in all tumor regions for case 2 and 3, respectively. Conclusion: Significant intratumoral heterogeneity was observed. These findings have significant implications not only for diagnostic testing of lung cancer but also for clinical trial design. Prospective clinical trials incorporating assessment of both geographic and temporal intratumoral heterogeneity will help explore the implications of this phenomenon on patient treatment. Acknowledgements: We acknowledge the patients, nurses, and staff of The Prince Charles Hospital for their contributions to this project. Funding: MD supported by Cancer Council Queensland and NHMRC PhD Scholarships. Supported by funding from NHMRC, Cancer Australia, TPCH Foundation, Queensland Health, Cancer Council Queensland. Citation Format: Marissa G. Daniels, Lutz Krause, Jonathan J. Ellis, Ian A. Yang, Rayleen V. Bowman, Vandana Relan, Kelly Chee, Felicia Goh, Brielle Parris, Leanne Morrison, Maria Martins, Linda Passmore, Elizabeth McCaul, Deborah Courtney, Edwina Duhig, Morgan Windsor, Rishendran Naidoo, Kwun M. Fong. Intratumoral genomic heterogeneity of primary pulmonary adenocarcinoma in never smokers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 24.

The Queensland Lung Cancer Screening Study: enhanced population risk stratification increases effectiveness of low dose CT screening for lung cancer

Background : The current guidelines for acceptable levels of ambient PM ( Methods : The PM fraction was extracted from surface soil samples from 4 communities across Western Australia. BALB/c 10 mice were intranasally exposed to 100 µg of PM . Control mice received 100 µg of polystyrene beads (2.5 µm) or vehicle 10 alone. Mice were assessed for inflammation (cellular influx, MIP-2, IL-6 and IL-1β), lung volume (plethysmography) and lung mechanics (forced oscillation technique) 6, 24 or 168 hours post-exposure. The physical and chemical characteristics of the particles were assessed by cascade impactor and ICP-MS/OES respectively. Principal component analysis of the outcome measures were used to construct lung impairment scores. Multivariate linear regression models were then used to identify the characteristics of the particles driving the lung responses. Results : Exposure to geogenic particles caused an acute inflammatory response (6 hours post-exposure), an acute impairment in lung mechanics (24 hours post-exposure) and a long term deficit in lung volume (168 hours post-exposure). Both the inflammatory response and long term deficits in lung volume were associated with the concentration of Fe and variability in particle size (GSD) while the impairment in lung mechanics was associated with Fe and particle size (MMAD). Conclusions : Despite the complex physico-chemical characteristics of geogenic dusts we were able to identify the concentration of Fe and physical dimensions of the particles as the key drivers of lung responses. Using these data we may be able to predict which communities are at greatest risk of adverse respiratory health due to high geogenic particle loads.

Telomere length in pleural fluid is not different between malignant and benign effusions

068-P12 LUNG ADENOCARCINOMA BIOMARKER SCREENING IN AN AUSTR`ALIAN POPULATION Ainge Allen H, Stone E, Plit Ml, Havryk A, Goldrick A, Field A, Mead S, Qiu M, Chou A, Morey A Department of Respiratory Medicine, St Vincent’s Hospital, Sydney, New South Wales/AUSTRALIA; Department of Anatomical Pathology, Sydpath, St Vincent’s Hospital, Sydney, New South Wales/AUSTRALIA Aims: The emergence of new targeted therapies and clinical trials in lung adenocarcinoma suggest that routine molecular biomarker testing is warranted to detected subtypes of adenocarcinoma which will benefit from new therapies. Australian data is still emerging. We aim to detect the incidence of treatment specific molecular subtypes of lung adenocarcinoma in the Australian population. Methods: This is a single centre prospective pilot study of patients with a histopathological diagnosis of adenocarcinoma who have been presented at a Lung Cancer multidisciplinary meeting. Formalin fixed paraffin embedded tissue of 22 tumour samples were submitted for targeted PCR-based EGFR and KRAS mutation analysis; and EGFR amplification, MET amplification and EML4-ALK rearrangement testing by in-situ hybridization. one sample had insufficient tissue for analysis. Results: So far 22 patients received a definite tissue diagnosis of lung adenocarcinoma, 8/22 with Stage III and 10/22 with Stage IV disease. 4/22 (19%) are positive for an EGFR mutation, with activating EGFR mutations in 3/22 (14%) and an EGFR mutation of uncertain clinical significance in one patient (5%). EGFR amplification was detected in 2/22 patients (9%), KRAS mutations were detected in 5/22 patients (23%) patients, of which none carried EGFR mutations. EML4-ALK translocation was seen in 2/22 patients (9%) and MET amplification in 1/22 (5%) patients. Conclusions: Preliminary results indicate that treatment specific molecular subtypes of lung adenocarcinoma occur more commonly in the Australia population than other Western populations. Though further investigation is required, routine molecular biomarker testing is warranted. Disclosure: This study was funded by an unrestricted grant from Roche.