Morgan Windsor

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.

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.

Prostate-specific membrane antigen avidity on positron emission tomography scan in malignant pleural mesothelioma: Images for Surgeons

Prostate‐specific membrane antigen (PSMA) is a type II transmembrane glycoprotein encoded by the folate hydrolase 1 (FOLH1) gene. The enzyme is overexpressed in prostate cancer cells.1 A recent systematic review showed 68Ga‐labelled PSMA–positron emission tomography (PET) as having a sensitivity of 66% and specificity of 99% for primary lymph node metastases in high‐risk prostate cancer patients.2 PSMA‐PET is also positive in renal cell carcinoma, hepatocellular carcinoma, non‐small cell lung cancer, squamous cell (lung and other), gynaecological and colorectal malignancies.3-7

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.