Vandana Relan

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis

Abstract The new peptide hormone insulin-like peptide 3 (INSL3) is a member of the insulin-relaxin family, yet, unlike insulin, it signals through a new G-protein coupled receptor, LGR8, distantly related to the receptors for LH and FSH. INSL3 is produced in large amounts by the Leydig cells of the testis in both fetal and adult mammals. Using a combination of mRNA analysis by RT-PCR, immunohistochemistry, ligand-binding, and/or bioactivity assays, the distribution of LGR8 expression was assessed in testicular tissues and cells and in the epididymis. There was consistent agreement that LGR8 was expressed in meiotic and particularly postmeiotic germ cells and in Leydig cells, though not in Sertoli or peritubular cells. Leydig cells appear to express only a low level of the LGR8 gene product; other transcripts may be present, representing nonfunctional products. Messenger RNA analysis suggested that LGR8 transcripts in germ cells represented mostly full-length forms. LGR8 mRNA was also expressed in the epididymis, though no function can yet be ascribed to this expression. Therefore, the INSL3/LGR8 system represents a further paracrine hormone-receptor system in the testis, which conveys information about Leydig cell status to germ cells, and possibly as part of an autocrine feedback loop.

Constitutive regulation of the Insl3 gene in rat Leydig cells.

Insulin-like factor 3 (Insl3) is a major new product of the Leydig cells in all mammalian species so far examined. The rat Insl3 gene is encoded by two exons in close juxtaposition to the Jak3 gene. Using RT-PCR analysis we now show that in the rat testis it is expressed as both major and minor splice variants, the former encoding the normal protein, the latter a truncated peptide comprising a C-terminally extended B-domain. Both transcripts are produced in constant relative amounts uniquely in the Leydig cells of the postnatal testis and in no other testicular cell type. Rat Insl3 protein is also expressed only in Leydig cells after postnatal day 30. Although specific mRNA is present at earlier times, corresponding protein is not detected. Semi-quantitative RT-PCR analysis of Insl3 transcripts in the mouse MA-10 tumour Leydig cell-line under a wide range of stimulation regimes shows that in an acute context, the Insl3 gene is expressed absolutely constitutively. This is confirmed by transfection and electrophoretic mobility shift (EMSA) analysis of the rat Insl3 gene promoter, wherein the importance of three putative SF-1 responsive elements is underscored, although these appear to differ in their relative importance from their counterparts in the mouse Insl3 gene.

Common pathogenic mechanisms and pathways in the development of COPD and lung cancer

Introduction: Lung cancer and COPD commonly coexist in smokers, and the presence of COPD increases the risk of developing lung cancer. In addition to smoking cessation and preventing smoking initiation, understanding the shared mechanisms of these smoking-related lung diseases is critical, in order to develop new methods of prevention, diagnosis and treatment of lung cancer and COPD. 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.

Diagnostic molecular biomarkers for malignant pleural effusions

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.

Whole genome sequencing for lung cancer

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.

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.

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.

Age-related changes in DNA methylation in the lung

Despite the prevalence of acute cough in children (<2 weeks duration), the burden to parents and families is largely unknown. The objectives of this study were to determine the parental burden of children’s acute cough, and to evaluate psychological and other infl uences on the reported burden of acute cough in children. Methods Parents of children with a current acute cough (<2 weeks) at enrolment completed 4 questionnaires (state trait anxiety inventory (STAI); short form health survey (SF-8); depression, anxiety and stress 21-item scale (DASS21); and our preliminary 48-item parent acute cough specifi c quality of life (PAC-QOL48) questionnaire). In PAC-QOL48, lower scores refl ect worse QOL. Results Median age of the 104 children enrolled was 2.63 (IQR 1.42, 4.79) years, 54 were boys. Median length of cough at enrolment was 3 (IQR 2, 5) days. Median total PAC-QOL48 score of parents enrolled at presentation to the emergency department (n = 70) was signifi cantly worse than of parents enrolled through the community (n = 24) (p < 0.01). More than half (n = 55) had sought medical assistance more than once for the current acute coughing illness. PAC-QOL48 score was signifi cantly negatively correlated to verbal category descriptive and visual analogue scale cough scores (Spearman r = −0.26, p = 0.05 and r = −0.46, p = 0.01 respectively) and DASS21 total score (r = −0.36, p = 0.01), but not to child’s age. Conclusions Consistent with data on chronic cough, stress was the predominant factor of parental burden. This study highlights the ongoing need for clinicians to be cognizant of parental worries and concerns when their children are coughing, and for further research into safe and effective therapies for acute cough in children.

Discovery of new active drugs for treatment of malignant mesothelioma by screening the JHCCL clinical compound library

Abstract S-227 for Poster Session: Reproductive Immunology (Saturday, 3/24/2012, 9:00 AM - 11:00 AM)