Marissa Daniels

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.

An emerging place for lung cancer genomics in 2013.

Lung cancer is a disease with a dismal prognosis and is the biggest cause of cancer deaths in many countries. Nonetheless, rapid technological developments in genome science promise more effective prevention and treatment strategies. Since the Human Genome Project, scientific advances have revolutionized the diagnosis and treatment of human cancers, including thoracic cancers. The latest, massively parallel, next generation sequencing (NGS) technologies offer much greater sequencing capacity than traditional, capillary-based Sanger sequencing. These modern but costly technologies have been applied to whole genome-, and whole exome sequencing (WGS and WES) for the discovery of mutations and polymorphisms, transcriptome sequencing for quantification of gene expression, small ribonucleic acid (RNA) sequencing for microRNA profiling, large scale analysis of deoxyribonucleic acid (DNA) methylation and chromatin immunoprecipitation mapping of DNA-protein interaction. With the rise of personalized cancer care, based on the premise of precision medicine, sequencing technologies are constantly changing. To date, the genomic landscape of lung cancer has been captured in several WGS projects. Such work has not only contributed to our understanding of cancer biology, but has also provided impetus for technical advances that may improve our ability to accurately capture the cancer genome. Issues such as short read lengths contribute to sequenced libraries that contain challenging gaps in the aligned genome. Emerging platforms promise longer reads as well as the ability to capture a range of epigenomic signals. In addition, ongoing optimization of bioinformatics strategies for data analysis and interpretation are critical, especially for the differentiation between driver and passenger mutations. Moreover, broader deployment of these and future generations of platforms, coupled with an increasing bioinformatics workforce with access to highly sophisticated technologies, could see many of these discoveries translated to the clinic at a rapid pace. We look forward to these advances making a difference for the many patients we treat in the Asia-Pacific region and around the world.

A randomized multicentric study comparing alternating combination chemotherapy (VCMP/VBAP) and melphalan-prednisone in multiple myeloma

SummaryBetween January 1985 and December 1988, 386 patients with multiple myeloma were randomized to receive either MP or combination chemotherapy based on alternating cycles of VCMP and VBAP. The major prognostic parameters did not differ significantly between both treatment groups. A significantly higher proportion of objective responses was observed with combination chemotherapy as compared to MP (47.8 vs 32.2,P = 0.01). The median survival for all patients was 33.5 months. So far no significant differences were found when comparing the survival curves from both groups of patients. However, the median survival of MP-treated patients is 26.8 months, whereas the median survival of patients receiving VCMP/VBAP has not yet been reached. The definitive analysis must await the evaluation of all patients entered into the study and a longer follow-up time.

Impact of sex on prognostic host factors in surgical patients with lung cancer

Lung cancer has markedly poorer survival in men. Recognized important prognostic factors are divided into host, tumour and environmental factors. Traditional staging systems that use only tumour factors to predict prognosis are of limited accuracy. By examining sex‐based patterns of disease‐specific survival in non‐small cell lung cancer patients, we determined the effect of sex on the prognostic value of additional host factors.

Sex-Dependent Staging in Non–Small-Cell Lung Cancer; Analysis of the Effect of Sex Differences in the Eighth Edition of the Tumor, Node, Metastases Staging System

&NA; Because men with lung cancer have poorer survival, we examined the effect of sex on the prognostic value of the eighth edition of tumor, node, metastases staging (TNM). Men had worse 5‐year survival within each stage. This suggests that outcomes in TNM staging should be quoted separately for men and women to improve the prognostic, research, and clinical accuracy. Introduction: Non–small‐cell lung cancer (NSCLC) has disproportionately negative outcomes in men compared with women. The importance of the relationship between sex and tumor, node, metastases (TNM) staging system remains unknown. The objective of this study was to investigate the effect of sex on NSCLC survival for each stage in the eighth edition of the TNM staging system in NSCLC. Patients and Methods: Two cohorts treated surgically with curative intent between 2000 and 2010 were analyzed. The primary cohort was from Australia with a second population set from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate analyses of putative and validated prognostic factors were undertaken to investigate sex‐dependent prognostication with detailed analyses of sex differences in each TNM stage. The primary outcome was disease‐specific survival (DSS) at 5 years. Results: Inclusion criteria were met by 555 patients in the Australian cohort, 335 men (60.4%) and 220 (39.6%) women; and 47,706 patients from the SEER cohort, 24,671 men (51.7%) and 23,035 women (48.3%). Five‐year DSS was significantly worse for men in multivariate analyses for the Australian (hazard ratio [HR], 1.44; 95% confidence interval [CI], 1.04‐1.98; P = .026) and SEER (HR, 1.24; 95% CI, 1.20‐1.28; P < .001) cohorts. Detailed analysis of TNM stage sex differences revealed a consistent pattern of men having worse survival than women across stages in both cohorts. Conclusion: The poorer survival in men with NSCLC presents research and clinical communities with an important challenge. This studys findings suggest that for men and women diagnosed with NSCLC, and managed surgically, stage‐specific outcomes should be quoted separately and consideration to a rapid prognostic score with sex combined with staging as a key element.

Molecular basis of lung carcinogenesis

Lung cancer is the most prevalent cancer and the largest cause of cancer deaths in the Western world. Smoking is the major carcinogen for lung cancer but only approximately 15 % of smokers develop lung cancer, suggesting individual differences in susceptibility to tobacco smoke carcinogens. Once invasive, survival from lung cancer is poor which necessitates the prompt development of new therapeutic strategies in addition to effective methods for early detection, chemoprevention, and smoking cessation. Modern diagnostic and therapeutic strategies are becoming more realistic with the greater understanding of the molecular basis of lung cancer including the role of genomics and epigenomics in its pathogenesis. One such example of the benefits that result from expanded knowledge includes the advent of new targeted therapies, which are becoming increasingly used in clinical practice.

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.

Screening lung Tumour tissue for whole genome sequencing

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.

From evidence to practice: Audit of adjuvant chemotherapy after complete resection of non-small cell lung cancer in an Australian lung cancer center

Objective:  To investigate patterns in the rate of referral and uptake of platinum‐based adjuvant chemotherapy following resection of non‐small cell lung cancer (NSCLC) after recent landmark evidence‐based publications.