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Kwok-kin Wong
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MA04 - Models and Biomarkers (ID 122)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 12
- Now Available
- Moderators:Montse Sanchez-Cespedes, Kwok-kin Wong
- Coordinates: 9/08/2019, 13:30 - 15:00, Interlaken (1988)
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MA04.01 - Development of an in Vivo Platform to Identify Novel Mechanisms Governing Lung Cancer Response to Immunotherapy (Now Available) (ID 714)
13:30 - 15:00 | Presenting Author(s): Kelsie L Thu | Author(s): Shawn P Kubli, Andrew C Wakeham, Andrew J Elia, Tak Mak
- Abstract
- Presentation
Background
Immune checkpoint inhibitors (ICIs) have emerged as a promising therapy for the treatment of advanced stage lung cancer. While these agents elicit durable responses in some patients, most do not respond. An improved understanding of the mechanisms governing ICI response in a complex in vivo setting has potential to reveal novel therapeutic combinations to enhance ICI efficacy and associated biomarkers indicative of response. We have developed a syngeneic lung tumour model for in vivo CRISPR/Cas9 screens to deduce novel tumour-intrinsic mediators of response to anti-PD1 therapy.
Method
To delineate physiologically relevant ICI response mechanisms, an in vivo, syngeneic and orthotopic lung tumour model with an intact immune system is required. For this purpose, we have developed the KRAS-mutant Lewis Lung Carcinoma (LLC) model. Flow cytometry and immunohistochemistry were used to characterize immune cell composition in resected tumours and in vivo experiments were conducted to determine the effects of anti-PD1 treatment on LLC tumour growth.
Result
Luciferase-tagged LLC cells were implanted into the lungs of syngeneic C57BL/6 mice using orthotopic injection and mice reached humane endpoints 10-14 days post injection. Immunophenotyping of dissociated tumours revealed changes in the proportions of myeloid and lymphocyte populations relative to tumour-naïve lungs. CD8+ T-cells were present and tumour cells expressed PDL1 suggesting LLC has the capacity to respond to ICI. Consistent with these observations, orthotopic LLC growth was delayed in mice treated with anti-PD1 therapeutic antibody compared to anti-IgG2a isotype control, demonstrating that LLC is an appropriate model for identifying mechanisms that confer sensitivity and/or resistance to ICI therapy. Based on these findings, we generated LLC-Luciferase cells stably expressing Cas9. Since genome-wide screens are not feasible with this in vivo tumour model, we are synthesizing a custom, focussed guide RNA (gRNA) library. Genomic analyses have identified ~500 candidate immunomodulatory genes expressed in LLC and clinical lung tumours that will be targeted to determine the effects of inactivating these candidates on anti-PD1 response.
Conclusion
This platform will enable high-throughput genetic screens to elucidate novel tumour-intrinsic determinants of ICI response in vivo. Our discoveries will have potential to inform novel biomarkers predictive of response, and putative targets for new combination therapies to enhance the anti-tumour effects of ICIs. Collectively, this work will improve our understanding of the biological mechanisms governing ICI sensitivity, thereby stimulating the development of new strategies to maximize therapeutic benefit from ICIs in lung cancer patients.
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MA04.02 - Molecular Profiling of Adenocarcinoma and Squamous Cell Lung Cancer at Single Cell Resolution (Now Available) (ID 1358)
13:30 - 15:00 | Presenting Author(s): fengying Wu | Author(s): Yayi He, Zhou Yiqi, Anwen Xiong, Jia Yu, Wei Li, Nan Fang, Caicun Zhou
- Abstract
- Presentation
Background
Adenocarcinoma and squamous are two main subgroups of lung cancer: adenocarcinoma (ADC) accounts for 30-50% and squamous cell carcinoma (SCC) accounts for nearly 30% of all cases respectively. ADC and SCC have different pathological phenotypes and respond differently to various therapies, including immunotherapy. However, the underlying molecular mechanism of such differentiated drug responses still needs to be further characterized.
Method
To achieve high resolution of both tumor cells and their tumor microenvironment (TME), we used single cell RNA sequencing method to characterize ADC and SCC tumors from Stage IV NSCLC patients. Tissue biopsy samples from 21 patients (12 patients with ADC, 9 with SCC) were collected. For each sample, single cell RNA sequencing was performed on an average of 1930 cells. A graph-based clustering approach was used to classify cells into different cell types based on their gene expression patterns. The cellular subtypes of both cancer cells and TME in ADC and SCC samples were analyzed.
Result
ADC and SCC show distinct patterns at single cell resolution. Cancer cells from all ADC patients form two closely related clusters, while cancer cells from SCC patients show high intra-and inter-patient heterogeneity. Gene Ontology (GO) analysis demonstrated that ADC samples are enriched in genes of neutrophil degranulation and activation, while SCCs are enriched in genes related to epidermal cell differentiation and glutathione metabolic process. Genes related to cancer progression and metastasis, such as LSD1 and FASCIN, are normally expressed at higher level in SCC than in ADC. Furthermore, ADC samples contain higher percentage of a specific myeloid cell population, while SCC has higher percentage of fibroblasts, demonstrating the difference also in TMEs of ACD versus SCC.
Conclusion
The significantly higher level of heterogeneity for SCC can be a possible reason for poor responses to standard lung cancer therapies, including immunotherapy. Accurate characterization of SCC with single cell resolution could hold the key to more effective therapeutic strategies.
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MA04.03 - Lung Tumorspheres Characterization Reveals Cancer Stem-Like Cells Potential Targets and Prognostic Markers in Non-Small Cell Lung Cancer (Now Available) (ID 2269)
13:30 - 15:00 | Presenting Author(s): Alejandro Herreros-Pomares | Author(s): Eloisa Jantus-Lewintre, Silvia Calabuig-Fariñas, Juan Diego De-Maya-Girones, Rut Lucas, Ana Blasco, Ricardo Guijarro, Miguel Martorell, Eva Escorihuela, María Dolores Chiara, Elena Durendez-Saez, Carolina Gandia, Rafael Sirera, Rosa Farràs, Carlos Camps
- Abstract
- Presentation
Background
Non-small cell lung cancer (NSCLC) is the leading cause of death cancer-related worldwide due to late diagnosis and high resistance against treatments. This resistance has been associated to cancer stem-like cells (CSCs), a highly tumorigenic subpopulation for which the identification of targets and biomarkers is still under development.
Method
Tissue samples from 8 NSCLC patients were successfully established and cultured using a sphere-forming assay for CSCs enrichment. Adherent counterparts were used as differentiated control cells. Proliferation, chemorresistance, invasion and differentiation capacities were tested in vitro, whereas tumor initiation capacity was determined in vivo. The expression of 44 CSCs-related genes was assessed by qPCR and protein expression of the best contributors to distinguish adherent cells from tumorspheres was determined by immunoblot and immunofluorescence. The prognostic role of these genes was evaluated in a cohort of 661 resected NSCLC patients from TCGA and validated in an independent cohort of 114 resected lung adenocarcinoma patients.
Result
Patient-derived tumorspheres showed unlimited exponential growth, high resistance against chemotherapy, great invasion and differentiation capacities in vitro in addition to a higher tumorigenic potential than adherent cells in vivo. The expression of 17 genes was significantly overexpressed in lung tumorspheres, being NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 the best contributors. Proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The expression of CDKN1A, SNAI1 and ITGA6 was associated to prognosis based on Cox regression analysis (Z-score > 1.5), so their absolute regression coefficients from a multivariate model were used to calculate a gene expression score. Kaplan-Meier survival analysis showed that patients with high score have shorter OS in the entire cohort [37.7 vs. 60.4 mo., p = 0.001] and the adenocarcinoma subcohort [36.6 vs. 53.5 mo., p = 0.003], but not in squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score was an independent biomarker of prognosis for OS in both, the entire cohort [HR: 1.498; 95% CI, 1.167-1.922; p = 0.001] and the adenocarcinoma subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. The prognostic value of this score was confirmed in an independent cohort of 114 lung adenocarcinoma patients (42.90 vs. NR mo, p = 0.020).
Conclusion
Proteins encoded by NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 are potential targets against lung CSCs. Elevated gene expression levels of CDKN1A, SNAI1 and ITGA6 are associated with worse prognosis.
Funded by CB16/12/00350 from CIBEROnc, PI12-02838, and PI15-00753 from ISCIII and Fundacion Arnal Planelles.
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MA04.04 - Discussant - MA04.01, MA04.02, MA04.03 (Now Available) (ID 3730)
13:30 - 15:00 | Presenting Author(s): David Santamaria
- Abstract
- Presentation
Abstract not provided
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MA04.05 - Deciphering the Molecular Mechanisms Underlying the Progression of Bronchial Premalignant Lesions (Now Available) (ID 2108)
13:30 - 15:00 | Presenting Author(s): Evgeny V. Denisov | Author(s): Polina A. Gervas, Anastasia A. Ponomaryova, Anastasia A. Schegoleva, Tatiana S. Gerashchenko, Artem M. Kiselev, German M. Demidov, Alexey A. Zarubin, Liliya S. Lyapunova, Sergey P. Kovalenko, Olga V. Cheremisina, Sergey A. Tuzikov, Olga V. Pankova, Nadezhda V. Cherdyntseva, Vladimir M. Perelmuter
- Abstract
- Presentation
Background
The mechanisms underlying the progression of bronchial lesions to squamous cell lung cancer remain undefined. Previously, we hypothesized that bronchial lesions presented individually or combined with each other in the bronchi of non-small cell lung cancer (NSCLC) patients mirror the different scenarios of the premalignant process: individual basal cell hyperplasia (iBCH) – the stoppage at hyperplasia, BCH plus squamous metaplasia (SM) – the progression of hyperplasia to metaplasia, and SM plus dysplasia – the progression of metaplasia to dysplasia. In this study, we aimed to assess the molecular profile of BCH, SM, and dysplasia depending on their co-occurrence in the bronchi of NSCLC patients and to identify mechanisms that are involved in the different scenarios of the premalignant process.
Method
The samples of lung tissue were obtained at a distance of 4-5 cm from the tumor during surgery of 21 NSCLC patients. Normal bronchial epithelium, BCH, and SM, as well as dysplasia, were isolated from tissue sections using laser microdissection PALM (Carl Zeiss). The microdissected samples underwent whole genome (One Step WGA, Bioron) and transcriptome (Ovation PicoSL WTA System V2, Nugen) amplification and sequenced using the SeqCap EZ Human Oncology Panel (Roche) and profiled using SurePrint G3 Human GE v2 8x60K microarrays (Agilent). Additionally, the samples were sequenced using the Pico Methyl-Seq Library Prep Kit (Zymo Research). Changes in gene expression were confirmed using immunohistochemical staining.
Result
Genetic alterations were observed already at the early stages of the premalignant process in the bronchial epithelium; however, their number varied from sample to sample. For example, one case of BCH showed more than 10 deleterious mutations in the GRM5, MAML2, SP1, ETV4, and other genes, whereas other BCHs carried single alterations. No significant differences were found in the mutational landscape between the iBCH and BCH combined with SM. Bisulfite sequencing demonstrated significant changes in the methylation status of the SAPCD2 and ST14 genes in BCH. Importantly, these changes differed between various forms of BCH. Sequencing of SM and dysplasia is in progress and results will be presented later. Gene expression profiling showed differences in the activity of immune response genes between the iBCH and BCH combined with SM and the cell cycle and cilium assembly genes between SMs co-presented with BCH and dysplasia. Overall, the transcription profile of SM co-presented with BCH was closer to BCHs, whereas SM co-detected with dysplasia was similar to dysplasia. Several genes were identified to be expressed specifically in different forms of BCH and SM, among which CCDC114, MAP7D2, and LIFR were confirmed by immunohistochemistry. The loss of CCDC114 and MAP7D2 in SM may serve as an indicator of its progression to dysplasia.
Conclusion
Taken together, this study demonstrates the significant differences between various types of BCH and SM. These differences support the hypothesis that the isolated and combined forms of the bronchial lesions mirror the different scenarios of the premalignant process as well as explore the mechanisms underlying the progression of hyperplasia and metaplasia to dysplasia.
The study was supported by RFBR (#17-29-06002) and the Russian President Fellowship (#SP-1549.2018.4).
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MA04.06 - Lung Epithelium Whole Transcriptome Signatures That Reflect Incident Lung Cancer Case-Control Status (Now Available) (ID 2526)
13:30 - 15:00 | Presenting Author(s): Simon D Spivack | Author(s): Xiao Dong, Miao Kevin Shi, Weiguo Han, Steven Keller, Alex Maslov, Yousin Suh, Jan Vijg
- Abstract
- Presentation
Background
BACKGROUND: Focusing early detection and prevention efforts on those at high risk for lung cancer is central to leveraging such strategies. Notably, that risk persists even after removal of a lung cancer, as reflected in lung recurrences, which are common, and usually occur remote from the surgical removal site. This implies risk for future incident lung cancer is represented in the biology of broad areas of lung epithelium, before, during, and after diagnosis. Given that somatic mutations in the bronchial tree are known to persist for decades, there is a suggestion that smoking transforms the entire epithelium at several somatic and gene regulatory levels. We hypothesize that the normal lung epithelium contains expression and epigenetic epithelial signatures that are representative of donor case-control status, that is poised for carcinogenesis.
Method
METHODS: As a first step, in order to identify differentially expressed genes (DEGs) associated with aging, smoking and cancer case-control status, we analyzed RNA-seq transcriptome data of laser capture microdissected (LCM) bronchial and alveolar epithelium separately, in paired tissue sets of 40- and 74 respective individuals, summarized here. Read count was the main normalization variable. We also measured differentially methylated sites (DME) by whole genome bisulfite genome sequencing (WGBSeq), covering >60% of the genome/methylome [as of this deadline, these methylome data are not yet fully analyzed].
Result
RESULTS: Mean subject age for 77 total subjects was 65 (+/-9.9), 19% current-, 76% former, 5% never smokers. For each cell type, we modeled gene expression level as a result of aging, gender, smoking and case-control status. We put all four clinical variables age, gender, smoking status, case-control status) along with cell type (alveolar/bronchial) into the model, to avoid potential confounding effects. We discovered 175 DEGs discriminating case-control status (FDR p<0.05) in alveolar and bronchial cells combined, and 420 case-control DEGs with bronchial cells alone. Bronchial cells displayed 31 DEGs discriminating current versus former smokers (FDR-adjusted P<0.05). Gene ontology (David) clusters for case-control discrimination in these “normal” bronchial epithelia included energetics pathways (GO 0042776/0006754; ATP biosynthesis) as well as transcriptional and translational regulation pathways; KEGG clusters also included oxidative phosphorylation pathways (hsa00190), among others.
Conclusion
CONCLUSION: There is a donor case-control discriminant expression signature for human lung bronchial captured cells, emphasizing bioenergetically-deranged metabolic pathways, among others. If confirmed in larger studies that measure deranged metabolites directly, metabolomics biomarkers representing bioenergetics and other pathways may serve to define those individuals whose epithelia is tilted toward carcinogenesis, and therefore are at increased risk for lung cancer.
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MA04.07 - Inhibition of CXCR2+ Neutrophil Migration as a Targeted Therapy in KRAS-Driven Lung Adenocarcinoma (Now Available) (ID 2914)
13:30 - 15:00 | Presenting Author(s): Meghan De Meo | Author(s): Roni F. Rayes, Simon Milette, Mara Vagai, Mariana Usatii, Arvind Chandrasekaran, Betty Giannias, France Bourdeau, Christopher Moraes, Sidong Huang, Daniela Quail, Logan Walsh, Veena Sangwan, Nicholas Bertos, Sophie Camilleri-Broet, Philippe Broet, Pierre O Fiset, Jonathan Cools-Lartigue, Lorenzo E. Ferri, Jonathan D. Spicer
- Abstract
- Presentation
Background
Lung adenocarcinoma (LUAD) accounts for 40% of all lung cancer cases. Although driver mutations in the K-RAS oncogene occurs in 25% of all LUAD cases, to date, there are no available targeted therapies. Infiltrating neutrophils in LUAD are indicative of the worst survival outcomes. The C-X-C motif chemokine receptor 2 (CXCR2) mediates their recruitment to the tumour microenvironment where they promote a pro-tumorigenic environment. CXCR2 ligand expression is higher in KRAS-driven LUAD compared to the other most frequently mutated oncogenes. Therefore, we hypothesize that K-RAS-driven LUAD may be the best candidate for a CXCR2 targeted treatment strategy.
Method
The PREdiction of Clinical Outcomes from Genomic Profiles (PRECOG) is a dataset of gene expression and survival outcome. The dataset includes data from approximately 18 000 human patients with 39 different malignancies. The dataset was used to determine whether high neutrophil infiltration, CXCR2 expression and CXCR2 ligand expression were associated with poor survival outcomes in LUAD. A 100 patient LUAD tissue microarray was built and stained for neutrophil elastase and CXCR2 by immunohistochemistry. Kaplan-Meier curves were used determine the effect of high neutrophil or CXCR2+ cell infiltration in the LUAD tumour microenvironment on survival outcome. The Cancer Cell Line Encyclopedia (CCLE) is an online dataset that provides gene expression and genotype data from 947 human cancer cell lines (36 cancer types). Expression data of all LUAD cell lines (n=70) from CCLE was obtained for all known CXCR2 ligands. The expression of CXCR2 ligands in K-RAS, EGFR, ALK and ROS-1-driven LUAD cell lines was compared. Microfluidics devices were used to compare the neutrophil recruitment to K-RAS, EGFR, ALK and ROS-1-driven LUAD cell lines. The neutrophil recruitment to each of the cell lines was compared in the presence and absence of CXCR2 inhibition.
Result
Using the PRECOG dataset, we found that CXCR2 expression in neutrophils is at least 18-fold greater than its expression in other immune cell types. Using all the LUAD cell lines (n=70) available on the CCLE, we found that K-RAS-driven LUAD is the highest CXCR2 ligand expresser as compared to EGFR, ALK and ROS1-driven LUAD. Moreover, using PRECOG, we found that poorer survival outcome is associated with high expression of eight out of nine known CXCR2 ligands (p < 0.05). In addition, high neutrophil infiltration in LUAD is associated with the worst survival outcome compared to other immune cell infiltrates (p < 0.001). In accordance with the PRECOG data, the presence of infiltrating neutrophils in a 100 patient LUAD tissue microarray is associated with poorer survival outcome when compared to patients with no infiltrating neutrophils (p < 0.05). Neutrophil migration to K-RAS, EGFR, ALK and ROS1-driven LUAD cell lines was examined in microfluidics devices and found to be highest in K-RAS-driven LUAD. CXCR2 inhibition reduced neutrophil migration only in K-RAS-driven lung adenocarcinoma (p < 0.05).
Conclusion
CXCR2 inhibition could be an exciting potential targeted treatment for patients with K-RAS-driven LUAD. CXCR2 inhibition is in clinical trials for metastatic melanoma, pancreatic, breast and head and neck cancer. Current evidence suggests that CXCR2 inhibition is safe and tolerable.
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MA04.08 - Discussant - MA04.05, MA04.06, MA04.07 (Now Available) (ID 3731)
13:30 - 15:00 | Presenting Author(s): Triantafillos (Lakis) Liloglou
- Abstract
- Presentation
Abstract not provided
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MA04.09 - Study of Exosomes in NSCLC for Biomarkers Searching (Now Available) (ID 2417)
13:30 - 15:00 | Presenting Author(s): Elena Durendez-Saez | Author(s): Silvia Calabuig-Fariñas, Cristian Suarez, Marais Mosqueda, Sandra Gallach, Eva Escorihuela, Andrea Moreno, Susana Torres, Alejandro Herreros-Pomares, Álvaro González, Ernesto De La Cueva, Eva Serna, Jesus M Paramio, Eloisa Jantus-Lewintre, Carlos Camps
- Abstract
- Presentation
Background
Exosomes are small membranous vesicles secreted by a most type of cells (especially in tumoral processes), around 40-130 nm of size, that carry relevant information to distant tissues and being able to modulate its physiology. Exosomes have been detected in different clinical samples and may play a key role in NSCLC, participating in several processes such as horizontal transfer of RNA from tumor to microenvironmental cells, angiogenesis, pre-metastatic niche formation, immunosuppression; and could also be key elements in stem cell differentiation (from different origins).
The principal objective of this study was to analyze the exosomes cargo from NSCLC cell lines and primary cultures with diverse characteristics under different growth conditions: suspension cultures with cancer stem cells (CSCs) features and monolayer cultures.
Method
Primary cultures from resected NSCLC patients and NSCLC cell lines were successfully established. Differentiated tumor cells were cultured under adherent conditions (2D) whereas CSCs were established in suspension cultures (3D tumorspheres). Exosomes isolation was performed by ultracentrifugation. Exosomes characterization was carried out through nanovesicles tracking analysis (NTA) and electron microscopy; and the determination of surface markers through immunoblot and flow cytometry. Exosomal DNA was extracted in order to determine the mutational status of the EGFR and RAS genes by BEAMing Digital PCR (Sysmex). Transcriptomic analysis has been carried out from exosomal RNA through whole genome gene expression microarrays, (Affymetrix). The data was normalized by Robust Multi-Array Average (RMA) and analyzed using Transcriptome Analysis Console (TAC), MultiExperiment Viewer (MeV) software and Partek Genomics Suite. Statistical significance was established at (p ≤ 0.01).
Result
In reference to the characterization, NTA and electron microscopy showed that exosomes were obtained free of cellular debris and their size ranges from 108-125 nm, according to the size of tumor-derived microvesicles. Exosomal surface markers analyzed by immunoblot and flow cytometry were detected in samples, confirming proper isolation. Mutational analysis of EGFR and RAS genes performed on exosomal DNA shown the same pattern displayed by the origin cells. Transcriptomic analysis of the exosomal content showed that the expression of mRNAs, miRNAs and precursors were significantly different between 3D and 2D-derived exosomes. Finally, a pathway enrichment analysis was carried out to know in which biological processes (cancer-related) are involved.
Significant differential expressions were also found between mRNAs, miRNAs and pre-miRs present in exosomes from adenocarcinoma (ADC) vs. squamous cell carcinoma (SCC). Interestingly, 7 miRNAs differentially expressed in exosomes (miR-200c; miR-29a; miR-339; miR-224; miR-31; miR-21; miR-33a) had already been identified as overexpressed in tumor tissue from NSCLC patients by our group. Moreover, miR-339 y miR-21 were related to prognosis (p < 0.05) in ADC group.
Conclusion
Differences in exosomal mRNA, miRNAs and pre-miRs expression have been observed between: i) lung-tumorspheres vs. more differentiated tumor cells and ii) ADC vs. SCC cultures. In addition, the same mutational pattern was detected in exosomes as compared with their parental cultures. Therefore, exosomes can be a useful source for biomarkers analysis in NSCLC.
Supported by grant GV/2018/026, PI18/00266, & Asociación Española Contra el Cáncer (AECC Valencia).
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MA04.10 - Development and Validation of a Gene Expression-Based Prognostic Signature in Early-Stage Squamous Cell Carcinoma of the Lung (Now Available) (ID 2643)
13:30 - 15:00 | Presenting Author(s): Pinaki Bose | Author(s): Anthony Boylos, Lars Frederick Petersen, Olga Kovalchuk, Igor Kovalchuk, Michelle L Dean, Doha Itani, Karen Kopciuk, Gwyn Bebb
- Abstract
- Presentation
Background
Squamous cell carcinoma of the lung (SqCCL) accounts for about 30% of all lung cancers and is usually associated with smoking. The clinical outcomes of early stage SqCCL are heterogeneous; while 60% of Stage I and II SqCCL patients never present with recurrence after surgery, the remaining will ultimately succumb to the disease. Therefore, a robust prognostication tool is an unmet clinical need. Here, we describe the development and validation of a gene expression-based prognostic signature in Stage I and II SqCCL patients.
Method
A total of 673 primary tumour samples obtained from surgically resected Stage I and II SqCCL patients were included in this study. The Cancer Genome Atlas (TCGA) cohort contained 365 patients with gene expression data generated using RNA sequencing (RNAseq). Five data sets (GSE30219, GSE37745, GSE50081, GSE4573, GSE14814) containing 308 patients profiled using Affymetrix microarrays were obtained from the Gene Expression Omnibus (GEO) database; batch effect mitigation of gene expression data was performed using ComBat. An additional cohort of consecutive Stage I and Stage II SqCLC patients was assembled at the Tom Baker Cancer Centre (TBCC), University of Calgary and gene expression was profiled using RNAseq. We performed a two-stage development of the gene signature by performing penalized elastic net Cox regression analysis in the TCGA training cohort followed by refinement of the gene list in the compiled GEO database patients. Final validation was performed using the in-house TBCC cohort. Progression-free survival (PFS) and overall survival (OS) were the primary and secondary outcomes of interest, respectively.
Result
All datasets used in this study were found to consist of patients with comparable clinical characteristics. A gene expression signature associated with PFS was developed in TCGA cohort that significantly stratified patients into high and low risk groups. The signature was refined in the complied GEO database cohort and validated in the U of C cohort. The signature also effectively stratified patients into high and low risk groups based on OS. We are currently performing multivariable analysis of the refined gene signature, adjusting for covariates of known prognostic value.
Conclusion
Our signature, if prospectively validated, will guide clinical decision making in SqCCL. Effective risk stratification using our signature may identify Stage I patients that will benefit from adjuvant therapy and stage II patients that could be spared adjuvant treatment following surgical resection.
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MA04.11 - Biological and Prognostic Implications of the Long Non-Coding Transcriptome in Tumour-Infiltrating Immune Cells (Now Available) (ID 2838)
13:30 - 15:00 | Presenting Author(s): Kevin W Ng | Author(s): Adam Sage, Erin A Marshall, Katey SS Enfield, Wan Lam
- Abstract
- Presentation
Background
The lung tumour microenvironment is defined by complex infiltration patterns of immune cells which can contribute to both tumour progression and rejection. The advent of targeted immunotherapies has transformed cancer therapy, leading to durable regression even in late-stage lung tumours. Single-cell RNA sequencing and deconvolution of bulk tumour samples have provided insight into the transcriptomes of tumour-infiltrating immune populations and the regulatory networks that promote cytotoxicity and exhaustion transcriptional programs. Long non-coding RNAs (lncRNAs) have emerged as master regulators of gene expression in tumour cells, but their role in immune cells remains undercharacterized. We sought to delineate lncRNA expression profiles in healthy and lung tumour-infiltrating immune cells in order to better understand transcriptional reprogramming in tumour-infiltrating immune cells and to explore their potential as biomarkers of patient outcome and response to immunotherapy.
Method
RNAseq profiles of flow-purified adaptive and innate immune subsets were analysed for lncRNA expression, yielding 4919 expressed lncRNAs. Immune lncRNAs were then mapped to tumour and paired non-malignant lung adenocarcinoma samples (TCGA n=108, BCCA n=72) and associated with infiltrating immune populations by deconvolution and methylation-based purity scores. Associations with tumour immunogenicity were assessed by somatic mutational load and expression of tumour-associated antigens. Immune-specific expression of lncRNAs was confirmed in an external single cell RNAseq dataset of lung adenocarcinomas (n=5).
Result
We found that lncRNA expression patterns display markedly greater cell-type specificity than protein-coding genes in healthy samples, supporting their role in cell-intrinsic transcriptional regulation. 323 immune lncRNAs were differentially expressed in lung tumours compared to matched non-malignant tissue, with enriched expression of immune lncRNAs in tumours with high antigenic load. Many of these genes were positively correlated with CD45 expression and negatively correlated with tumour purity, suggestive of immune cell-restricted expression patterns. Furthermore, a substantial proportion of these genes showed decreased expression in microdissected tumour samples, suggesting that immune-derived lncRNAs may account for gene expression patterns observed in bulk tumour data. We validated these findings in a scRNAseq dataset and analysed co-expression patterns of immune lncRNAs with immune cell markers in order to identify specific immune cell phenotypes and assess the interaction of immune lncRNAs with cytotoxicity and exhaustion transcriptional networks. We identify immune lncRNAs which may regulate expression of important immune genes related to NK and CD8+ T cell cytotoxicity, as well as immune lncRNAs which predict patient outcome and response.
Conclusion
We present an atlas of lncRNAs expressed in innate and adaptive immune cells, emphasizing the multifaceted roles of lncRNAs in homeostasis and anti-tumour immunity. We highlight the potential of immune infiltrate to confound differential expression analysis of bulk tumour RNAseq data, with consideration needed for tumour purity and immune infiltration levels. Our data provide a resource that will facilitate further identification of functionally and clinically useful lncRNAs.
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MA04.12 - Discussant - MA04.09, MA04.10, MA04.11 (Now Available) (ID 3732)
13:30 - 15:00 | Presenting Author(s): Alfonso Calvo
- Abstract
- Presentation
Abstract not provided
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Author of
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MS05 - Novel Biological Pathways and Druggable Targets (ID 68)
- Event: WCLC 2019
- Type: Mini Symposium
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Jin Jen, Marisol Arroyo-Hernandez
- Coordinates: 9/09/2019, 11:00 - 12:30, Vancouver (2003)
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MS05.01 - Epigenetic Therapeutics in Lung Cancer (Now Available) (ID 3461)
11:00 - 12:30 | Presenting Author(s): Kwok-kin Wong
- Abstract
- Presentation
Abstract
Epigenetic Targets and Drugs to enhance immune response in Lung Cancer
Kwok-Kin Wong
Effective therapies for non–small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with ability to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. We have previously demonstrated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we have shown that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T-cell activation and improved function of antigen-presenting cells. The pan-bromodomain inhibitor JQ1 attenuated CD4+FOXP3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Finally, we have recently performed in vivo CRISPR screens to identify additional novel epigenetic targets that would synergize with PD1 blockade in KRAS driven NSCLC.
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