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Katey S.S. Enfield
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MA 10 - Immunotherapy I (ID 664)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Immunology and Immunotherapy
- Presentations: 1
- Moderators:S. Wang, Robert Pirker
- Coordinates: 10/17/2017, 11:00 - 12:30, Room 303 + 304
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MA 10.09 - Increased T Follicular Helper Cell Infiltration in Lung Adenocarcinoma Tertiary Lymphoid Organs (ID 8487)
11:00 - 12:30 | Author(s): Katey S.S. Enfield
- Abstract
- Presentation
Background:
T follicular helper cells (Tfh) are an antigen-experienced CD4+ T cell subset that have been found to play crucial roles in the development of humoral immunity. In particular, their presence in the B cell-rich germinal centre of secondary and tertiary lymphoid tissue aids in B cell maturation through selection of B cells producing high-affinity antibodies. Tfh cells have known roles in autoimmune disease and B cell malignancies; however, their role in many solid tumours, including those of the lung, remains unclear.
Method:
We analyzed 83 paired tumour-normal lung adenocarcinoma samples from the BC Cancer Agency (BCCA) as well as 576 unpaired samples from The Cancer Genome Atlas (TCGA). Relative immune cell content was obtained from gene expression data using a linear support vector regression deconvolution approach (CIBERSORT). Spatial positioning of B and T cells within selected tumour sections was examined through IHC. The impact of Tfh infiltration on patient survival was analyzed using a Cox Proportional Hazard model.
Result:
T follicular helper cells are increased in tumour tissue, accompanied by global upregulation of Tfh markers PDL1 and CXCR5 in both the BCCA and TCGA cohorts. Histological analysis revealed localization of Tfh cells within tertiary lymphoid organs, with direct contact with B cells in the follicular zone observed. Importantly, Tfh recruitment appears to be an early event in tumour development and a function of neoantigen exposure, indicative of an active anti-tumour response rather than a result of chronic inflammation of the tumour microenvironment.
Conclusion:
T follicular helper cells are required for B cell maturation and subsequent antibody responses. As such, it is not surprising that Tfh infiltration in tumour-resident ectopic lymphoid structures correlates with patient survival in various cancer types. Given the importance of tumour-specific antibody responses in natural and therapeutic immunity, further investigation of Tfhs may show prognostic utility and be a marker of early-stage lung tumours.
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MA 15 - Lung Cancer Biology II (ID 670)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Biology/Pathology
- Presentations: 1
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MA 15.14 - Long Non-Coding RNA Disruption in Lung Adenocarcinoma Reveals Novel Mechanisms of Metastasis (ID 8659)
15:45 - 17:30 | Author(s): Katey S.S. Enfield
- Abstract
- Presentation
Background:
Identifying the drivers of metastasis will yield new molecular targets for prognostics and therapeutics. Long non-coding RNAs (lncRNAs) are known to regulate gene transcription through their influence on the expression of nearby (cis) and distant (trans) genes. Emerging evidence suggests that lncRNAs are involved in key cellular processes, presenting an opportunity for large-scale identification of lncRNA genes critical to lung cancer progression. Here we investigate the contribution of this class of non-coding RNA to lung adenocarcinoma (LUAD) metastasis.
Method:
Stage T1 and T2 tumours with (N≥1 and/or M≥1) and without (N=0 and M=0) metastasis were examined for expression comparisons. Sequencing data from 265 non-metastatic and 130 metastatic tumours obtained from The Cancer Genome Atlas were used as our discovery cohort. Results were validated in 20 non-metastatic and 10 metastatic tumour samples microdissected to 90% purity and sequenced using the Illumina Hi-Seq platform. Normalized sequence read count comparisons were performed (Mann Whitney U-Test, FDR-BH p<0.05) to identify lncRNAs significantly deregulated in metastatic samples. LncRNAs over- and under-expressed in metastatic LUAD were compared to nearby protein-coding-target genes to identify putative mechanisms of regulation in cis.
Result:
We discovered 150 lncRNAs to be significantly differentially expressed between metastatic and non-metastatic tumours, including lncRNAs with previously described oncogenic roles in lung cancer, such as Lung Cancer Associated Transcript 1 and H19. As individual lncRNAs can positively or negatively regulate target-gene expression, it is noteworthy that we identified potential protein-coding-target genes that display both concordant and discordant expression patterns with specific lncRNAs. For example, we discovered the upregulation of linc00942 in metastatic LUAD (FDR-BH p=0.001) and the concordant overexpression of its corresponding protein-coding-target gene, ELKS/RAB6-Interacting/CAST Family Member 1 (ERC1) (FDR-BH p=0.02). Further, metastatic LUAD samples stratified by linc00942 expression also display corresponding elevation of ERC1 (p=0.0002), which holds true in the validation cohort. ERC1 (an upstream member of the NF-κB signaling pathway) is implicated in cell migration and focal adhesion, and displays deregulated expression in a number of cancer types. Thus, overexpression of linc00942 may act as a novel positive cis-regulator of ERC1, promoting metastasis.
Conclusion:
This work has led to the discovery of a large number of lncRNA genes deregulated in metastatic LUAD, suggesting that altered lncRNA expression contributes functionally to malignant progression. Understanding cis- or trans-mediated mechanisms of gene deregulation enacted by metastasis-associated lncRNAs will present novel opportunities for diagnosis and treatment.
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OA 07 - Biomarker for Lung Cancer (ID 659)
- Event: WCLC 2017
- Type: Oral
- Track: Biology/Pathology
- Presentations: 1
- Moderators:Philip Christopher Mack, Shinichi Toyooka
- Coordinates: 10/16/2017, 15:45 - 17:30, Room 503
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OA 07.07 - Inhibition of the Novel Oncogene ELF3 Abolishes Lung Adenocarcinoma Growth (ID 8408)
15:45 - 17:30 | Presenting Author(s): Katey S.S. Enfield
- Abstract
- Presentation
Background:
Oncogenic reactivation of transcription factors involved in fetal lung development is integral to lung adenocarcinoma (LUAD) biology, as observed with TITF1/NKX2-1 and the ETS transcription factors ETV4 and ETV5. ELF3 is an uncharacterized ETS family member implicated in fetal lung development encoded at 1q32.1. Interestingly, chromosome 1q is a region of frequent gain in LUAD that lacks a bona fide oncogene. We hypothesize that ELF3 is a novel oncogene and putative therapeutic target in LUAD.
Method:
Multiple independent datasets encompassing 1,685 clinical samples of LUAD, lung squamous cell carcinoma (LUSC), small cell lung cancer, and non-malignant lung tissues were analyzed to establish the frequency of ELF3 overexpression and underlying genetic mechanisms of selection. Protein-protein interaction (PPI) networks were constructed around ELF3, and integrated pathway analysis was performed to decipher the signaling network disruptions resulting from ELF3 overexpression. Isogenic cell lines were established to assess the ability of ELF3 to regulate oncogenic phenotypes. The effect of ELF3 loss on tumour growth was assessed in xenograft mouse models.
Result:
Strong ELF3 overexpression was frequently observed in LUAD (>2-fold: TCGA 40% p=1.5E-07; BCCA 73% p=1.6E-21), but was not observed in other lung cancer subtypes. Similarly, high ELF3 expression was significantly associated with poor overall survival of LUAD patients (all Stages p<0.0001, Stage I p<0.0001), but not LUSC patients (p>0.05). These clinical associations prompted further examination of ELF3 in the LUAD subtype of lung cancer. While mutations in ELF3 were rare, up to 80% of LUAD patients harboured focal amplification, DNA gain, and/or promoter hypomethylation at the ELF3 locus, which resulted in transcript overexpression. ELF3 overexpression induces remodeling of 23 direct PPI networks, resulting in loss of interaction with proteins such as MYC and GLI2, while forming new interactions with NKX2-1, HOXA5 and CDK8, among others. This reprogramming of PPI networks affects multiple oncogenic pathways including MAPK, TGF-beta and WNT. ELF3 knockdown in LUAD cell lines resulted in significantly reduced proliferation, viability, and anchorage-independent growth, demonstrating ELF3 has oncogenic properties. Loss of ELF3 abolished the ability of LUAD cells to establish tumours in xenograft mouse models, demonstrating the requirement of ELF3 expression for tumour growth.
Conclusion:
ELF3 is a novel LUAD oncogene encoded on chromosome 1q, activated in up to 73% of patients, and strongly associated with poor overall survival. As ELF3 inhibition abolished tumour growth, therapeutic targeting of ELF3 could benefit LUAD patient outcome.
Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 3
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-017 - Aberrant Expression of Long Non-Coding RNAs from Pseudogene Loci Highlights Alternative Mechanisms of Cancer Gene Regulation (ID 10231)
09:30 - 16:00 | Author(s): Katey S.S. Enfield
- Abstract
Background:
Less than half of lung adenocarcinoma (LUAD) patients harbour clinically actionable driver genes, emphasizing the need to explore alternative mechanisms of cancer gene deregulation. Long non-coding RNAs (lncRNAs) have emerged as important players in cell biology, and can be exploited by tumours to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional protein-coding parent genes but retain high sequence homology. Interestingly, several lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans due to sequence complementarity. We hypothesize that this phenomenon occurs more broadly than previously realized, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumourigenesis that has clinical implications.
Method:
Illumina HiSeq reads were processed and aligned to the ENSEMBL annotation file in order to derive the most complete set of both protein-coding and non-coding genes. Two datasets were selected due to their paired nature, complete with both LUAD and non-malignant lung profiles (TCGA n=108, BCCA n=72). LncRNAs were filtered based on positional overlap within pseudogene loci, and a Wilcoxon sign-rank test was run to identify lncRNAs with significantly altered expression between paired tumour and normal tissues (FDR p<0.05). To identify lncRNAs that likely regulate protein-coding parent gene expression in trans, tumours were ranked by lncRNA expression, and protein-coding parent gene expression of top and bottom ranked tertiles was compared by Mann Whitney U-test (p<0.05). Survival analysis was performed using a Cox proportional hazard model.
Result:
Our analysis has identified 129 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD in both datasets. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) protein-coding parent gene expression was significantly associated with survival. For example, RP11-182J1.1 is a lncRNA expressed from a pseudogene to EGLN1, a previously described cancer gene involved in regulation of tumour hypoxia. RP11-182J1.1 was underexpressed in LUAD and significantly positively correlated with EGLN1 expression. In addition, EGLN1 was significantly associated with patient survival (p=1.2e-08) emphasizing the clinical potential of these lncRNAs.
Conclusion:
This work uncovers evidence to suggest the lncRNA-pseudogene-protein-coding gene axis is a prominent mechanism of cancer gene regulation. Further characterization of this understudied gene regulatory mechanism could lead to novel therapies that silence oncogenes or reactivate tumour suppressor genes.
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P2.02-022 - Alternative Regulation of Cancer-Associated Genes through Modulation of Long Non-Coding RNAs (ID 8658)
09:30 - 16:00 | Author(s): Katey S.S. Enfield
- Abstract
Background:
Uncovering novel mechanisms of cancer-gene regulation may reveal new actionable targets to direct the treatment of patients who do not harbour targetable molecular drivers of lung cancer. Long non-coding RNAs (lncRNAs), are a class of transcripts that hold an emerging role in cell biology, particularly in gene regulation. These genes have since been implicated in cancer-associated phenotypes, and may represent attractive therapeutic intervention points; however, prediction of downstream regulatory targets of lncRNAs has been impeded due to their complex tertiary structure. Recently, a subset of lncRNAs has been shown to regulate the expression of neighbouring protein-coding genes in cis. Here we take a novel approach to identify lncRNAs deregulated in lung adenocarcinoma (LUAD) and examine their roles in the expression modulation of their cancer-associated protein-coding cis-partner genes.
Method:
RNA-sequencing was performed on 36 LUAD tumour samples with matched adjacent non-malignant tissue obtained via microdissection to 90% purity. Significantly deregulated lncRNAs and neighbouring protein-coding genes were identified by comparison of matched tumour and non-malignant normalized read counts (Wilcoxon Signed-Rank Test, FDR-BH<0.05). Fifty LUAD tumours with paired normal tissue from The Cancer Genome Atlas (TCGA) were used to validate these findings. Cox-Proportional Hazard analysis was performed on both datasets to assess survival associations of significantly deregulated lncRNAs.
Result:
Our approach revealed greater than 500 lncRNAs that were significantly deregulated between LUAD and matched normal tissues. Many of these lncRNAs have neighbouring protein-coding genes that also display deregulated expression patterns. Of particular interest are the protein-coding-target genes that have been previously implicated in cancer, including OIP5, which is involved in chromatin segregation, as well as HMGA1, which contributes to cell transformation and metastasis. In both of these cases, the neighbouring lncRNA is significantly underexpressed while the protein-coding gene is significantly overexpressed, suggesting a negative regulatory function of the lncRNA. Moreover, survival analyses revealed that patients with high expression of either OIP5 or HMGA1 had significantly shorter overall survival. Strikingly, patients with low expression of the lncRNA near OIP5 also displayed poorer overall survival, illustrating the clinical opportunity that these genes present.
Conclusion:
Our results highlight the landscape of lncRNA deregulation in LUAD and uncover a role of these non-coding transcripts in the cis-regulation of neighbouring protein-coding genes, many of which have been described in cancer and predict patient survival. Further characterization of this alternative lncRNA-mediated cancer-gene regulatory mechanism may reveal novel therapeutic targets that may improve treatment for LUAD patients without well defined molecular drivers.
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P2.02-038 - Imaging Platform for the Quantification of Cell-Cell Spatial Organization within the Tumour-Immune Microenvironment (ID 9605)
09:30 - 16:00 | Presenting Author(s): Katey S.S. Enfield
- Abstract
Background:
The contribution of the tumour-immune microenvironment to tumour progression and patient outcome has become increasingly evident. Newly developed genomic tools have enabled the study of immune cell composition from bulk tumour data. However, such tools (e.g. CIBERSORT) do not provide the key spatial information that is crucial to understand tumour-immune cell interactions. To this end, we have developed a multispectral imaging platform that improves upon traditional analysis methods of cell segmentation and cell density calculations by further quantifying nearest-neighbour interactions (cell-cell spatial relationships). We apply this technology to investigate tumour-immune cell spatial relationships and their clinical significance to discover novel biological insights.
Method:
Whole tissue sections from 20 lung adenocarcinomas were stained for CD3, CD8, and CD79a and counterstained with haematoxylin. Multispectral images were acquired for five fields of view and analyzed to quantify cell types. Regions of Interest (ROIs) were then identified for the characterization of intra-tumoural and dense inflammatory regions. Image files including ROIs were analyzed in order to quantify cell-cell spatial relationships. Non-random patterns of immune cell distributions were identified using the Monte Carlo re-sampling method (500 iterations). Immune cell counts, densities, spatial relationships, and significant immune cell distributions were associated with clinical features by two-group comparison (Kruskal-Wallis p<0.001).
Result:
Our analysis generated 234 image files for analysis, including ROIs. Each field of view contained an average of 16,400 cells. The densities of intra-tumoural CD3+CD8+ and CD3+ T cells were significantly lower in recurrent cases, agreeing with literature reports. Following Monte Carlo analysis, non-random cell-cell spatial proximities emerged that were not observed at a cell density level. For example, an increased proximity of CD3+ T cells and B cells was observed in never smokers, while a decreased proximity was observed in ever smokers.
Conclusion:
While immune cell densities are of clinical prognostic importance, their spatial organization within the tumour architecture is of functional importance (e.g. the inhibition of cytotoxic T cell activity by adjacent PD-L1 expressing cells). In addition to cell densities, our platform is capable of quantifying cell-cell spatial relationships, thereby providing further information for clinical associations and for the identification of novel prognostic interactions. This automated quantification could be used to complement visual diagnostics and improve prognostic interpretation of histology specimens.
