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S. Lam
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MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 4
- Moderators:E. Brambilla, M. Noguchi
- Coordinates: 12/05/2016, 14:20 - 15:50, Stolz 2
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MA02.01 - Extracellular Vescicle miRNAs Regulate Gene Expression in Local Lung Adenocarcinoma Endothelial Cells (ID 4655)
14:20 - 15:50 | Author(s): S. Lam
- Abstract
- Presentation
Background:
Extracellular vesicles are small vesicles released from all cell types which can be used as a form of cell to cell communication. Recently these extracellular vesicles have been shown to play a key role in cancer development, growth, progression and angiogenesis. These extracellular vesicles are loaded with functional mRNAs, miRNAs and proteins which can be transferred from one cell to another. Extracellular vesicles have been known to enter neighboring cells including the surrounding stroma, and even enter biofluids. Our research shows that miRNAs transferred from lung adenocarcinoma cells through extracellular vesicles influence gene expression in endothelial cells and enhance their ability to form new blood vessels.
Methods:
Using 5 lung adenocarcinoma cell lines (H1395, H1437, H2073, H2228 and H2347) we isolated extracellular vesicles using differential ultracentrifugation. RNA was extracted from the extracellular vesicles as well as the cells from which they were derived and profiled for 742 miRNAs using the miRCURY LNA[TM] Universal RT miRNA PCR system (Exiqon) to identify miRNAs that were enriched by at least 4-fold in the extracellular vesicles. Tube formation assays were conducted on a commonly used endothelial cell line HMEC-1.
Results:
We found an enrichment of a select set of miRNAs within lung adenocarcinoma extracellular vesicles. These miRNAs have previously been identified as tumor suppressors: miR-142-3p, miR-143-3p, miR-144-3p, miR-145-5p, miR-150-5p, miR-223-3p, miR-451a, miR-486-5p, miR-605-5p in various cancer types. When extracellular vesicles are isolated from miR-143 and miR-145 over expressing adenocarcinoma lines they contain an increase in their over expressed miRNAs. When these miRNA enriched exosomes were incubated with HMEC-1 cells, we observed an increase in their ability to form new blood vessels and a decrease in the expression of CAMK1D in the endothelial cells. miR-143-3p and miR-145-5p were also found to be enriched in serum samples draining directly from lung adenocarcinoma tumors compared to arterial serum.
Conclusion:
Extracellular vesicles originating from lung adenocarcinoma cells can enter into endothelial cells and increase their ability to form new blood vessels through extracellular vesicle transfer of miR-145/miR-143 suggesting that this form of communication increases angiogenesis within lung adenocarcinoma tumors.
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MA02.03 - Expression of Oncofetal miRNAs Inactivates NFIB, a Developmental Transcription Factor Linked to Tumour Aggressiveness in Lung Adenocarcinoma (ID 5224)
14:20 - 15:50 | Author(s): S. Lam
- Abstract
- Presentation
Background:
Fetal and tumour development share striking similarities, such as intense cell proliferation, angiogenesis, increased cell motility, and immune evasion. Molecular regulators, including microRNAs (miRNAs), play important roles in both fetal lung development and in the malignant transformation of adult lung cells. Consequently, investigation of lung tumour biology in the context of lung development may reveal key regulatory mechanisms that tumours hijack from normal development, which potentially play critical roles in the pathology of lung cancer.
Methods:
131 pairs of non-small cell lung cancer (NSCLC) tumour and non-malignant lung tissues and 15 human fetal lung tissue samples were profiled by miRNA-sequencing. Genes controlled by the oncofetal miRNAs identified were first investigated by miRNA-Data-Integration-Portal (mirDIP) prediction, followed by luciferase-reporter assays. Associations between patient survival and mRNA expression of oncofetal miRNA-gene targets were evaluated in independent samples (>1,400 cases) across multiple NSCLC cohorts. Immunohistochemical analysis of oncofetal miRNA targets was performed on 96 lung adenocarcinoma (LUAD) specimens.
Results:
We describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, and identified numerous miRNAs that recapitulate their fetal expression patterns in NSCLC. Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, was identified as being frequently targeted by these oncofetal miRNAs. Overexpression of the oncofetal miRNA miR-92b-3p, significantly reduced NFIB levels in vitro. Concordantly, analysis of NFIB expression in multiple NSCLC cohorts revealed its frequent underexpression in tumours (~40-70%). This is in contrast with its recurrent oncogenic overexpression recently reported in SCLC. Low expression of NFIB was significantly associated with poorer survival in LUAD patients but not in squamous cell carcinoma patients, consistent with the functional role of NFIB in distal lung cell differentiation (i.e., precursor cells of LUAD). Furthermore, an NFIB-related gene signature was identified in LUAD tumours, comprising several well-known lung differentiation markers (e.g., TTF-1, SFTPB, ABCA3). The underexpression of NFIB protein was ultimately validated in LUAD specimens, which also revealed that tumours presenting lower levels of this transcription factor are associated with higher grade, biologically more aggressive LUAD (invasive mucinous, micropapillary and solid subtypes).
Conclusion:
This work has revealed a prominent mechanism for the downregulation of NFIB, a transcription factor essential for lung differentiation, which we found to be associated with aggressive phenotypes of LUAD and consequently, poor patient survival. Restoration of NFIB expression, specifically in LUAD, has the potential to induce lung cell differentiation and thereby reduce tumour aggressiveness.
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MA02.08 - Deregulation of Cis-Acting Long Non-Coding RNAs in Non-Small Cell Lung Cancer (ID 6303)
14:20 - 15:50 | Author(s): S. Lam
- Abstract
- Presentation
Background:
Lung cancer remains the cause of the most cancer-related deaths each year, with a 5 year survival rate of less than 17%. Targeted therapeutics have been developed against drivers of the lung adenocarcinoma (AC) subtype, but are relevant only to the proportion of patients harbouring these genetic aberrations, emphasizing the need to explore alternative mechanisms of AC development. Natural antisense transcripts (NATs) are long non-coding RNA (lncRNA) products expressed from the opposite strand of coding mRNAs. NATs can function in cis or trans to regulate the transcriptional activity of their cognate gene partner in either a positive or negative fashion. Here we take a novel approach to identify cis- NATs deregulated in lung AC, and explore the function of these genes with regards to their protein coding partner genes.
Methods:
We performed RNA-sequencing on a set of 36 lung AC and matched non-malignant lung tissues. A sign-rank test was used to identify NATs and partner genes with significantly altered expression between tumor and matched normal tissues. These findings were validated in an external dataset of 50 lung AC tumors with matched non-malignant tissue obtained from The Cancer Genome Atlas (TCGA). Survival analysis was performed using a Cox Proportional hazard model, as well as the log-rank method.
Results:
Analysis of Illumina Hi-seq data from TCGA revealed the majority (79%) of deregulated sense-antisense partnerships observed in AC displayed concordant regulation. However, several discordant cis-NAT pairs were identified including an antisense to OPA INTERACTING PROTEIN 5 (OIP5), a gene required for chromatin segregation, as well as an antisense to HIGH MOBILITY GROUP A1 (HMGA1) a gene involved in the metastatic progression of many cancer types. Both the antisense to OIP5 (OIP5-AS1) as well as the antisense to HMGA1, (HMGA1-AS1) were significantly underexpressed in AC, while we find the overlapping protein coding partner genes to be significantly overexpressed, suggesting that these genes may negatively regulate their sense counterparts. In addition both OIP5 and HMGA1 are significantly associated with 5-year survival. Patients with higher expression levels of either of these genes had a significantly shorter overall survival time than patients with low expression levels, highlighting the potential clinical importance of these genes.
Conclusion:
This study characterizes the landscape of antisense expression in AC and highlights novel mechanisms of oncogene regulation through natural antisense transcripts. Characterizing these oncogene regulatory mechanisms could uncover therapeutic intervention points and further our understanding of AC biology.
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MA02.09 - Long Non-Coding RNA Expression from Pseudogene Loci as a Novel Mechanism of Cancer Gene Regulation (ID 6287)
14:20 - 15:50 | Author(s): S. Lam
- Abstract
- Presentation
Background:
The advent of next generation sequencing has lead to the discovery of the functional importance of non-coding RNAs (ncRNAs) in a wide variety of cellular processes, and these genes can be exploited by tumours to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional parent genes but retain high sequence homology. Long non-coding RNAs (lncRNAs) have been shown to regulate protein-coding genes; however, complex folding patterns make lncRNA function difficult to predict. 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. The biological impact of this mechanism has not been investigated in lung adenocarcinoma (LUAD). We hypothesize that expression changes in lncRNAs expressed from pseudogene loci can affect the expression of corresponding protein-coding parent genes in trans, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumourigenesis.
Methods:
We analysed RNA-seq data from 50 LUAD with matched non-malignant tissue obtained from the TCGA for both protein-coding and non-coding gene expression. Significantly differentially expressed lncRNAs located within pseudogene loci were identified by sign-rank test (p<0.001). Mann Whitney U-tests were used to identify lncRNA-parent gene pairs which significantly correlated expression, and survival analysis was performed using a Cox proportional hazard model.
Results:
Our analysis has identified 172 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD. Remarkably, many of these lncRNAs were expressed from the loci of pseudogenes related to known cancer genes. One of these lncRNAs, CTD-2583A14.8, was expressed from a pseudogene to ubiquitin-conjugating enzyme E2C (UBE2C), which regulates tumor growth, apoptosis, and angiogenesis through phospho-ERK1/2. We find CTD-2583A14.8 as well as the UBE2C parent gene to be significantly upregulated in LUAD tumours compared to matched normal tissue. Furthermore, tumours with higher levels of CTD-2583A14.8 have significantly higher levels of UBE2C expression than tumours with low levels of CTD-2583A14.8, indicating that CTD-2583A14.8 may positively regulate UBE2C in trans.
Conclusion:
Here we show expression of lncRNAs within pseudogene loci is deregulated in LUAD, and can correlate with the expression of their protein-coding counterparts. Many of these genes associated with this putative lncRNA-pseudogene-protein-coding axis have previously been implicated in cancer. Therefore, this represents an alternative mechanism of cancer gene deregulation, and may represent novel therapeutic intervention points for the treatment of LUAD.
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-052 - Signal Regulatory Protein a (SIRPA): A Key Regulator of the EGFR Pathway Demonstrates Both Tumor Suppressive and Oncogenic Properties (ID 6061)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
The epidermal growth factor receptor (EGFR) signaling pathway is one of the most frequently deregulated pathways in non-small cell lung cancer. While targeted therapy prolongs survival in patients harbouring EGFR mutations, resistance to treatment eventually develops in all cases. As multiple genetic and epigenetic alterations are known to disrupt signaling pathways, the objective of this study is to perform a multidimensional analysis of signaling pathways to identify alterations essential to tumorigenesis that are overlooked when assessing a single genomic dimension.
Methods:
Multidimensional integrative analysis of copy number, DNA methylation, and gene expression profiles of 77 lung adenocarcinomas and matched non-malignant tissues identified Signal Regulatory Protein A (SIRPA) as a novel candidate tumor suppressor gene. Following validation of genomic findings in multiple external data sets, the tumor suppressive effects of SIRPA were assessed in vitro and in vivo with a panel of lung cancer cell lines.
Results:
SIRPA negatively regulates receptor tyrosine kinase signaling through activation of the protein phosphatases SHP1 and SHP2 and was found to be underexpressed in 70% of lung tumours, ranking it in the 95[th] percentile of altered genes within the EGFR pathway. Immunohistochemistry (IHC) confirmed reduced protein expression in tumors, which was found to correlate with EGFR mutation and adenocarcinoma histology. In vitro, SIRPA knockdown promoted migration while simultaneously inducing a dramatic senescent phenotype, suggesting SIRPA may act as a barrier to tumorigenesis. This phenotype is dependent upon upregulation of the CDK inhibitor p27, which hypophosphorylates RB leading to cell cycle blockade and reduced tumor growth in vivo. Importantly, increased expression of p27 resulted in mis-localization into the cytoplasm where it is known to promote an invasive phenotype. Inhibition of p27 confirmed previous findings and emphasized the importance of this pathway in lung tumorigenesis. Surprisingly, overexpression of SIRPA increased cell growth and migration, suggesting SIRPA may also possess oncogenic properties due to its regulation of multiple signaling pathways. Overexpression of SHP2 following ectopic expression of SIRPA promotes migration through the inhibition of focal adhesions. This phenotype is abrogated upon siRNA knockdown of SHP2.
Conclusion:
SIRPA is an important player in lung tumor biology, capable of acting as both an oncogene and tumor suppressor due to its ability to regulate multiple signaling pathways. Due to the complex nature in its signaling, future work should focus on elucidating how the timing of alterations to SIRPA affects tumorigenesis to design treatment strategy.
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P1.02-073 - Characterizing the Genomes of Lung Adenocarcinomas from Never Smokers Reveals SHPRH as a Novel Candidate Tumour Suppressor Gene (ID 4772)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
Approximately 15 to 25% of lung adenocarcinomas (LAC) arise in never smokers. They develop through mechanisms distinct from those that affect smokers and are associated with unique histological and molecular characteristics. A significant fraction of LACs in never smokers do not have mutations in known oncogenic driver genes such as EGFR/ALK/KRAS. Furthermore, mutations in oncogenic driver genes appear to be insufficient for tumorigenesis, suggesting that additional alterations are required.
Methods:
To address these issues, we used whole-exome sequencing to comprehensively study 15 LACs from never smokers - seven “triple negative” tumors (with normal EGFR/ALK/KRAS) and eight EGFR-mutant tumors - with the goal of identifying novel mutant genes in these subsets. To identify mutated genes that confer a selective advantage a multistep approach was used to filter variants based on gene expression level, background mutation rate and gene size. Targeted sequencing of 180 genes in the original 15 and an extended panel of 85 tumor/normal pairs validated these alterations and indicated their prevalence in LAC. Sequence data was integrated with copy number and gene expression levels to determine mechanisms, and consequences, of gene disruption. Animal and cell models were used to functionally validate identified genes of interest and explore their role in LAC biology.
Results:
32 unique genes demonstrated significant evidence of conferring a selective advantage including known oncogenes (EGFR/ERBB2/MET) and tumor suppressor genes (p53/RB1/ATM). In addition, RNA-seq revealed fusions involving RET or ROS1 in one tumour each. The variations in MET consisted of truncating and splice-site mutations that we are currently investigating in transgenic mouse models. Pathway analysis indicated frequent mutation in genes implicated in PI3-kinase signaling, RNA splicing and histone modification. Importantly, we identified the hemizygous and homozygous loss of multiple genes from chromosome arm 6q - a genetic locus associated with familial lung cancer susceptibility - including a novel candidate tumor suppressor gene, SHPRH, based on its high frequency of biallelic disruption. SHPRH is an evolutionarily conserved E3-ligase that mediates crucial processes related to DNA repair. We found that SHPRH silencing increased transformation of normal lung cells, increased DNA damage and induced cell cycle changes while SHPRH inhibition sensitized LAC cells to topoisomerase II and PARP inhibitors.
Conclusion:
SHPRH inactivation may induce genetic alterations that cooperate with mutations in driver oncogenes to promote LAC development. Together, this work will expand our understanding of LAC initiation and progression in never smokers and may offer new biomarkers for response to therapy.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 4
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-022 - A PIWI-Interacting RNAs Co-Expression Networks as a Prognostic Factor in Lung Cancer (ID 5812)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
PIWI-interacting RNAs (piRNAs) are small (24-32 nucleotides) non-coding RNAs. Their functions, widely conserved across species, are associated to epigenetic control of gene expression and maintenance of genomic stability by the repression of mobile elements. In humans, >23,000 piRNAs are known, showing tissue-specific expression patterns. While the aberrant expression of individual piRNAs has been identified in some cancer types, the role of piRNA co-expression networks in the development of lung tumors and their utility as molecular markers remains unexplored. By analyzing over 7000 piRNA transcriptomes from human tumors and non-malignant tissues, we have identified lung cancer (LC) specific expression networks associated with clinically-relevant tumor features and patient prognosis.
Methods:
We developed a custom small-RNA sequence analysis pipeline to generate >7,000 human piRNA transcriptomes. piRNA expression baseline was deduced from 6,378 piRNA transcriptomes (non-malignant/tumors) from 11 organ sites. In lungs, we analyzed 1,082 tumors and 209 non-malignant samples from two cohorts: BC Cancer Agency (BCCA) and The Cancer Genome Atlas (TCGA). Network analysis was performed using the weighted gene co-expression network analysis (WCGNA). We evaluated tumour aggressiveness by considering correlation to several clinical parameters, including stage, number of mutations, nodal/distant metastasis, and overall/disease-free survival. piRNA survival signatures were identified using a Cox Proportional Hazard model.
Results:
A subset of piRNA showed robust expression in somatic tissues. Expressed piRNAs display organ-specific patterns and mainly map to coding transcripts, suggesting a role in regulation of gene expression. In lungs, 204 piRNAs were consistently expressed in both LC cohorts. Tumor piRNA expression profiles are markedly different from their non-malignant counterparts (133 piRNAs were differentially expressed). The patterns differ between the adenocarcinoma and squamous cell carcinoma, and were influenced by smoking status. Network-based analysis identified piRNA expression changes in two modules of piRNAs are associated with aggressiveness tumor features, such as increased number of mutations, tumor size and nodal metastasis. Finally, combined expression of piRNAs define signatures associated with patient overall and recurrence free survival.
Conclusion:
We provide evidence of somatic, tissue-specific human piRNA expression. In lungs, aberrant expression patterns are associated with well-established etiological factors of cancer and seem to contribute to lung cancer subtype-specific biology. We discover that specific piRNA-based expression patterns characterize aggressive lung tumors and also exhibit prognostic value. The unique expression patterns of piRNAs offer an opportunity to better understand lung cancer-specific biology as well as develop novel prognostic markers for clinical application.
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P2.01-024 - Expression of miR-106 Paralogs Improves Prognostic Value of Mesenchymal Signatures but Only miR-106b Promotes Invasiveness (ID 6250)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
Improved understanding of the molecular mechanisms driving lung cancer progression can lead to novel therapeutic strategies to improve the currently poor patient treatment outcome. Deregulation of microRNA (miRNA) expression in malignant cells activates molecular pathways that drive tumor progression such as epithelial-mesenchymal transition (EMT). We identify miRNA paralogs, miR-106a and miR-106b, to be elevated in metastatic lung adenocarcinoma (LUAD). We assess whether these two highly similar miRNAs share the same functions in vitro, and measure how their elevated expression increases invasiveness or induces EMT in LUAD tumor.
Methods:
MiRNA expression was obtained from small RNA sequencing data derived from clinical primary LUAD specimens and paired non-malignant tissues (60 localized, 27 with lymph node invasion). Non-invasive, epithelial LUAD cell lines with low endogenous miR-106a/b levels were transfected and co-transfected with overexpression vectors for miR-106a and miR-106b. Invasiveness of experimentally-modulated tumor cells was assessed in vitro by Boyden chamber assay and in vivo using a zebrafish model, and expression of EMT markers was determined by Western Blot. Predicted miRNA targets were identified using mirDIP portal. To identify putative genetic mechanisms of mir-106a/b overexpression, DNA copy number, methylation, and Gene Set Enrichment Analysis (GSEA) were performed. Clinical associations were computed in an independent cohort of TCGA LUAD samples.
Results:
Both miR-106 paralogs were significantly overexpressed in LUAD samples with lymph node invasion. However, increased expression of miR-106b alone or together with miR-106a, but not miR-106a alone, enhanced metastatic phenotypes, and correlated with increased mesenchymal and decreased epithelial marker expression. Predicted targets include EP300, a transcriptional activator of E-cadherin, and members of the TGFβ signaling pathway. Copy number and methylation status did not correlate with miRNA expression; however, GSEA analysis revealed enrichment of E2F transcription factor targets in LUAD with high expression of either miR-106 paralogs. Furthermore, expression of miR-106 paralogs was significantly positively correlated with E2F1 and E2F2, suggesting that upstream regulation by E2F is a potential mechanism. Interestingly, miR-106a and miR-106b expression was associated with poor survival and advanced stage when stratified by mesenchymal marker vimentin.
Conclusion:
Although both miR-106a and miR-106b are overexpressed in metastatic LUAD, the strongest prognostic association was found in LUAD with a mesenchymal expression signature and high expression of both miRNAs. Our cell models suggest that miR-106b may play a direct role in EMT, with miR-106a influencing tumor progression via alternative mechanisms. Inhibition of one or both of these miRNAs may provide a strategy for treating advanced stage disease.
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P2.01-037 - Molecular Biology Underlying COPD and Lung Cancer Converge on FOXM1 Network (ID 5773)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease associated with an up to 10-fold increased risk of lung cancer (LC). COPD and LC share common etiologies including genetic susceptibilities and risk factors, such as smoking. This study systematically characterizes the molecular overlap between COPD and LC.
Methods:
Small airway gene expression data was obtained from subjects with spirometry measures (n=267) (GSE37147). Genome-wide, multi-omics data for lung adenocarcinoma (LUAD) tumor and non-malignant lung tissues from two cohorts (TCGA, n=515; BCCA, n=90) was analyzed. Weighted correlation network analysis (WGCNA) was applied to identify clusters (modules) of highly correlated genes across airway expression profiles. Combined module expression (eigengene scores) were used to: 1) identify modules negatively associated with FEV~1~ and 2) calculate module preservation in lung tumors. Signaling network, pathway and gene ontology analyses were performed using IID, pathDIP, ClueGo and PARADIGM. Known and predicted protein-protein physical interactions (PPIs) were obtained from IID. Network analysis and visualization was performed in NAViGaTOR.
Results:
A module of 31 genes significantly co-expressed across small airways was negatively associated with FEV~1~ and preserved in LUAD tumors. Genes in this module were enriched in functions associated with cell cycle progression, and known and/or predicted to physically interact in the protein complex critical to mediating G2/M progression. The forkhead transcription factor FOXM1 network was the most highly perturbed entity across 515 LUAD tumors. FOXM1 is an essential mitotic protein, known to regulate expression of genes involved in cell cycle progression, as well as stress response to ROS and DNA damage, angiogenesis and metastasis. COPD-related airway mRNA changes and genes highly altered at the DNA and mRNA level in LUAD tumors directly converge on the FOXM1 regulated mitotic complex proteins and/or FOXM1 transcription factor network.
Conclusion:
FOXM1 is overexpressed in multiple cancer types where it is correlated with poor prognosis and oncogenic transformation of epithelia through induction of genomic instability. The convergence of COPD and LUAD changes on this network may underlie increased LC risk in COPD patients, warranting further exploration as a target for COPD treatment and/or LC prevention or treatment.
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P2.01-065 - Quantification of Tumour-Immune Cell Spatial Relationships in the Lung Tumour Microenvironment Using Single Cell Profiling (ID 6104)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
How clinical-genomic features of the lung tumour microenvironment (TME) influence immune-checkpoint-blockade therapy is not well understood. Immunohistochemistry (IHC) is necessary to decipher cell-cell relationships that cannot be observed by bulk tumour profiling. In this pilot study, we assess whether immune cell phenotypes and spatial relationships differ between lung adenocarcinoma (LUAD) from smokers/non-smokers, KRAS/EGFR mutation, or with stage and tumour size using a novel multicolour IHC quantitative pathology method that enables in situ single cell profiling within the TME.
Methods:
Two consecutive sections from 21 cases of LUAD were stained with multicolour IHC panels to assess immune cell composition (CD8, CD3, CD79a) and T-cell exhaustion (CD8, PD1, PDL1). Hyperspectral images were captured as directed by a pathologist and analyzed using software developed in-house. The software segments individual cell boundaries based on haematoxylin stain. IHC stain positivity thresholds were applied based on intensity. Tumour and immune cells were classified into groups based on IHC staining. Interactions between specific groups were quantified by assessing the frequency and variance of the spatial relationship of each group vs. all other groups. Voronoi tessellation, based on cell centres, was used to define “next to”. Group counts and relationships were then compared with clinical features using a Student’s t-test or Kruskal-Wallis test.
Results:
A greater number of cells expressed PDL1 in KRAS+ LUAD. While the total number of CD8+PD1+ T-cells did not differ between KRAS+ and EGFR+ LUAD, there was an observed increased proximity between PDL1+ cells and CD8+PD1+ T-cells in KRAS+ LUAD. In EGFR+ LUAD, CD8+ T-cells that did not express PD1 were primarily localized in PDL1 negative regions. Both EGFR+ LUAD and never smokers harbored a higher proportion of CD8- T-cells and CD3-CD8+ immune cells. Both immune cell types were frequently localized in clusters with CD8+ T-cells. KRAS+ LUAD and smokers had increased B-cell counts. No significant associations of PD1 and PDL1 expression were found with stage; however, there was a statistically significant increase in proximity between varied immune cell types as stage and tumour size increased.
Conclusion:
Our method enabled identification of specific cell-cell spatial relationships within LUAD that are associated with smoking history and KRAS/EGFR mutation. Despite limited sample size, we observed an increased proximity between PDL1+ cells and CD8+PD1+ T-cells in KRAS+ LUAD. TME single cell profiling and cell sociology is a promising method to improve stratification of patients for immune-checkpoint-blockade therapies and opens new avenues to explore the complex cell-cell interactions within the TME.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-049 - ELF3 Overexpression Leads to Oncogenic Reprogramming of Protein Interactions Exposing Therapeutically Actionable Targets (ID 5807)
14:30 - 15:45 | Author(s): S. Lam
- Abstract
Background:
Emerging evidence has implicated ELF3 involvement in cancer signaling pathways. To determine the biological basis to pursue ELF3 as a novel therapeutic target, we investigated the role of ELF3 in lung adenocarcinoma (LUAD). Using a multi-omics approach in two independent cohorts of LUAD we (a) discover genetic mechanisms driving aberrant expression of this oncogene, (b) identify the protein-protein-interaction (PPI) partners of ELF3, and (c) determine the specific functions of ELF3 in LUAD using model systems.
Methods:
Comprehensive, multi-omic data was collected from the BC Cancer Research Centre (BCCRC), The Cancer Genome Atlas (TCGA), and several mouse models of LUAD tumourigenesis. ELF3 cellular localization was visualized by immunofluorescence. ELF3 knock-down and overexpression was achieved by lentiviral vector delivery for in vitro and in vivo assays. Physical protein-protein interaction (PPI) networks obtained from IID were overlaid onto cancer and non-malignant gene expression data from TCGA and 11 restructured datasets from Gene Expression Omnibus. PPIs were interrogated to investigate malignancy-associated ELF3 interactions. Pathway analysis was performed using pathDIP. Survival analysis was performed using the log-rank method.
Results:
ELF3 was significantly overexpressed in both cohorts, remarkably in >70% of cases (p=1.64E-21). However, mutation of known upstream regulators was not sufficient to explain the frequency of ELF3 overexpression. Instead, the ELF3 locus underwent frequent (>80%) genetic alteration including focal amplification and promoter hypomethylation, which corresponded with increased expression. ELF3 was predominantly localized to the nucleus, consistent with its transcription factor function. Analysis of PPI networks indicated highly LUAD-specific ELF3 interactions whereby loss and gain of interactions lead to reprogramming of LUAD transcriptional networks, including loss of TNFα pathway, and gain of TGFβ pathway, PI3K pathway, and translesion (DNA repair) pathway interactions. Furthermore, EGFR, KRAS, and MYC transgenic models of LUAD tumourigenesis all displayed a marked increase (6 to 8-fold) in ELF3 expression signifying its importance to LUAD of varied genetic backgrounds. In culture, ELF3 regulated proliferation, viability and anchorage-independent growth. In animal models, ELF3 knock-down cells underwent negative clonal selection, suggesting ELF3 expression is beneficial to tumour growth. Clinically, high expression of ELF3 was associated with poor survival regardless of tumour stage.
Conclusion:
Overexpression of ELF3 reprograms protein-protein-interactions in LUAD leading to the activation of cancer-specific pathways, and producing oncogenic phenotypes. Depletion of ELF3 with shRNAs reverses tumour cell growth, suggesting ELF3 is a promising therapeutic target. In addition to ELF3, interruption of cancer-specific PPIs also represents a therapeutically actionable strategy.
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SC13 - Interaction of COPD and Lung Cancer - Consequences for Early Diagnosis and Management (ID 337)
- Event: WCLC 2016
- Type: Science Session
- Track: Radiology/Staging/Screening
- Presentations: 1
- Moderators:A. Meert, K. Zarogoulidis
- Coordinates: 12/06/2016, 11:00 - 12:30, Stolz 1
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SC13.02 - Increased Risk for Lung Cancer in COPD (ID 6650)
11:00 - 12:30 | Author(s): S. Lam
- Abstract
- Presentation
Abstract:
Globally, chronic obstructive pulmonary disease (COPD) and lung cancer are among the top 5 causes of death. The two diseases share common risk factors such as tobacco smoking, outdoor and household air pollution. COPD is associated with a two to four fold increased risk for lung cancer independent of smoking.[1,2] COPD can be defined by symptoms, lung function criteria (forced expiratory volume in one second (FEV~1~) <80% predicted for age, gender and height, FEV~1~/ forced vital capacity (FVC) <0.7, or diffusing capacity <80%.[3,4] COPD has also been defined by computed tomography (CT) changes such as pulmonary emphysema by visual examination or by quantitative measurement (percent voxels < -950 HU based on a threshold of 4.8%) or air trapping from comparison between inspiratory and expiratory CT scans.[5,6] In the PLCO~m2012 ~risk prediction model[7], self-reported COPD was significantly associated with lung cancer risk (OR 1.45, 95% CI: 1.25-1.67). The area under the ROC curve (AUC) was 0.559. The AUC of the prediction model with and without a history of COPD was 0.800 and 0.799 respectively (M. Tammemagi, personal communication). A study in British Columbia examined the incremental value of pulmonary function test in 2,596 ever smokers above 40 years of age who had smoked ≥20 pack-years. One hundred and thirty-nine participants developed lung cancer after a median follow-up of 7.7 years.[8. ]Lower FEV~1~% increased the lung cancer risk for both men and women, but did so more strongly for men than in women (interaction P <0.001). FEV~1~% was found to substantially improve lung cancer prediction in a risk prediction model that included age, sex, education level, body mass index, family history of lung cancer and smoking. In this study, although CT detected emphysema was significantly associated with lung cancer in unadjusted analysis (OR 2.22, 95% CI: 1.17 – 3.78; P=0.012), in the fully adjusted model, neither emphysema nor a history of chronic bronchitis, which were significant in other studies, approached significance. A recent case-control study showed that only air trapping on quantitative CT imaging and FEV~1~/FVC <0.7 were independent predictors of lung cancer risk in a multivariable model.[9] Thirty-five percent of lung cancer cases and 55% of the controls had no evidence of COPD on spirometry or air trapping on quantitative CT. Forty-nine percent of lung cancer patients had FEV~1~/FVC >0.7. In the Pan-Canadian lung screening study of 2,537 ever smokers between the age of 50 to 75 with a 6 year lung cancer risk of ≥2% using a prototype PLCO prediction model, 50.6% of the participants had FEV~1~/FVC <0.7 and 42.8% had FEV~1~<80%. Among those who were found to have lung cancer, 63% had FEV~1~/FVC <0.7 and 54.5% had FEV~1~<80%. In those without lung cancer, the corresponding figures were 49.5% and 42% respectively. While COPD detected by pulmonary function test(s) and/or thoracic CT identify smokers at higher risk of lung cancer, a strategy to focus CT screening using these criteria may miss a significant proportion of lung cancers. Quantitative CT imaging of air trapping requires an inspiratory and expiratory CT with added cost and radiation exposure. The US Preventive Services Task Force recommended against screening for COPD in asymptomatic adults.[10 ]Whether identification of COPD by pulmonary function test and/or quantitative thoracic CT imaging can benefit a sub-population who would otherwise not meet the current criteria for lung cancer screening with CT or reduce the frequency and duration of follow-up CTs in a population based lung cancer screening program require further study. References 1. Skillrud DM, Offord KP, Miller RD. Higher risk of lung cancer in chronic obstructive pulmonary disease. A prospective, matched, controlled study. Ann Intern Med 1986;105:503-7. 2. Turner MC, Chen Y, Krewski D, Calle EE, Thun MJ. Chronic obstructive pulmonary disease is associated with lung cancer mortality in a prospective study of never smokers. Am J Respir Crit Care Med. 2007 Aug 1;176(3):285-90. 3. Vestbo J, Hurd SS, Agustí AG, Jones PW, Vogelmeier C, Anzueto A, Barnes PJ, Fabbri LM, Martinez FJ, Nishimura M, Stockley RA, Sin DD, Rodriguez-Roisin R. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2013 Feb 15;187(4):347-65. 4. de-Torres JP, Marin JM, Casanova C, et al. Identification of COPD patients at high risk of lung cancer mortality using the COPD-LUCSS_DLCO. Chest 2016; 149:936-42. 5. Lynch DA, Austin JH, Hogg JC, Grenier PA, Kauczor HU, Bankier AA, Barr RG, Colby TV, Galvin JR, Gevenois PA, Coxson HO, Hoffman EA, Newell JD Jr, Pistolesi M, Silverman EK, Crapo JD. CT-Definable Subtypes of Chronic Obstructive Pulmonary Disease: A Statement of the Fleischner Society. Radiology. 2015 Oct;277(1):192-205. 6. COPDGene CT Workshop Group, Barr RG, Berkowitz EA, Bigazzi F, Bode F, Bon J, et al. A combined pulmonary-radiology workshop for visual evaluation of COPD: study design, chest CT findings and concordance with quantitative evaluation. COPD. 2012 Apr;9(2):151-9. doi: 10.3109/15412555.2012.654923. 7. Tammemagi MC, Katki HA, Hocking WG, Church T, Caporaso N, Kvale P, et al. Selection criteria for lung-cancer screening. N Engl J Med 2013;368:728-36. 8. Tammemagi M, Lam S, McWilliams A, Sin D. Incremental value of pulmonary function and sputum DNA image cytometry in lung cancer risk prediction. Cancer Prev Res (Phila). 2011 Apr;4(4):552-61. 9. Schwartz AG, Lusk CM, Wenzlaff AS, Watza D, Pandolfi S, et al. Risk of Lung Cancer Associated with COPD Phenotype Based on Quantitative Image Analysis. Cancer Epidemiol Biomarkers Prev. 2016 Sep; 25(9):1341-7. 10. US Preventive Services Task Force (USPSTF). Screening for chronic obstructive pulmonary disease. US Preventive Services Task Force recommendation statement. JAMA April 5, 2016; 315(13):1372-1377. Supported by the Terry Fox Research Institute.
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