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MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 1
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): J.C. English
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.
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.
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).
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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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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): J.C. English
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.
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.
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.
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.