Start Your Search
MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 1
MO01.01 - Next generation sequencing of circulating tumour cells captured by antibody-independent enrichment and matched primary tumours/metastases in patients with non-small cell lung cancer (ID 3311)
10:30 - 12:00 | Author(s): J.K.C. Tam
Circulating tumour cells (CTCs) are considered the seeds of metastasis, and characterization of CTCs promises novel insights into metastasis, new targets for intervention, and less-invasive samples for assessing tumour status. CTCs however are rare in circulation and thus highly sensitive tools are required for their reliable capture and analysis. Antibody-based platforms using candidate gene-based approaches have begun to provide insights into CTCs. However tumour heterogeneity and the dependence of these methods on antigen expression has made antibody-independent methods of interest. The Clearbridge ClearCell System is a microfluidic-based platform that enables antibody-independent capture and retrieval of CTCs based on differences in the biomechanical characteristics of blood cells and CTCs. Next Generation Sequencing (NGS) has emerged as a tool to perform massive parallel sequencing of genomic regions with high efficiency and accuracy. The aim of this study was to perform NGS analysis of CTCs captured by antibody-independent methods, and their matched primary tumour or metastases samples, in patients with NSCLC.
Three matched CTC and primary tumour samples and three matched CTC and metastases samples were obtained from patients with NSCLC. Whole blood samples were also obtained from the patients for germline DNA. Five patients had adenocarcinoma and none of the patients had received targeted therapy prior to biospy of the metastatic lesions. CTCs were captured and retrieved from 2ml whole blood using the Clearbridge ClearCell System near the time of tumour sampling. DNA was extracted from CTCs, tumour tissue, and whole blood using the Qiagen QiaAMP DNA Micro Kit, DNAeasy Blood and Tissue kit , and Biorobot EZ1 workstation respectively. NGS was performed on the Ion Torrent PGM Sequencer using the AmpliSeq Comprehensive Cancer Panel targeted to 409 genes prominent in cancer. DNA variants were identified using Ion Torrent Software Suite v3.4, and pathway analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID).
After subtraction of DNA variants found in whole blood, the average number of variants in CTC, primary tumour and metastases samples was 283 (range: 110-470), 433 (70-1002), and 242 (81-166) respectively. The concordance in variants between CTC and primary tumour samples was 22% (15-29%) and between CTC and metastases samples was 29% (20-38%). Genes frequently mutated in matched CTCs and primary tumours/metastases included NOTCH2, AKT1, and RET. Pathway analysis of genes with DNA variants revealed an enrichment of genes involved in mTOR signalling in both CTC/primary and CTC/metastases samples. In CTC/metastases samples, pathways including the JAK-STAT and B-cell receptor pathways were additionally enriched.
Our results have highlighted a high level of genetic variability between CTCs and their matched tumours, reflective of high tumour heterogeneity. Preliminary analysis has identified genes and pathways with alterations in CTCs that could be potential targets for systemic treatment.
Only Active 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 or select "Add to Cart" and proceed to checkout.
P1.01 - Poster Session 1 - Cancer Biology (ID 143)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
P1.01-018 - Epidermal growth factor up-regulates CD151 protein expression to promote non-small cell lung cancer proliferation (ID 1780)
09:30 - 16:30 | Author(s): J.K.C. Tam
CD151 is a member of the tetraspanin superfamily. It is expressed on the surface of a variety of cell types and is involved in cellular processes such as cell motility, adhesion, proliferation, differentiation and signal transduction. CD151 is a positive regulator of tumor progression; metastasis, tumor cell growth and survival. It is over-expressed in various cancers, including non-small cell lung cancers (NSCLCs). The 5-year survival rate for NSCLC patients with CD151 gene-positive tumors has been shown to be lower than that of those with CD151 gene-negative tumors. Most CD151 studies to date have compared the cellular functions in the presence or absence of CD151 via over-expression and knockdown techniques respectively. However, the endogenous mechanisms by which CD151 protein is up-regulated in cancer cells remain unknown. The epidermal growth factor (EGF) is a distinct ligand for EGF receptors (EGFRs). Over-expression of EGFRs has been observed in more than 60% of NSCLCs. EGF is secreted by NSCLCs, causing the hyperactivity of EGFRs and the activation of its downstream signaling pathways to promote cell proliferation. In this study, we investigated the impact of EGF stimulation on CD151 protein expression in NSCLC cell proliferation.
A549 cells (a NSCLC cell line) were stimulated with increasing concentrations of EGF for 48hr and the CD151 protein abundance was measured via immunoblotting. To examine whether the effect of EGF on CD151 expression was a class-effect of growth factors, we also stimulated A549 cells with various stimulants (IL-1b, TNF-a, TGF-b1 and VEGF). To determine whether the effect of EGF on CD151 protein expression in NSCLC cells was cell-type selective, we examined the effects of EGF stimulation on CD151 protein abundance in other NSCLCs (H358, H1975), colon cancer (SW620) and breast cancer (MDA-MB-231) cell lines. Subsequently, silencing of CD151 via siRNA was carried out to investigate the effect of CD151 on EGF-stimulated A549 cell proliferation. Cell number was measured via trypan blue exclusion after 72hr of EGF stimulation, and phospho-ERK1/2 levels were examined via immunoblotting after 6hr of EGF stimulation.
In A549 cells, EGF induced significant up-regulation of CD151 protein expression and this effect was not a class effect of growth factors. The up-regulation of CD151 protein by EGF was observed in more than one type of NSCLCs and in other cancer cell types. Silencing of CD151 down-regulated EGF-stimulated increase in A549 cell number and this effect was dependent on ERK1/2 phosphorylation.
This is the first study to show that EGF up-regulates CD151 protein expression to promote NSCLC cell proliferation, possibly via the MAPK (ERK1/2) pathway.