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T. Agasthian

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    MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      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): T. Agasthian

      • Abstract
      • Slides

      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.

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