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B.T. Hennessy

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    MO04 - Lung Cancer Biology I (ID 86)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO04.03 - Chromosomal and mutational analysis of the cisplatin resistant phenotype in NSCLC cells (ID 3313)

      16:15 - 17:45  |  Author(s): B.T. Hennessy

      • Abstract
      • Presentation
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      Primary and acquired resistance to platinum agents such as cisplatin have become a major obstacle in the management of lung cancer patients, in particular non-small cell lung cancer (NSCLC). The availability of comprehensive genomic data on DNA copy number changes in cisplatin resistant NSCLC is limited, and little is known about the genes driving this chemoresistant phenotype. Detailed molecular portraits through high density genomic DNA arrays and genome wide mutation profiles will aid in understanding the molecular basis of individual responses to new molecular therapies.

      A panel of cisplatin resistant (CisR) NSCLC cell lines were recently generated and characterised in our laboratory. In this study, high resolution array-based comparative genome hybridization (aCGH) was performed on a panel of five CisR NSCLC cell lines to examine DNA copy number gains, losses and amplifications. Cellular DNA (500ng) and control DNA was differentially labelled with Cy3 and Cy5, respectively. Labelled test (4µg) and reference DNA were hybridised to a 12-plex 135,000 probe array (Roche NimbleGen) for 18 hours in a MAUI hybridisation station (BioMicro Systems) at 42°C. Fluorescent intensities were extracted and log 2 ratios calculated and normalized using NimbleScan Software (version 2.4). Chromosomal aberrations were identified using the CGH-segMNT algorithm (NimbleScan 2.4). A significance log 2 ratio threshold of <−0.25 for loss and >0.25 for gain was used to identify DNA copy number imbalances. For mutational analysis, Sequenom®, a mass-spectrometry-based SNP genotyping technology, was used to identify mutations in our panel of resistant cell lines. Using a literature search and the Catalogue of Somatic Mutations in Cancer (COSMIC) database, a mutation panel was identified for the detection of 547 frequently occurring and potentially clinically relevant mutations in 49 cancer-related genes. Some of these include KRAS, NRAS, BRAF, PIK3CA, MET, CTNNB1, STK11, AKT, and EGFR. Matrix chips were analysed on a Sequenom® MassArray MALDI-TOF system. Visual inspection and Sequenom® typer software were used to perform genotyping based on mass spectra.

      Using aCGH arrays, a number of gains, losses and amplifications of various chromosomes were found across a panel of CisR cell lines, relative to corresponding PT cells. The most frequently occurring of these chromosomal imbalances included gains, losses and homozygous deletions on chromosome 3 (MOR, A549, H1299), deletions and amplifications on chromosome 7 (H460, A549, H1299) and deletions and gains on chromosome 15 (MOR, A549, H1299) and chromosome X (MOR, H460, SKMES-1). Deletions on chromosomes 4, 6, 11, 12, 14, and amplification of chromosome 5, were also identified among the different CisR cell lines. The collation and analysis of data arising from mutation analysis of CisR cells using the Sequenom® platform are currently being completed.

      High-resolution mapping of chromosomal imbalances may offer potential in the identification of genes, including oncogenes and tumour suppressor genes, affected by these imbalances. These findings may further contribute to the delineation of the genomic profile of cisplatin resistant lung cancer, and offer perspectives for the identification of genes contributing to this disease phenotype and in assessing the response to new molecular treatments.

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