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K. Marini



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    MO15 - Novel Genes and Pathways (ID 89)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO15.11 - Using synthetic lethal screening to identify therapeutic targets for innately platinum resistant lung cancer (ID 2629)

      17:15 - 17:20  |  Author(s): K. Marini

      • Abstract
      • Presentation
      • Slides

      Background
      Although platinum-based chemotherapy is the standard of care for most cases of advanced lung adenocarcinoma, its effectiveness is limited by the frequent incidence of innate chemoresistance. As a result, response rates rarely exceed 20%, even though cis-platinum and carboplatin are highly effective in other settings such as small cell lung, ovarian and testicular cancers. We hypothesized that innate chemoresistance in lung adenocarcinoma is mediated by one or more signalling pathways dependent on the expression of a single gene, and that these pathways could ultimately be targeted therapeutically.

      Methods
      To address this question, we developed a synthetic-lethal high throughput siRNA screen using the innately resistant A549 lung adenocarcinoma cell line. Optimisation of the screen was performed using a siRNA death control (PLK1), which induced cell death in the absence of platinum, and a sensitization control (MTOR), which enhanced cell death only in combination with a sublethal concentration of carboplatin. These independent controls revealed that the screening protocol performed within acceptable limits of variability, quality and reproducibility as determined by Z’ factor analysis. Screening was then performed using a pool of four siRNAs targeting a single gene in conjunction with vehicle treatment, or with carboplatin.

      Results
      After screening siRNAs targeting the 720 kinases, 256 phosphatases and 4794 “druggable” targets of the human genome, we identified 50 candidate targets based on fold change difference between platinum and vehicle treatments, and statistical significance determined by multiple t-test corrected for false discovery rate. Preliminary pathway analysis revealed a highly significant enrichment for genes in previously identified pathways as well as novel pathways.

      Conclusion
      These data demonstrate that a synthetic-lethal approach can be used to identify therapeutic targets that could potentially sensitize lung adenocarcinoma to platinum-based chemotherapy.

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    P2.03 - Poster Session 2 - Technology and Novel Development (ID 151)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.03-006 - Determining Read Origin of Next-Generation Sequencing Datasets from Lung Cancer Xenografts (ID 2647)

      09:30 - 09:30  |  Author(s): K. Marini

      • Abstract

      Background
      Next-generation sequencing (NGS) studies in cancer are often limited by the amount, quality and purity of tissue samples obtained from patients. In this situation, primary xenografts have proven useful in providing preclinical models. Although xenograft lines are maintained in immunodeficient mice, we and others have shown that they retain important characteristics that are irreversibly lost in cell culture. Since the stromal component of xenograft tumors is derived from the host, the presence of mouse DNA and RNA has the potential to limit the use of these models for next-generation sequencing (NGS) analysis.

      Methods
      We prospectively addressed this question in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy that is almost always diagnosed using small biopsies or needle aspiration cytology. We first developed an in-silico strategy that separates human and mouse reads with at least 97% accuracy. We then compared NGS data from a series of primary xenograft models with clonally derived, stroma-free cell lines, and with published datasets derived from the same models.

      Results
      Starting with the NCI-H209 cell line as a reference sample, we show that low coverage whole genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. NGS analysis of exon-capture DNA revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene signatures, whereas mouse- transcript profiles correlated with published datasets from human cancer stroma.

      Conclusion
      Primary xenograft models may therefore be a useful NGS platform for cancers where tissue samples are limiting.