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Y.Y. Cheng



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    MO14 - Mesothelioma II - Surgery and Multimodality (ID 121)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Mesothelioma
    • Presentations: 1
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      MO14.07 - Elevated tumour expression of miR-210 is associated with short survival in malignant pleural mesothelioma patients undergoing extrapleural pneumonectomy (ID 1491)

      10:30 - 12:00  |  Author(s): Y.Y. Cheng

      • Abstract
      • Presentation
      • Slides

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive cancer with a median survival of around one year and a 5 year survival rate of less than 10%. A selected group of patients with a potentially resectable tumour mass and good performance status may be considered for extrapleural pneumonectomy (EPP). However the results of this form of treatment are variable. Several prognostic markers have been explored to assist with patient selection including histological subtype, neutrophil-to-lymphocyte ratio (NLR), calretinin and microRNA miR-29c* expression in tumour tissue. In the present study we used microarray profiling to identify other microRNAs which might have the potential to serve as a prognostic biomarker.

      Methods
      The study used 60 formalin-fixed paraffin embedded (FFPE) tumour tissues from MPM patients who underwent EPP and had sufficient tumour for RNA extraction, a series which had been previously used to assess the prognostic value of the NLR. MicroRNA microarray profiling was performed on RNA from the 8 patients with longest (median: 53.7 months) and the 8 patients with shortest (median: 6.4 months) survival. Candidate microRNAs were selected on a basis of biological (>2-fold difference) and statistical (p<0.05) significance, and selected candidates were independently validated in the initial profiling samples using TaqMan assay-based microRNA-specific RT-qPCR. Levels of validated candidates were then assessed by RT-qPCR in 44 additional tumour samples. Overall survival (OS) was calculated from date of EPP and date of death or last follow-up, with patients still alive at last follow-up censored. The median relative expression level of each candidate was used as cut-off to determine high and low expression for examination using the Kaplan-Meier method. Individually significant (p<0.05) variables were entered into a multivariate model together with the established risk factors age, gender, histological subtype, NLR.

      Results
      Microarray profiling identified 12 microRNAs with lower expression in long-term survivors and 4 microRNAs with higher expression in long-term survivors. None of the microRNAs with higher expression in long-term survivors could be validated using RT-qPCR. Of the microRNAs with lower expression in long-term survivors, miRs-30e, -93, -106b, -210, and -222 were validated by RT-qPCR in the same samples used for the profiling and found to be significantly different between long-term and short-term survivors. The expression levels of miR-30e and miR-210 showed a significant association with survival. MiR-30e: median OS of 24.2 months for low expression vs 13.3 months for high expression (p=0.03); miR-210: median OS of 24.2 months for low expression vs 13.7 months for high expression (p=0.008). In addition, both gender and histological subtype were significant prognostic factors using a univariate model. Multivariate analysis with age, gender, histological subtype, NLR and microRNA expression included as variables revealed that miR-210 was the only factor remaining significant (p= 0.006; hazard ratio: 0.41; 95% CI: 0.2-0.85).

      Conclusion
      This study has identified expression of miR-210 as a potential new prognostic factor for patients undergoing EPP. Further validation is needed, but this marker has the potential to assist in better selection of patients considered for radical surgery of MPM.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-006 - Targeted delivery of RRM1-specific siRNA leads to tumour growth inhibition in malignant pleural mesothelioma (ID 1508)

      09:30 - 16:30  |  Author(s): Y.Y. Cheng

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy with poor prognosis. MPM is typically recalcitrant to treatment and new therapies are urgently needed. Multiple genes involved in proliferation and metabolic activity are upregulated in MPM and these represent attractive targets for an siRNA-based therapeutic intervention.

      Methods
      We carried out an RNAi-based screen of 40 target genes previously shown to be upregulated in MPM to identify candidate genes with roles in cell growth and survival in MPM cell lines. Effects of target gene silencing were measured using standard in vitro proliferation assays. Lead candidates were further assessed with siRNA dose response experiments. The specificity of siRNA-mediated growth inhibition was confirmed by assessing gene knockdown by real-time qPCR and Western blotting. The effects of the most potent siRNAs on xenograft tumour growth were assessed in vivo by delivery using EGFR-targeted, siRNA-loaded, minicells.

      Results
      All 40 genes were effectively silenced, and for 6 genes (PLK1, CDK1, NDC80, RRM1, RRM2 and BIRC5) knockdown with 2 independent siRNAs resulted in significant growth inhibition over time in multiple cell lines. Dose response experiments revealed that siRNAs specific for RRM1 and RRM2 were the most effective at inhibiting growth with IC50 values in the low nanomolar range. Intravenous administration of RRM1 siRNA packaged in minicells targeted with EGFR-specific antibodies (2x10[9] minicells per dose, 4 times per week for 3 weeks) led to consistent and dose-dependent inhibition of MPM tumor growth compared with treatment with an inactive siRNA. Reducing the dose and number of administrations did not reduce growth inhibition; as little as 1x10[9] minicells administered once a week were sufficient to completely inhibit MPM tumour growth.

      Conclusion
      RRM1 is an attractive target for siRNA-based inhibition, and siRNA delivery with EGFR-targeted minicells represents a novel therapeutic approach for MPM.

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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-017 - Long non coding RNAs (lncRNAs) are dysregulated in malignant pleural mesothelioma (MPM) (ID 1524)

      09:30 - 16:30  |  Author(s): Y.Y. Cheng

      • Abstract

      Background
      Malignant Pleural Mesothelioma (MPM) is an aggressive disease, often diagnosed at an advanced stage. It is characterized by a long latency period and prior asbestos exposure. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates rarely exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) plays an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. The aims of this study were to characterize the expression and function of these lncRNAs in MPM.

      Methods
      To identify novel lncRNAs involved in MPM, microarray profiling was performed on five cell lines - the immortalized normal mesothelial cell line (MeT-5A) and four MPM lines (two epithelioid H28 and H226 and two biphasic MM05 and MSTO) using Invitrogen’s NCode lncRNA microarrays. These allow simultaneous assessment of mRNA and lncRNA content. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological (>3-fold difference) significance. Expression of high priority candidates were technically validated using RT-qPCR, and biologically validated in three independent test sets. Pathway analyses were performed to interrogate the relationship between lncRNA and mRNA expression. Cell proliferation and colony formation assays were used to investigate lncRNA function.

      Results
      Microarray profiling and real-time qPCR validation identified 9 lncRNA candidates with significant differential expression in MPM compared with normal mesothelial cells Validation in three independent test sets by RT-qPCR analysis demonstrated consistent up-regulation of four of these lncRNAs. Receiver Operating Curve analysis showed that two of these candidates were able to separate benign pleura and MPM with high sensitivity and specificity. In addition, high expression of AK054908 was associated with nodal metastases with lower levels of AK130275 and AF268386 observed in patients receiving induction chemotherapy. Cases with higher EF177379 levels also demonstrated a trend to improved survival. The majority of mRNAs co-expressed with candidate lncRNAs were associated with cellular and metabolic processes including cell cycle, cell death and apoptosis. In functional studies, siRNA knockdown of AK130275 showed suppression of cell growth and colony formation in MPM cells with moderate changes observed following knockdown of EF177379.

      Conclusion
      To our knowledge this is the first systematic study of lncRNA expression profiles in MPM. We have found that lncRNA expression profiles can distinguish malignant mesothelium from normal pleural tissue, and that some lncRNAs are associated with nodal metastasis and long term survival. We also demonstrate that lncRNAs have potential prognostic and diagnostic utility with functional roles in regulating cell growth. Further work is required to evaluate whether these lncRNAs are capable of differentiating mesothelioma from lung cancer and benign asbestos-related diseases, and to reveal their specific functions in MPM pathogenesis.