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M.B. Kirschner



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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 - 09:30  |  Author(s): M.B. Kirschner

      • 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: 2
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      P2.06-015 - Novel plasma proteins associated with prognosis in malignant pleural mesothelioma (ID 1337)

      09:30 - 09:30  |  Author(s): M.B. Kirschner

      • Abstract

      Background
      The search for novel biomarkers to define more successful and individual treatment approaches represent an important challenge for those involved in the care for patients with malignant pleural mesothelioma (MPM). In this exploratory study, we have systematically investigated the proteins present in plasma of MPM patients and correlated their levels with disease outcomes.

      Methods
      Plasma samples from twelve MPM patients (6 ‘short-’ and 6 ‘long-term’ survivors from parallel phase II studies investigating thalidomide) were used for proteomic analyses. Our series included samples from 9 patients with epithelial MPM and 3 patients with biphasic MPM. Plasma samples were immuno-depleted of the 14 most abundant proteins prior to labelling for isobaric tag for relative and absolute quantitation (iTRAQ) analysis using mass spectrometry. The most promising candidates and mesothelin were chosen for selected reaction monitoring mass spectroscopy (SRM-MS) quantification and enzyme-linked immunosorbent assay (ELISA) validation. Statistical analyses using T-Test of peak areas were used to identify proteins that were differentially expressed between the short- and long-term survivor groups.

      Results
      Median survival of short- and long-term survivors (1.2 and 38.3 months, respectively) differed significantly (p = 0.001). This was also the case for the neutrophil-to-lymphocyte ratio (NLR) that was significantly higher in the group of short-term survivors (p=0.03). Other baseline characteristics did not reveal major differences between the short- and long-term survivors. The total number of proteins identified was 226 (1% false discovery rate) in iTRAQ. A number of those were found to be differentially expressed between short- and long-term survivors (≥1.2-fold change; p≤0.05) by iTRAQ: selenoprotein P; tetranectin; insulin-like growth factor-binding protein 2 (IBP2); osteonectin (SPARC); platelet basic protein (CXCL7); and attractin. Mesothelin was assessed to validate the proteomic methodology: SRM-MS quantification was highly correlated with the MESOMARK ELISA values with a Pearson correlation of 0.82 (p=0.001). SRM-MS quantification revealed that the concentrations of attractin (p=0.02), tetranectin (p=0.003) and selenoprotein P (p=0.001) were higher in long-term survivors. In contrast, there was a trend for an increase in the concentration of SPARC (p=0.32), IBP2 (p=0.12) and CXCL7 (p=0.19) to be correlated with shorter survival. Furthermore, quantification by ELISA demonstrated an association between long survival and low concentration of SPARC (p=0.07) as well as high tetranectin (p=0.13).

      Conclusion
      We have demonstrated the feasibility of using the iTRAQ and SRM-MS proteomic techniques to investigate potential prognostic protein markers in plasma of MPM patients. Potential prognostic biomarkers worthy of further studies include SPARC and tetranectin and we plan to validate these in a larger clinical cohort.

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      P2.06-017 - Long non coding RNAs (lncRNAs) are dysregulated in malignant pleural mesothelioma (MPM) (ID 1524)

      09:30 - 09:30  |  Author(s): M.B. Kirschner

      • 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.