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G. Reid



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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)

      11:10 - 11:15  |  Author(s): G. Reid

      • 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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    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): G. Reid

      • 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): G. Reid

      • 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.

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    P3.01 - Poster Session 3 - Cancer Biology (ID 147)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.01-019 - Analysis of Integrins in Malignant Mesothelioma (MM). (ID 1147)

      09:30 - 09:30  |  Author(s): G. Reid

      • Abstract

      Background
      Malignant mesothelioma (MM), strongly associated with exposure to asbestos, is a growing worldwide problem (1). This aggressive tumour is largely resistant to oncological treatments and new approaches to therapy are urgently needed. Integrins are a class of adhesion molecules composed of an α and a β chain. Combinations of 18 α and 8 β subunits form the 24 members of the integrin family. The αv subunit can dimerize with β1, β3, β5, β6 and β8. Aberrant expression of αv integrins was reported in MM, and the integrins αv β3 and αv β5 have been implicated in tumour progression and metastasis. We have investigated the expression and function of αv integrins in MM cell lines and the effect of gene knockdown on cell invasion.

      Methods
      Expression of the integrin (ITG) genes was analysed by qPCR in 7 MM cell lines. Expression of the heterodimers was determined by Western blot, immunofluorescence and immunocytometry (monoclonal antibodies kindly provided by Simon Goodman, 2). In addition, we knocked down the genes potentially involved in tumour progression (ITGB3 and ITGB5) and analysed the in vitro 2D and 3D invasiveness with an agarose spot invasion assay (3) and MM spheroids.

      Results
      All 7 MM cell lines showed high ITGB1 expression, moderate ITGB5 expression, and a general low ITGB6 and ITGB8 expression. ITGB3 was expressed in one cell line, which accordingly had high αv β3 expression. ITGB3 knockdown of this cell line resulted in suppression of invasion both in 2D and 3D cultures.

      Conclusion
      We have found evidence that integrin αv β3 may play a role in MM invasion. Presently, we are testing cilengitide, a peptide antagonist of integrin αv, in MM cell lines. References: 1. Delgermaa F, Takahashi K, Park EK, Le GV, Hara T, Sorahan T. Global mesothelioma deaths reported to the World Health Organization between 1994 and 2008. Bull World Health Organ. 2011, 89:716-724. 2. Goodman SL, Grote HJ, Wilm C. Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors. Biol Open. 2012, 1:329-340. 3. Wiggins H and Rappoport J. An agarose spot assay for chemotactic invasion. Biotechniques. 2010, 48:121-124.