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ED13 - Treatment of Malignant Pleural Mesothelioma (ID 282)
- Event: WCLC 2016
- Type: Education Session
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:H. Lee Kindler, N. Van Zandwijk
- Coordinates: 12/07/2016, 11:00 - 12:30, Hall C1
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ED13.02 - Tissue-Based Biomarkers (ID 6496)
11:15 - 11:30 | Author(s): G. Reid
- Abstract
- Presentation
Abstract:
Introduction: Malignant pleural mesothelioma (MPM) is difficult to diagnose and accurate prediction of patient outcomes still relies on a range of clinical scores. Despite extensive efforts in the last decade, there are few tumour-based molecular markers that can accurately contribute to diagnosis and prediction of disease course. Recent reports describing the mutational and transcriptional landscape of MPM tumours have revealed a number of changes that may yield clinically useful biomarkers following further development and validation studies. Diagnosis: The definitive MPM diagnosis relies on a tissue biopsy and demonstration of invasion. Diagnostic markers consist of a combination the expression of mesothelial-specific proteins and absence of markers of adenocarcinoma. Recent advances have shown that the mutation of the tumour suppressor BAP1 leads to loss of nuclear staining, and that this is highly specific for discriminating mesothelioma from benign conditions. As in some cases MPM has neither BAP1 mutation nor loss of nuclear staining, sensitivity is lacking, but this can be improved by incorporating detection of CDKN2A genomic loss using FISH. Assessment of additional mutations and fusion genes recently identified in MPM may represent useful markers for future development. Characteristic changes in microRNA expression are present in MPM, and these form the basis of a highly accurate molecular test for the differential diagnosis of MPM from other tumours affecting the pleura. Prognosis: Clinical and pathological parameters remain the best predictors of disease outcome, and although some molecular markers have demonstrated prognostic significance, these are yet to be validated. Histopathological subtype is an accurate prognostic indicator, with the epithelioid subtype associated with significantly better outcomes than the non-epithelioid biphasic and sarcomatoid types. The variation within epithelioid tumours is well recognised, and epithelioid tumours with a pleomorphic morphology have poor prognosis, similar to patients with non-epithelioid tumours. Recent results from transcriptomic analyses have revealed subsets within epithelioid and non-epithelioid tumours which more accurately describe prognosis. These include the two-cluster C1/C2 classification system based on a 3 gene predictor, and the 4 clusters (sarcomatoid, epithelioid, biphasic-epithelioid and biphasic-sarcomatoid) derived from RNA-seq analysis. MicroRNA expression has also been linked to outcome. Early studies revealed prognostic significance of miR-29c-3p, with higher levels corresponding to longer survival. More recently, microRNA expression profiles differing between long and short survivors yielded a 6-microRNA score that predicted outcome in two surgical series. Whether TCGA data confirm these observations remains to be determined. In addition to RNA and protein biomarkers, the cellular composition of tumours influences patient outcomes. It is likely that the mix of cell types within tumour samples also contributes to biomarker expression, especially for RNA extracted from whole tumours. For some proteins, differential expression in the stromal and tumour compartments is of prognostic value, for example in the case of SPARC expression. The importance of the immune cell infiltrate was recently investigated in a large number of epithelioid samples revealing that greater numbers of tumour-infiltrating CD4+ and CD8+ T lymphocytes (TILs), as well as fewer tumour-associated macrophages (TAMs) of the M2-type correlate with survival. In addition, the ratio of the TAMs/TILs was also shown to predict outcome in epithelioid MPM. Other cell populations associated with vascular and lymphatic invasion are also linked to survival. Prediction: Unlike lung cancer, few actionable mutations are present in MPM that predict sensitivity to targeted agents, and clinical trials with these drugs have yielded disappointing results. Markers for single agent chemotherapy and the standard cisplatin/pemetrexed doublet have also been investigated in retrospective studies attempting to link patient outcomes with gene (mRNA and protein) expression and polymorphisms. Multiple reports have linked levels of TS protein, but not mRNA, to outcomes with pemetrexed-based chemotherapy. As expected from a multi-targeted agent, other levels of other proteins such as folypoly-glutamate synthase (FGPS) and the reuced folate carrier (RFC) were also associated with tumour response and patient outcomes. However, a subsequent study with a similar number of patients suggested that both TS and FPGS lack predictive value. With respect to DNA repair genes involved in cisplatin activity, ERCC1 and others have been evaluated, but results are again inconclusive. The picture is complicated by assessment of target genes in patients treated with two interacting agents (with or without subsequent surgery), and the true value of these genes awaits carefully controlled prospective analyses. The recent breakthrough success of immune checkpoint inhibiting antibodies targeting CTLA4 and the PD-1/PD-L1 axis in melanoma and lung cancer has seen these agents applied to MPM patients. With response rates of around 25% for PD-1 targeting antibodies pembrolizumab and nivolumab in MPM, new predictive markers are needed to improve patient selection and for health economics reasons. Although the Keynote trial included patients based on positivity of PD-L1 staining, PD-L1 status appears to have little value in predicting response rate. Ongoing research into immune cell involvement may shed more light on this. Future directions: Continuing research in this area should learn from limitations of the biomarker studies of the last decades to improve the search for useful molecular markers. Large prospective trials are needed to carefully evaluate predictive markers. Alternative approaches such as the analysis of live cell populations taken from fine-needle aspirates and investigation of circulating tumour cells and tumour-derived markers in the circulation (DNA, exosomes) may yield novel markers. Conclusions: Extensive research into tumour-based markers for MPM is gradually making progress. New markers to assist in diagnosis and prognosis have been identified, but the selection of accurate predictive markers has so far remained elusive. Next-generation sequencing has identified multiple new candidate markers requiring further investigation, and may provide breakthroughs in the future.
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MTE29 - Advances in Malignant Pleural Mesothelioma (Ticketed Session) (ID 322)
- Event: WCLC 2016
- Type: Meet the Expert Session (Ticketed Session)
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 07:30 - 08:30, Stolz 1
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MTE29.02 - Advances in Malignant Pleural Mesothelioma (ID 6593)
08:00 - 08:30 | Author(s): G. Reid
- Abstract
- Presentation
Abstract:
Epidemiology: MPM, representing around 90% of all mesothelioma cases diagnosed, is an aggressive tumour with a poor prognosis, and relatively few treatment options. The association of mesothelioma with asbestos exposure is well established. The latency period, the interval between first asbestos exposure to the diagnosis is long (around 40 years), and explains why in many instances the effect of banning asbestos from the workplace has yet to be seen. At the same time there is evidence accumulating that non-occupational asbestos exposure may significantly contribute to mesothelioma incidence [1] and it is most worrying that unrestricted use of this carcinogen is allowed in Russia and most Asian, African and South American countries. Unfortunately the multilateral treaty to promote shared responsibilities in relation hazardous chemicals (Rotterdam convention) has become paralyzed by the veto of asbestos producers and considering the rapid surge of asbestos consumption in developing countries the end of the mesothelioma epidemic is not in sight [2]. Molecular biology: Major efforts have been undertaken to explore the genomic alterations responsible for the development of malignant pleural mesothelioma. Recent next-generation sequencing efforts have confirmed the frequent loss of tumour suppressor genes identified in earlier studies. Deletion and loss of function mutation of CDKN2A, NF2 and BAP1 are common molecular events in MPM, but the overall mutational load tends to be lower in MPM than in lung cancer. Mutation, aberrant splicing, and gene fusions occur in additional genes such as SF3B1, TRAF7 and SETD2, but at lower frequency [3]. Expression analyses suggest that there are subgroups of tumours both within and between the traditional histopathological subtypes of MPM [3, 4], and this has potential implication for prognosis. Although still to be published, the results from a TCGA mesothelioma study paint a similar picture of the mutational and transcriptional landscape. Investigation of microRNA expression reveals a general downregulation of microRNAs with tumour suppressor activities. In addition to miR-31, frequently co-deleted with CDKN2A, the miR-15/16 family is consistently downregulated in MPM tumours. This family controls expression of targets such Bcl-2, CCND1 and VEGF, and thus plays a role in the regulation of proliferation, apoptosis and angiogenesis. Recent data suggest that these microRNAs also play role in controlling the levels of PD-L1 expression in MPM cells [5], targeted by immune checkpoint inhibitors. Treatment Options: MPM is notoriously refractory to localized and systemic treatment. Meta-analyses (multivariate analyses) of large series of patients confirm that the prognosis of the select group of patients able to undergo radical surgery is significantly better than without surgery [6]The debate about the extent of radical surgery has for some time been governed by the significant risks associated with radical surgery as noted in the MARS trial. Therefore, when radical multimodality treatment approaches are considered, it seems prudent to involve an experienced team at a high volume centre. While the important palliative role of radiotherapy in MPM has been accepted by the oncological community, consolidation radiotherapy after radical surgery [7] has not been shown to provide major benefits in terms of local control. To define the role of (intensity modulated) accelerated radiotherapy in MPM comparative studies are needed. The impressive data (median overall survival of 51 months) from the SMART study [8] combining pre-operative intensity modulated radiation therapy (IMRT) immediately followed by extra-pleural pneumonectomy in 62 patients MPM patients with epithelial histology suggests that such an approach may have the potential to become an alternative for induction chemotherapy followed by radical surgery. Almost every chemotherapy agent has been tested in MPM. Cisplatin, methotrexate, pemetrexed and the anthracyclines doxorubicin and daunorubiucin were most active, but single agent activity seldomly exceeded a 20% response rate. A systematic review of the chemotherapy literature carried out in the early 2000s concluded that combination therapy was likely to be more effective than single agent therapy [9]and shortly thereafter Vogelzang’s randomized comparison between cisplatin and cisplatin/pemetrexed confirmed cisplatin/pemetrexed as the new therapy standard. Thirteen years later this standard has been augmented by a large comparative French intergroup study revealing that the addition of bevacizumab to the cisplatin/pemetrexed standard is associated with a 2.7 months advantage in median overall survival [10]. However, it important to note that a not insignificant number of negative phase II and III studies with a range of inhibitors of growth factors including EGFR, VEGF and PDGF had preceded this positive result. Other targeted agents investigated in phase II and III studies including bortezomib, vorinostat, everolimus, and defactinib, the inhibitor of the NF2/mTOR/FAK pathway, have also failed to show notable activity in pre-treated MPM patients [11]. It has become clear that MPM is an immunogenic tumour type and the preliminary data showing responses after immune checkpoint (PD-L1) inhibition [12] seem to indicate that reversing the immunosuppression induced by advancing disease is likely to represent a major step forward. However, monotherapy with Tremelimumab, inhibitor of CTLA-4 and considered active in phase II studies, failed to produce a survival benefit over placebo in 2[nd] and 3[rd] line, underlining the importance of comparative studies [13]. Independent research groups have reported ‘spontaneous’ regression of MPM, revealed a relation between infiltrating lymphocytes and plasma cells and prognosis and presented promising early clinical results with mesothelin-targeting antibodies [11]. Most recently dendritic cell vaccination combined with pulsed (metronomic) cyclophosphamide to deplete regulatory T cells resulted in prolonged tumour control in a limited group of MPM patients [14]. It is not excluded that targeting multiple compartments involved in immune surveillance will lead to increased efficacy. Early signs of efficacy of experimental treatment with tumour suppressive microRNAs packaged in minicells [15, 16] and the interaction between the microRNA 15/16 family and PD-L1 expression point to the complexity of immune checkpoint regulation and underlines the need for additional translational studies to unravel the resilient drug resistance mechanisms operable in MPM. 1. Marinaccio, A., et al., Malignant mesothelioma due to non-occupational asbestos exposure from the Italian national surveillance system (ReNaM): epidemiology and public health issues. Occup Environ Med, 2015. 72(9): p. 648-55. 2. Takahashi, K., P.J. Landrigan, and R. Collegium, The Global Health Dimensions of Asbestos and Asbestos-Related Diseases. Ann Glob Health, 2016. 82(1): p. 209-13. 3. Bueno, R., et al., Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations. Nat Genet, 2016. 48(4): p. 407-16. 4. de Reynies, A., et al., Molecular classification of malignant pleural mesothelioma: identification of a poor prognosis subgroup linked to the epithelial-to-mesenchymal transition. Clin Cancer Res, 2014. 20(5): p. 1323-34. 5. Williams, M., et al., Tumour suppressor microRNAs regulate PD-L1 expression in malignant pleural mesothelioma., in International Mesothelioma Interest Group (iMig) 2016. 2016: Birmingham 6. Linton, A., et al., Factors associated with survival in a large series of patients with malignant pleural mesothelioma in New South Wales. Br J Cancer, 2014. 111(9): p. 1860-9. 7. Stahel, R.A., et al., Neoadjuvant chemotherapy and extrapleural pneumonectomy of malignant pleural mesothelioma with or without hemithoracic radiotherapy (SAKK 17/04): a randomised, international, multicentre phase 2 trial. Lancet Oncol, 2015. 16(16): p. 1651-8. 8. de Perrot, M., et al., Accelerated hemithoracic radiation followed by extrapleural pneumonectomy for malignant pleural mesothelioma. J Thorac Cardiovasc Surg, 2016. 151(2): p. 468-73. 9. Berghmans, T., et al., Activity of chemotherapy and immunotherapy on malignant mesothelioma: a systematic review of the literature with meta-analysis. Lung Cancer, 2002. 38(2): p. 111-121. 10. Zalcman, G., et al., Bevacizumab for newly diagnosed pleural mesothelioma in the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS): a randomised, controlled, open-label, phase 3 trial. Lancet, 2016. 387(10026): p. 1405-14. 11. Schunselaar, L.M., et al., A catalogue of treatment and technologies for malignant pleural mesothelioma. Expert Rev Anticancer Ther, 2016. 16(4): p. 455-63. 12. Alley, E.W., et al., Clinical safety and efficacy of pembrolizumab (MK-3475) in patients with malignant pleural mesothelioma: Preliminary results from KEYNOTE-028. Cancer Research, 2015. 76(18): p. CT 103. 13. Kindler, H.L., et al., Tremelimumab as second- or third-line treatment of unresectable malignant mesothelioma (MM): Results from the global, double-blind, placebo-controlled DETERMINE study. Journal of Clinical Oncology, 2016. 34(15 (May Suppl)): p. #8502. 14. Cornelissen, R., et al., Extended Tumor Control after Dendritic Cell Vaccination with Low-Dose Cyclophosphamide as Adjuvant Treatment in Patients with Malignant Pleural Mesothelioma. Am J Respir Crit Care Med, 2016. 193(9): p. 1023-31. 15. Reid, G., et al., Clinical development of TargomiRs, a miRNA mimic-based treatment for patients with recurrent thoracic cancer. Epigenomics, 2016. 8(8): p. 1079-85. 16. Kao, S.C., et al., A Significant Metabolic and Radiological Response after a Novel Targeted MicroRNA-based Treatment Approach in Malignant Pleural Mesothelioma. Am J Respir Crit Care Med, 2015. 191(12): p. 1467-9.
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OA02 - Novel Targets and Biomarkers in Malignant Pleural Mesothelioma (ID 369)
- Event: WCLC 2016
- Type: Oral Session
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 3
- Moderators:L.E. Raez, M. Jakopovic
- Coordinates: 12/05/2016, 11:00 - 12:30, Stolz 2
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OA02.01 - The microRNA-15/16 Family Regulates Tumour Cell Growth via Fibroblast Growth Factor Signals in Malignant Pleural Mesothelioma (ID 5395)
11:00 - 11:10 | Author(s): G. Reid
- Abstract
- Presentation
Background:
Malignant pleural mesothelioma (MPM) is a highly aggressive, asbestos-related malignancy characterized by poor outcome and limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth and malignant behavior and their inhibition leads to reduced tumor growth. MicroRNAs (miRNAs) are conserved noncoding RNAs controlling gene expression via translational repression of target mRNAs. The miR-15/16 family is downregulated in MPM and has tumor suppressor functions. Several FGFs/FGFRs are predicted miR-15/16 targets. The aim of this study was to explore the link between the miR-15/16 and the FGF/R family in MPM.
Methods:
Gene and microRNA expression was determined by RT-qPCR or Taqman Low Density Arrays (TLDAs). Mimics were used for restoring microRNA expression. Stimulation or inhibition of FGF signals or bcl-2 was achieved by recombinant FGF2, siRNAs, or small-molecule inhibitors, respectively. A SYBR green-based proliferation assay and colony formation assays were used to monitor effects on cell growth.
Results:
Expression analysis showed a consistent downregulation of target FGF/FGFR genes after transfection with miRNA mimics. Restoration of miR-15/16 led to dose-dependent growth inhibition, which significantly correlated with sensitivity to the specific FGFR1 inhibitor PD166866. Re-expression of microRNAs in combination with FGFR knock-down or pharmacological inhibition resulted in reduced activity, indicating target competition. Combined inhibition of the FGF-axis and bcl-2, another established target of miR-15/16, resulted in enhanced activity. Treatment with recombinant FGF2 further reduced mature as well as pri-microRNA levels and also could prevent/reduce growth inhibition by mimics, but only when added within 24 hours after transfection. TLDA screens after stimulation/inhibition of FGF signals identified regulation of several other miRNAs involved in pathways relevant for tumour growth and aggressiveness.
Conclusion:
Our data shows that the post-transcriptional repression of FGF-mediated signals contributes to the tumour-suppressor function of the microRNA-15/16 family. Impairing hyperactivated FGF signals as well as the anti-apoptotic protein bcl-2 through the restoration of this miRNA family might serve as a novel therapeutic strategy in mesothelioma.
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OA02.03 - Circulating Fibroblast Growth Factor 18 is Elevated in Malignant Pleural Mesothelioma Patients - A Multi-Institutional Study (ID 5988)
11:20 - 11:30 | Author(s): G. Reid
- Abstract
- Presentation
Background:
Malignant pleural mesothelioma (MPM) is a rare but devastating malignancy. Despite the search for new promising treatment approaches, the outcome for most MPM patients remains dismal. Therefore, the identification of novel biomarkers is urgently needed in order to identify patients with a better prognosis and to support personalized therapeutic decisions. In our previously published study, we were able to show that fibroblast growth factor 18 (FGF18) is overexpressed in MPM tissue specimens and cell models. The objective of this study was the evaluation of FGF18 as a circulating biomarker in MPM.
Methods:
Plasma was collected from 107 MPM patients at the time of diagnosis or before surgical resection. Samples were included from the Medical University of Vienna, University Hospital Center in Zagreb and from The Concord Repatriation General Hospital and Strathfield Private Hospital in Sydney. Samples from 49 healthy volunteers and from 8 patients with non-malignant pleural diseases served as controls. Circulating FGF18 was measured by enzyme-linked immunosorbent assay and correlated to clinical, pathologic and radiologic parameters.
Results:
Plasma FGF18 level was significantly elevated in MPM patients vs. healthy controls (P<0.0001). A slight increase of circulating FGF18 level was also detected in patients with pleuritis or fibrosis (vs. control, P=0.0067). Sarcomatoid (n=7) morphology was associated with high FGF18 levels when compared to the epithelioid (n=77) histology (P=0.0064). Importantly, MPM patients presenting with FGF18 levels below the median had a significantly longer overall survival when compared to those with high FGF18 levels (median survival 625 versus 382 d, P=0.0038). Data on multivariate analysis, disease-free survival, correlation with other biomarkers and tumor volume will be presented at the conference.
Conclusion:
Our findings reveal that FGF18 is a promising blood-derived candidate biomarker in MPM. Furthermore FGF18 may support the histological classification of MPM and the identification of MPM patients with poor prognosis. .
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OA02.05 - Expression of miR-223 in Mesothelioma Xenografts Originates from Stromal Cells in the Tumour Microenvironment (ID 5875)
11:45 - 11:55 | Author(s): G. Reid
- Abstract
- Presentation
Background:
Malignant pleural mesothelioma (MPM) is an aggressive cancer caused by asbestos exposure with limited therapeutic options. Dysregulated microRNAs play an important role in MPM biology and candidate microRNAs have been investigated as diagnostic and prognostic biomarkers or as a potential treatment targets. The role of miR-223 has previously been investigated in MPM tumour cells and was shown to act as a tumour suppressor by regulating cell mobility. Previous research indicated miR-223 to be primarily expressed by myeloid progenitor derived cells during differentiation of granulocytes and monocytes. This suggests miR-223 might have a more significant role in the inflammatory response during tumourigenesis. In this study we aimed to investigate the origin of miR-223 using mesothelioma xenograft and syngraft models.
Methods:
Human and mouse mesothelioma cell line-derived xenograft (MSTO-H211 and H226) and syngraft (AB1) models were established. MicroRNA profiles of xenografts were compared against profiles of their corresponding in vitro cultured cells to determine candidates. RT-qPCR using TaqMan MicroRNA assays was used to validate expression levels of miR-143-3p, miR-214-3p and miR-223-3p in tumour xenografts and syngrafts with those in corresponding cell lines in vitro. Species-specific ddPCR analysis was performed on RNA from xenograft tumours to determine the expression of human and mouse pri-miR-223.
Results:
MicroRNA profiles of xenograft tumours showed significant upregulation (p < 0.05) of miR-143-3p, miR-214-3p and miR-223-3p compared to corresponding in vitro mesothelioma cell lines. Only miR-223 showed significant upregulation in both xenograft and syngraft tumours compared to corresponding in vitro mesothelioma cell lines (>10000-fold increase). Other microRNAs were not significantly different between cell lines and tumours. RNA isolated from xenograft tumours contained significantly more mouse pri-miR-223 than human pri-miR-223 (p < 0.001), with only minimal expression levels of human tumour pri-miR-223 within xenograft tumours.
Conclusion:
Mature miR-223 is significantly overexpressed in xenograft tumours compared to corresponding in vitro mesothelioma cell lines suggesting stromal contribution. Species-specific pri-miRNA confirmed miR-223 is almost exclusively expressed by the mouse stromal cells in xenograft tumours. Ultimately, localising the expression of miR-223 to specific cell types (such as myeloid derived cells) through in situ hybridisation should help identify a more biologically relevant role for miR-223 in the tumour microenvironment.
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OA22 - Novel Trials and Biomarkers in Malignant Pleural Mesothelioma (ID 403)
- Event: WCLC 2016
- Type: Oral Session
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:H. Pass, N. Van Zandwijk
- Coordinates: 12/07/2016, 14:20 - 15:50, Strauss 3
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OA22.06 - Refinement of the Prognostic miR-Score for Use in Diagnostic Specimens from Chemo-Naïve Malignant Pleural Mesothelioma Patients (ID 5045)
15:05 - 15:15 | Author(s): G. Reid
- Abstract
- Presentation
Background:
A 6-microRNA signature (miR-Score, Kirschner et al 2015) was previously demonstrated to show high prognostic accuracy in a series of surgical specimens (with and without induction chemotherapy). In the present study we investigated these microRNAs in an independent cohort of MPM patients all treated with induction chemotherapy followed by extrapleural pneumonectomy (EPP). The main focus of the study was to evaluate the possible effects of induction chemotherapy on microRNA expression and to refine and validate the miR-Score for use in chemo-naïve diagnostic specimens.
Methods:
We identified a cohort of 120 MPM patients who received chemotherapy followed by EPP between 1999 and 2014 at University Hospital Zurich. At present microRNA analysis (RT-qPCR) has been carried out in 34 pairs of chemo-naïve (diagnostic biopsy) and chemo-treated (EPP) specimens. Paired-samples t-test was employed to determine differences in microRNA expression pre- and post-chemotherapy. Accuracy of the miR-Score in predicting a good prognosis (>20 months survival post-surgery) was evaluated by ROC curve analysis. In addition, binary logistic regression modelling was used to build a refined miR-Score.
Results:
Applying the miR-Score to chemo-naïve diagnostic specimens revealed an area under the ROC curve (AUC) of 0.65 (95% CI: 0.46-0.84), and the same analysis on the EPP specimens gave an AUC of 0.57 (95% CI: 0.37-0.77). Therefore, the accuracy of the miR-Score was lower than observed in the previous study. However, pairwise comparison of microRNA expression before and after chemotherapy showed that although not reaching statistical significance, the levels of several microRNAs were lower following induction chemotherapy. We next employed binary logistic regression modelling on microRNA levels in chemo-naïve tissue to determine whether a refined microRNA signature less susceptible to chemotherapy-induced changes could be created. A refined miR-Score consisting of miR-221 and miR-30e, the two microRNAs least affected by chemotherapy, achieved AUCs of 0.77 (95% CI: 0.61-0.94) and 0.80 (95% CI: 0.64-0.96) in diagnostic and EPP specimens, respectively. When applied to samples from the previous study, the refined score resulted in an AUC of 0.72 (95% CI: 0.54-0.90).
Conclusion:
This validation and refinement study has shown that the expression of several miR-Score microRNAs appears to be affected by standard chemotherapy. A refined miR-Score was generated which is less susceptible to the effect of chemotherapy and may have prognostic value when applied to diagnostic specimens. Further validation in additional paired samples and investigation of the effect of cisplatin, pemetrexed and gemcitabine on microRNA expression are ongoing.
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P1.05 - Poster Session with Presenters Present (ID 457)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Early Stage NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.05-021 - circRNAs: Potential Novel Biomarkers for the Early Detection of Lung Cancer (ID 5020)
14:30 - 14:30 | Author(s): G. Reid
- Abstract
Background:
Lung cancer is the leading cancer killer globally. Cancers such as colon, breast, and prostate all have relatively reliable early detection tests. In contrast, lung cancer does not. If caught early, lung cancer has a much better prognosis. Non-invasive or minimally invasive tools to improve early detection of lung cancer represents a critical unmet need. Analysis of the human transcriptome indicates that a mere 2% of the genome corresponds to protein coding transcripts, yet ~ 75% of the genome is transcribed. It is now well established that these non-coding RNAs (ncRNAs) play important regulatory functions within the cell and their expression are often altered in cancer. Circular RNAs (circRNAs) are a species of ncRNAs. They are abundant, conserved and demonstrate cell-type specific expression patterns. Moreover, they are extremely stable with half-life’s greater than 48 hours, are resistant to degradation by RNA exonucleases, and have been shown to play important roles in cancer. Taken together these suggest that circRNAs could potentially be important biomarkers in early lung cancer diagnosis.
Methods:
Total RNAs isolated from a panel of matched normal/tumour NSCLC adenocarcinoma (Stage IA/IB) samples (n=6) were probed for circRNAs using the Arraystar circRNA microarray. Survival was assessed on linear mRNAs with associated circRNAs using KM-Plot.
Results:
Interim analysis of the data has identified n=206 circRNAs with a 2-fold difference in expression between their matched normal vs. tumour counterparts. Principal Component Analysis (PCA) demonstrated a clear separation of the samples (Tumour vs. Normal). Self-Organizing Maps (SOMs) analysis generated distinctive SOMS clusters of circRNAs, while associated linear pathway enrichment for microRNA and transcriptional binding motifs identified several additional potential networks. Moreover, an analysis of linear mRNAs associated with 10 circRNAs with altered expression in adenocarcinomas found that these mRNAs were linked to overall survival, and that the majority were adenocarcinoma specific.
Conclusion:
Altered levels of a number of circRNAs were associated with lung adenocarcinoma. A separate cohort of squamous cell carcinomas is currently being assessed for circRNAs. At present we are validating the expression of these circRNAs in a larger cohort of specimens, and assessing whether or not these are detectable in plasma/serum from the same individuals. Overall, circRNAs may represent novel potential biomarkers for the detection of NSCLC, and may provide additional critical basic knowledge regarding the development and biology of NSCLC.
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P3.03 - Poster Session with Presenters Present (ID 473)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 3
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.03-002 - Inducible Changes in Cell Morphology and Gene Expression Reflecting the Histological Subtypes of Mesothelioma (ID 5405)
14:30 - 14:30 | Author(s): G. Reid
- Abstract
Background:
Malignant pleural mesothelioma (MPM) represents an aggressive malignancy with dismal prognosis and limited therapeutic options. MPM occurs in three main histological subtypes: epithelioid, sarcomatoid and biphasic, which are characterized by differences in morphological growth pattern, aggressiveness and patient prognosis. However, the mechanisms and causes responsible for the different cell morphologies are poorly understood. Epithelial-mesenchymal transition (EMT) has been implicated in cancer progression and chemoresistance, but its role in MPM is not well understood. Fibroblast growth factor (FGF) signals promote cell growth, survival and aggressiveness in several tumors including mesothelioma. Aim of this study was to characterize growth factor-induced, EMT-like changes with respect to the MPM histological subtypes.
Methods:
Morphological and behavioral changes of treated cell models were analyzed by morphometry, immunoblotting and functional assays. Alterations in gene or microRNA expression were evaluated via qPCR and array hybridization. Pathway enrichment analysis was based on KEGG.
Results:
In several cell lines established from biphasic MPM, treatment with FGF2 and EGF induced morphological changes reminiscent of EMT and aggressive behavior such as scattering, increased migration, proliferation and invasiveness. Inhibition of the fibroblast growth factor receptors (FGFR) or the MAPK axis via small-molecule inhibitors could prevent these changes and, in cell lines with sarcomatoid-like shape, reverse scattering and induce a more epithelioid morphology. Comparable results were obtained using an engineered FGFR1 enabling contactless activation via blue light. Analyses of genes and microRNAs regulated by FGF2 or EGF showed an overlap with previously established EMT markers but also identified several novel potential markers such as MMP1, ESM1, ETV4, PDL1, ITGA6 or BDKRB2. Blocking the FGFR or MAPK pathways resulted in the opposite regulation of these genes. Inhibition of MMP1 via siRNAs or pharmacological inhibitors prevented FGF2-induced scattering and invasiveness. In unsupervised clustering, the gene expression profiles of solvent- or cytokine-treated cells were associated with those of epithelioid and sarcomatoid MPM, respectively. Immunohistochemistry showed an association of MMP1 as well as phospho-ERK with the sarcomatoid part of tissue specimens from biphasic tumors. Pathway enrichment analysis of differentially expressed genes as well as the targets of altered microRNAs after FGF2 treatment showed that the regulated genes are assigned to categories important for cell growth and aggressive behavior.
Conclusion:
Our data characterize FGFR-mediated signals as important players in MPM aggressiveness and the morphological and behavioral plasticity of mesothelioma cells, leading to a better understanding of the link between the MPM histological subtypes and their influence on patient outcome.
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P3.03-007 - miR-137 Acts as a Tumour Suppressor via the Down-Regulation of YB-1 in Malignant Pleural Mesothelioma (ID 5579)
14:30 - 14:30 | Author(s): G. Reid
- Abstract
Background:
Malignant pleural mesothelioma (MPM) continues to increase in incidence worldwide and has limited therapeutic options. MPM displays characteristic changes in gene expression, including noncoding RNAs such as microRNAs, which have potential therapeutic relevance. One such miRNA is miR-137, a tumour suppressor whose promoter region is frequently methylated in other cancers and lies in in a commonly deleted chromosomal region in MPM (1p21-23). A potential role for miR-137 has yet to be investigated in MPM. One known target of miR-137 is YB-1, a multifunctional protein often up-regulated in other aggressive cancers, where elevated YB-1 levels are linked to poor clinical outcomes. This study investigates the causes of miR-137 suppression, the relationship between miR-137 and YB-1, one of its targets, as well as their roles in MPM cell growth and malignant behaviour.
Methods:
Basal expression of miR-137 and YB-1 was determined in 13 MPM cell lines by RT-qPCR and immunoblotting. Cells were treated with 5’Aza-cytidine and RT-qPCR was conducted to link methylation with miR-137 suppression. Copy number variation (CNV) was investigated by ddPCR. Cells were transfected with miR-137 mimic and subsequent YB-1 expression was investigated using RT-qPCR. Proliferation, colony formation and wound-healing assays were conducted after transfection with miR-137 mimics or YB-1-specific siRNAs.
Results:
miR-137 was absent in 4 MPM cell lines (p<0.01) and was up-regulated in response to 5’Aza-cytidine treatment in these lines, as well as other lines with low basal expression. Copy-number loss was evident in 5 cell lines and gain was present in 2. Increasing levels of miR-137 generally inhibited MPM cell migration, proliferation and colony formation. miR-137 mimics significantly down-regulated YB-1 expression, while YB-1 protein was overexpressed in the majority of MPM cell lines, compared to MeT-5A. YB-1 knock-down resulted in dose-dependent growth inhibition over 120 hours, reduced colony formation and also decreased cell migration. Effects were more pronounced in those cell lines showing high YB-1 protein levels.
Conclusion:
Our results show that methylation and CNV are likely to play a role in miR-137 down-regulation in MPM and that miR-137 acts as a tumour suppressor in MPM through at least in part the down-regulation of YB-1. We also demonstrated that YB-1 is commonly overexpressed and plays a role in proliferation and migration. These results imply a direct relationship between miR-137 and YB-1 expression, a biological interaction that may prove a useful target in developing future therapeutic approaches in MPM.
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P3.03-008 - Hypoxia-Induced Changes in microRNA Levels Contribute to Drug Resistance in a 3D Model of Malignant Pleural Mesothelioma (ID 5867)
14:30 - 14:30 | Author(s): G. Reid
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related thoracic cancer. Chemotherapy is the most frequent treatment option but almost every patient will be confronted with recurrence of disease and drug resistance. Previous studies have used 3D spheroid cultures to investigate drug response in MPM. We showed that microRNAs are important players in MPM biology and that they contribute to the response of MPM cells to some chemotherapy drugs. In the current study we aimed to investigate the role of microRNAs in the drug resistance of a 3D spheroid model of MPM.
Methods:
MPM cells were grown in standard 2D culture or as 3D spheroids in low adherence round bottom multi-well plates. The structure of the spheroids was confirmed by conventional and scanning electron microscopy. MicroRNA expression was profiled using TaqMan Low Density Arrays. RT-qPCR and droplet digital PCR were used to validate candidate microRNAs. HIF1a expression was examined in MPM spheroids using immunofluorescence staining. Drug cytotoxicity was investigated in both 2D and 3D cultures using standard proliferation assays, and the effect of drugs on gene expression was analysed. MicroRNA mimics and siRNAs were used to determine the influence of microRNA and HIF1a expression on drug resistance.
Results:
In our adapted model of 3D cell growth, MPM cell lines formed spherical 3D structures, in contrast to the donut shapes reported with other models. MPM cells in these spheroids were more resistant to cisplatin and gemcitabine when compared to cells grown in 2D cultures. Immunofluorescence revealed a hypoxic gradient with high HIF1a expression observed in the centre of the spheroids. Spheroids also exhibited a significant up-regulation of miR-210, miR-21, miR-378a, miR-195 and miR-146b, and down-regulation of miR-320b and miR-1225b. Transfecting MPM cells in 2D culture with miR-210 or miR-21 mimics resulted in increased drug resistance, whereas HIF1a knockdown inhibited spheroid formation and decreased drug resistance. Spheroids displayed higher expression of the ABCG2 drug pump, and ABCG2 was also up-regulated in cisplatin and gemcitabine treated MPM cells.
Conclusion:
Our spheroid model revealed a clear impact of hypoxia on gene expression in MPM cells. Hif1a was highly expressed in the hypoxic centre of the spheroids and is an upstream regulator of the microRNAs we found to be differentially expressed. Pharmacologic and genetic modulation of microRNA and HIF1a levels altered drug resistance in MPM cells, suggesting a link between hypoxia, microRNAs and drug resistance in MPM.
