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M.B. Kirschner
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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: 4
- 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 - 12:30 | Author(s): M.B. Kirschner
- 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)
11:00 - 12:30 | Author(s): M.B. Kirschner
- 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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OA02.04 - Discussant for OA02.01, OA02.02, OA02.03 (ID 6952)
11:00 - 12:30 | Author(s): M.B. Kirschner
- Abstract
- Presentation
Abstract not provided
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OA02.05 - Expression of miR-223 in Mesothelioma Xenografts Originates from Stromal Cells in the Tumour Microenvironment (ID 5875)
11:00 - 12:30 | Author(s): M.B. Kirschner
- 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)
14:20 - 15:50 | Author(s): M.B. Kirschner
- 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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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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P3.03-001 - Targeting Cullin Ubiquitin Ligase Leads to Growths Arrest in Malignant Pleural Mesothelioma Cells (ID 5486)
14:30 - 15:45 | Author(s): M.B. Kirschner
- Abstract
Background:
Mutation of the tumor suppressor gene NF2 was detected in 30-40% of malignant pleural mesothelioma (MPM) patients. NF2 suppresses tumorigenesis in part by inhibiting Cullin4 ubiquitin ligase. Cullin4A (CUL4A) gene amplification and its’ overexpression has been detected in MPM cell lines and tumors. We hypothesized that cullin4 is a potential treatment target for MPM. Cullins’ activity can be blocked by the inhibition of neddylation, a post-translational modification for cullins. In this study we assessed the efficacy of pevonedistat, an inhibitor of protein neddylation.
Methods:
Thirteen MPM cell lines and 3 MPM primary cells grown in monolayer (2D) were employed to assess the efficacy of pevonedistat in vitro compared to normal mesothelial cells, using MTT assay. The expression of cullins was assessed by quantitative real time PCR and western blot. Cell cycle was analyzed by flow cytometry. Four cell lines were cultured in multicellular spheroid (3D) format and measured for viability by acid phosphatase assay.
Results:
Five MPM cell lines overexpressing CUL4A are highly sensitive to pevonedistat (figure1). The treatment induced G2 cell cycle arrest and accumulation of cells containing >4N DNA content, representing cells undergoing DNA re-replication. DNA re-replication is known to be mediated by the accumulation of a DNA replication licensing factor, CDT1. Indeed, higher CDT1 accumulation was detected in the sensitive compared to the resistant cell lines. All primary cells showed no CUL4A overexpression compared to normal mesothelial cells, nonetheless 2 of them were sensitive to pevonedistat. Interestingly, these cells exhibited higher levels of neddylated (activated) CUL4A and higher CTD1 accumulation following the treatment. Cells lines overexpressing CUL4A remained sensitive to pevonedistat when grown in 3D spheroids. Figure 1
Conclusion:
Inhibition of cullins by pevonedistat induced growth arrest preferentially in MPM cells overexpressing CUL4A in 2D and 3D cultures. The major mechanism seems to be mediated by DNA re-replication induced by CDT1 accumulation.
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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 - 15:45 | Author(s): M.B. Kirschner
- 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.
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P3.03-044 - Is Toxicity Increased by Adding Intraoperative Chemotherapy to Preoperative Induction Chemotherapy for Mesothelioma Patients? (ID 5945)
14:30 - 15:45 | Author(s): M.B. Kirschner
- Abstract
Background:
Intracavitary application of chemotherapy after mesothelioma resection is intended to prevent local recurrence. In the present study, we compared hematological and renal toxicity of patients treated with or without additional intracavitary cisplatin-fibrin after (extended) pleurectomy/decortication ((e)P/D) and previous i.v. induction chemotherapy with cisplatin/pemetrexed (CTX).
Methods:
Hemoglobin values, platelet count as well as urea, creatinine, sodium, potassium and magnesium values of 32 patients treated with (e)P/D were compared to the first five patients receiving 44mg/m[2] BSA intracavitary cisplatin-fibrin in our INFLuenCe-Meso phase II trial (www.clinicaltrial.gov NCT01644994). The median time between last cycle of CTX and surgery was 6 weeks (1-14 weeks). The blood values were measured on postoperative day (POD) 1 to 5, 7, 10 and 14 if available. For statistical comparison Mann-Whitney U test was used.
Results:
No significant difference between the 2 groups was observed in the preoperative baseline blood samples. On POD3 hemoglobin dropped significantly more in patients with cisplatin-fibrin application (Figure 1A). However, the use of blood transfusion was not significantly different in both groups. Also sodium, potassium (Figure 1B) and magnesium levels were significantly lower in the study patient group. Disorders in electrolytes were however never reflected in clinical symptoms and reached only in 3 patients a CTCAE level ≥3. There was no significant difference in platelet count, urea and creatinine levels. Figure 1
Conclusion:
The present analysis shows that additional intracavitary cisplatin-fibrin after eP/D and previous i.v. induction chemotherapy with cisplatin/pemetrexed can lead to electrolyte disorders and drop in hemoglobin concentration. However, none of the mentioned laboratory findings had a clinically significant impact on the patients’ postoperative course.
