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Michaela B Kirschner
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MA17 - Molecular Mechanisms and Therapies (ID 143)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Eloisa Jantus-Lewintre, Hongbin Ji
- Coordinates: 9/09/2019, 15:45 - 17:15, Melbourne (1991)
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MA17.03 - Importance of Cullin4 Ubiquitin Ligase in Malignant Pleural Mesothelioma (Now Available) (ID 2349)
15:45 - 17:15 | Author(s): Michaela B Kirschner
- Abstract
- Presentation
Background
Loss of the tumor suppressor NF2 is frequent in malignant pleural mesothelioma (MPM). NF2 suppresses tumorigenesis in part by inhibiting Cullin4 ubiquitin ligase (CUL4) complex. Here we aimed to evaluate an importance of CUL4 in MPM.
Method
We evaluated the expression of CUL4A and CUL4B in tissue microarrays using immunohistochemistry. We tested the efficacy of cullin inhibition by pevonedistat, a small molecule inhibiting cullin neddylation, in 13 cell lines and 3 primary cells in 2D and 3D culture. Four groups of SCID mice haboring intraperitoneal (ip.) pevonedistat sensitive (MSTO211H) or resistant (ACC-Meso1) cell lines were treated with pevonedistat (50 mg/kg; ip.) on a 5day on/5day off schedule for 3 cycles. Treatment efficacy was assessed by means of overall survival.
Result
CUL4B expression was associated with clinical outcomes (figure 1). Five MPM cell lines (38%) were highly sensitive to pevonedistat (IC50<500 nM). This remained true in 3D spheroid culture. The treatment induced S/G2 cell cycle arrest and accumulation of cells undergoing DNA re-replication (containing >4N DNA content) known to be mediated by p21 and CDT1 accumulation. Indeed the accumulation of p21 and CDT1 was more pronounced in pevonedistat sensitive cell lines after the treatment. Two of primary cells (67%) were sensitive to pevonedistat and also showed higher CDT1 accumulation following the treatment compared to the resistant cells. In vivo, pevonedistat treatment significantly prolonged survival of mice bearing both sensitive and resistant MPM tumors. Pevonedistat treatment reduced growth (phosphorylated histoneH3 positive) in pevonedistat sensitive tumor but increased apoptosis (cleaved–caspase3 positive) in pevonedistat resistant tumor.
Conclusion
High CUL4B expression may play a role in MPM progression. Inhibition of cullins by pevonedistat induced growth arrest and DNA re-replication strongly in a subset of MPM. The major mechanism seems to be mediated by p21 and CDT1 accumulation in vitro. Investigation of mechanisms in vivo is ongoing.
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P1.06 - Mesothelioma (ID 169)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Mesothelioma
- Presentations: 2
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.06-15 - Safety of Irradiation Combined with Intracavitary Cisplatin-Fibrin After Lung-Sparing Surgery in a Rat Model of Mesothelioma (ID 2372)
09:45 - 18:00 | Author(s): Michaela B Kirschner
- Abstract
Background
To investigate feasibility and toxicity of new localized therapeutic treatment combinations for malignant pleural mesothelioma (MPM), we performed lung-sparing surgery followed by cisplatin-fibrin application and hemithoracic irradiation in an orthotopic immunocompetent rat model of MPM.
Method
Male F344 rats (n=9) were implanted sub-pleurally (parietal pleura) with 1 million rat mesothelioma cells (IL45-luciferase). Formed tumor nodules confirmed by IVIS bioluminescence imaging (BLI) were resected on day 9 after implantation. Following resection, animals were treated with local-intracavitary cisplatin-fibrin or placebo (NaCl-fibrin). Three days later, CT guided local irradiation of the former tumor region, resembling IMRT in human patients, was performed. Irradiation was given in a single high dose application using the image-guided stereotactic small animal irradiation X-RAD SmART (small animal radiotherapy) and image guided biological irradiator PXi (precision X-Ray) with precise localization.
Treatment schemes after tumor resection were as followed:
i) Intracavitary cisplatin-fibrin application (n=2)
ii) Irradiation with 10 Gy (n=2)
iii) Irradiation with 20 Gy (n=1)
iv) Intracavitary cisplatin-fibrin plus 10 Gy radiotherapy (n=2)
v) Intracavitary cisplatin-fibrin plus 20 Gy radiotherapy (n=2)
Wellbeing of the animals was monitored daily until the predefined termination criteria were reached. Particular attention was given to possible irradiation toxicity related pulmonary side effects and weight loss.
Result
We successfully treated 1-2 animals per group according to the methods above. The irradiation was performed after visualization of the tumor with BLI- and CT-imaging to ensure an individual treatment plan. None of the animals, whether with radiotherapy alone or in combination with cisplatin-fibrin application, showed any signs of pulmonary side effects. In addition, none had reduced pulmonary functions, measured by increased breathing or the appearance of blue or white colored ear/extremities/eyes assuming desaturation. Furthermore, neither significant body weight loss of ≥ 15%, deterioration of body conditioning score nor of the activity score were observed in the immediate post-interventional phase. In all animals, termination endpoint was reached because of tumor relapse.
Conclusion
In this pilot study, we have shown that irradiation alone and in combination with local intracavitary cisplatin-fibrin application in rats is safe and feasible up to a dosage of 20 Gy. The efficacy of the various treatment schemes and a possible radio-sensitizing effect by intracavitary cisplatin is currently being evaluated in the same animal model.
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P1.06-18 - MicroRNA Expression Is Linked to Response of Malignant Pleural Mesothelioma to Cisplatin-Pemetrexed Chemotherapy (ID 2367)
09:45 - 18:00 | Presenting Author(s): Michaela B Kirschner
- Abstract
Background
Treatment of malignant pleural mesothelioma is difficult due to a high intrinsic drug resistance of these tumors. Currently, platinum-based chemotherapy represents the backbone of MPM treatment. However, only approximately 40% of patients respond to this therapy, and true predictors for response have yet to be identified. Towards this end, we here investigate the expression of microRNAs in responders and non-responders to chemotherapy.
Method
FFPE tumour samples were available from 32 MPM patients, who showed either partial response (PR, N=21) or progressive disease (PD, N=11) following 3-4 cycles of cisplatin-pemetrexed chemotherapy. RT-qPCR based microRNA profiling was performed on chemo-naïve tissue of 5 PD and 5 PR patients using TaqMan Low Density Arrays (TLDAs, Thermo Fisher), which cover the expression of 754 microRNAs. Candidate microRNAs with differential expression (P≤0.05 Mann-Whitney Test) were then measured in the remaining samples using microRNA-specific RT-qPCR. Expression of these microRNAs was also assessed in post-chemotherapy specimens (obtained during extrapleural pneumonectomy) and compared to that in chemo-naïve samples. In addition, for two candidates, preliminary in vitro experiments investigating the effect of microRNA overexpression (transfection with microRNA mimics) on cell growth were performed.
Result
TLDA-based profiling identified 35 microRNA with differential expression between patients with PD and PR following cisplatin-pemetrexed chemotherapy. The majority of these microRNAs showed higher expression in patients who showed no reponse to therapy. In an initial step, 8 candidates identified from the profiling (miR-145, miR-193a-3p, miR-30a-3p, miR-24, miR-380-5p, miR-494, miR-625-3p, miR-221-3p) were further evaluated in additional 16 PR and 6 PD samples. This confirmed a trend towards differential expression for miR-145 (p=0.08). Interestingly, when comparing expression pre- and post-chemotherapy, levels of miR-145 significantly decreased in patients with PD, while they remained stable in PR. Lack of validation of other microRNAs could be the result of the low number of cases with PD in this preliminary validation set, and additional samples will included. For miR-221-3p and miR-380-5p, preliminary analysis in vitro showed that overexpression in established MPM cell lines results in an increased sensitivity towards cisplatin.
Conclusion
Taken together, our data show that several microRNAs show trends towards differential expression between responders and non-responders to chemotherapy. Overall, higher expression appears to be linked to PD under cisplatin-pemetrexed, however further in-depth investigations are required. Furthermore, preliminary in vitro data suggest that altering expression of specific microRNAs has the potential to increase sensitivity of MPM to chemotherapy.
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PL04 - Food for Thought in the Management of Thoracic Malignancies (ID 91)
- Event: WCLC 2019
- Type: Plenary Session
- Track: Advanced NSCLC
- Presentations: 1
- Now Available
- Moderators:Daniel SW Tan, Ramon Rami-Porta
- Coordinates: 9/10/2019, 16:15 - 17:00, Barcelona (2005)
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PL04.04 - Prognostic Factors in Malignant Pleural Mesothelioma (Now Available) (ID 3598)
16:15 - 17:00 | Presenting Author(s): Michaela B Kirschner
- Abstract
- Presentation
Abstract
Malignant pleural mesothelioma (MPM) is a disease for which we are facing difficulties and challenges at many levels from diagnosis over treatment selection and prediction of treatment response to prediction of the prognosis of individual patients. Besides the necessities to obtain an accurate and early diagnosis and to be able to predict the response to a specific treatment, it is equally important to be able to estimate the overall prognosis of a patient as this will also affect treatment decisions.
Looking at the research efforts of the last two decades, we see an abundance of studies investigating prognostic markers for MPM, yet to date the most reliable predictors of disease outcome are still the clinical and pathological parameters, which have been used in the past. Probably the most accurate prognostic factor for MPM is the histopathological subtype, with epithelioid MPM being associated with the best prognosis followed by the biphasic subtype and sarcomatoid histology. In addition, it is well recognised that patients presenting with less advanced disease, of younger age and female gender are generally doing better. Some of these factors were combined with additional proposed predictors of outcome into scoring systems suggested by the European Organization for Research and Treatment of Cancer (EORTC, [1]) and the Cancer and Leukemia Group B (CALGB, [2]). Both scores have been subsequently validated and still hold true today, over 20 years later.
Since then, many studies have attempted to identify additional prognostic biomarkers. One area of extensive research is the investigation of changes in blood cell ratios, which can be linked to the inflammatory and/or nutritional status of the patients [3, 4]. In terms of inflammation-related indicators of poor outcome, elevated C-reactive protein (CRP), a high neutrophil-to-lymphocyte ratio, and a low lymphocyte-to-monocyte ratio (LMR) have been proposed. Some of these inflammatory markers have also been combined with factors reflecting the nutritional status of patients. For example, has the combination of CRP and albumin been suggested to have prognostic value, as shown in the CRP-to-albumin ratio (CAR) and the modified Glasgow Prognostic Score (mGPS). Another proposed combination is that of albumin and lymphocytes into the Prognostic Nutritional Index (PNI). Additionally, radiological factors, such as tumour volume or pleural thickness measured by CT or MRI alone or as part of prognostic scores (e.g. in combination with other factors such as in the multimodality prognostic score [5]), as well as radiomics approaches have shown prognostic potential [6]. While many of these proposed prognostic factors are often routinely collected during standard clinical work-up and blood tests, prospective testing in a clinical setting has yet to be attempted.
A second area of extensive research in the last decade has been molecular factors, namely the expression of proteins, genes, and microRNAs [3]. Initially, the majority of studies focused on the potential prognostic role of protein expression in tumour tissue. Here, rather frequently, the expression of tyrosine kinases such as epidermal growth factor receptor (EGFR) and c-Met has been investigated, due to the additional potential of targeting those using tyrosine kinase inhibitors. In addition, many cell cycle and apoptosis-related proteins such as p21, p53, survivin or PTEN were evaluated, but in many cases these proteins did not reach significance in multivariate analyses, highlighting that they do not represent independent markers. Other proteins, such as ERCC1 and TS, the target of the antimetabolite drug pemetrexed, did not show consistent results between various studies, hence none of these proteins are used routinely in the clinic. On the level of gene expression, already 15 years ago a 4-gene signature was proposed, which was subsequently independently validated, but never in a prospective fashion. In addition to gene expression, microRNAs have also been proposed to hold prognostic value, but again, independent validation is thus far lacking.
While many candidate prognostic biomarkers have been proposed, these tend to be dependent on the histological subtype, and the identification of factors predictive of outcome within the individual histological subgroups of MPM patients remains a major challenge. However, with more genetic profiling of larger datasets becoming available also in MPM, investigators have started to address this issue. By aiming to genetically subclassify pathologically purely epithelioid or sarcomatoid tumours, this resulted in the C1/C2 classification [7] and the e-score and s-score classification [8], as well as the 4 cluster iCluster classification generated based on TCGA data [9]. Besides providing potential novel prognostic factors, the molecular characterization of MPM can also provide us with urgently needed deeper insights into the biology of the different subgroups of MPM, which is likely to allow us to identify novel treatment targets together with respective markers predictive of response.
Nevertheless, these exciting recent findings remain to be independently validated, most importantly in a prospective fashion. A closer look at the literature of the last two decades, however, shows us that rather than aiming at validation of proposed prognostic factors, we are inclined to identify novel candidates. In order to move these promising novel candidates from the bench to the bedside, international collaboration to increase cohort sizes as well as prospective validation will be crucial.
References:
1. Curran, D., et al., J Clin Oncol, 1998. 16(1): p. 145-52.
2. Herndon, J.E., et al., Chest, 1998. 113(3): p. 723-31.
3. Davidson, B., Hum Pathol, 2015. 46(6): p. 789-804.
4. Yamagishi, T., et al., Lung Cancer, 2015. 90(1): p. 111-7.
5. Opitz, I., et al., J Thorac Oncol, 2015. 10(11): p. 1634-41.
6. Armato, S.G., 3rd, et al., Lung Cancer, 2019. 130: p. 108-114.
7. de Reynies, A., et al., Clin Cancer Res, 2014. 20(5): p. 1323-34.
8. Blum, Y., et al., Nat Commun, 2019. 10(1): p. 1333.
9. Hmeljak, J., et al., Cancer Discov, 2018. 8(12): p. 1548-1565.
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